Compositions and methods for expressing therapeutic agents

Abstract

Described herein are compositions and methods for expressing therapeutic agents.The compositions include engineered polypeptides that include natriuretic peptides linked to antibodies or fragments thereof.Also disclosed are AAV vectors that code for the engineered polypeptides and their therapeutic use for treating diseases such as glaucoma.

Description

[Technical field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 319,233, filed March 11, 2022; U.S. Provisional Patent Application No. 63 / 440,858, filed January 24, 2023; and U.S. Provisional Patent Application No. 63 / 441,643, filed January 27, 2023, the entireties of which are hereby incorporated by reference into this specification.

[0002] INCORPORATION BY REFERENCE

[0002] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, it is intended that the present specification supersede and / or take precedence over any such conflicting material. [Background technology]

[0003]

[0003] Natriuretic peptide precursor C is a prohormone composed of 126 amino acids. Natriuretic peptide precursor C can be further cleaved to produce a receptor-binding active form known as C-type natriuretic peptide (CNP), which can be one of three forms: CNP53 (CNP composed of 53 amino acids); CNP36 (CNP composed of 36 amino acids); or CNP22 (CNP composed of 22 amino acids). When CNP binds to its cognate receptors, natriuretic peptide clearance receptor B (NPR-B) and NPR-C, it activates membrane-associated guanylyl cyclase (GC), which catalyzes the enzymatic conversion of GTP to cyclic guanosine monophosphate (cGMP), a functional effector or second messenger. Due to its ability to activate cGMP, CNP has been utilized as a therapeutic agent. Summary of the Invention [Problem to be solved by the invention]

[0004]

[0004] However, truncated CNPs, such as CNP53, CNP36, or CNP22, have a half-life of only a few minutes, which limits their use to treat diseases or conditions. Therefore, there is a need to engineer CNPs to increase their stability. Similarly, there is still a need to engineer CNPs to treat diseases or conditions. [Means for solving the problem]

[0005]

[0005] Described herein in some aspects is an engineered polynucleotide comprising a viral vector, the viral vector comprising an expression cassette, the expression cassette encoding an engineered polypeptide comprising a natriuretic peptide covalently linked to an antibody or fragment thereof. In some embodiments, the natriuretic peptide comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 4. In some embodiments, the natriuretic peptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 4. In some embodiments, the natriuretic peptide comprises an amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or fragment thereof comprises a fragment crystallizable (Fc) region. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 6-8. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 6-8. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 6-8. In some embodiments, the natriuretic peptide is covalently attached to the N-terminus of the antibody or fragment thereof. In some embodiments, the natriuretic peptide is covalently attached to the C-terminus of the antibody or fragment thereof. In some embodiments, the natriuretic peptide is covalently attached to the antibody or fragment thereof by a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence comprising (GGGGS)n, where n is an integer between 0 and 20. In some embodiments, n is an integer between 4. In some embodiments, the engineered polypeptide comprises a protease cleavage site. In some embodiments, the protease cleavage site comprises a furin protease site.In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 131-140. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 131-140. In some embodiments, the engineered polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 131-140. In some embodiments, the natriuretic peptide comprises C-type natriuretic peptide (CNP) or a fragment thereof. In some embodiments, the CNP or a fragment thereof comprises 22 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises the C-terminal 22 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 36 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises the C-terminal 36 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 53 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises the C-terminal 53 contiguous bases of SEQ ID NO: 1. In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector comprises an AAV serotype comprising AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV serotype comprises AAV2. In some embodiments, the AAV vector encodes a modified AAV capsid. In some embodiments, the AAV vector comprises a second expression cassette. In some embodiments, the second expression cassette encodes a therapeutic agent. In some embodiments, the therapeutic agent comprises a hormone. In some embodiments, the therapeutic agent comprises an agonist of the natriuretic peptide receptor (NPR). In some embodiments, the therapeutic agent comprises an agonist of the cyclic GMP (cGMP) signaling pathway. In some embodiments, the therapeutic agent comprises a VEGF inhibitor.

[0006]

[0006] Described herein, in some aspects, is an engineered polypeptide comprising an antibody or fragment thereof operably linked to a natriuretic peptide, the antibody or fragment thereof comprising an amino acid sequence at least 80% identical to any one of SEQ ID NOs:6-8. In some embodiments, the natriuretic peptide comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs:1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs:1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence of any one of SEQ ID NOs:1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence at least 80% identical to SEQ ID NO:4. In some embodiments, the natriuretic peptide comprises an amino acid sequence at least 95% identical to SEQ ID NO:4. In some embodiments, the natriuretic peptide comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs:6-8. In some embodiments, the antibody or fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs:6-8. In some embodiments, the natriuretic peptide is covalently attached to the N-terminus of the antibody or fragment thereof. In some embodiments, the natriuretic peptide is covalently attached to the C-terminus of the antibody or fragment thereof. In some embodiments, the natriuretic peptide is covalently attached to the antibody or fragment thereof by a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence comprising (GGGGS)n, where n is an integer between 0 and 20. In some embodiments, n is an integer equal to 4. In some embodiments, the engineered polypeptide comprises a protease cleavage site. In some embodiments, the protease cleavage site comprises a furin protease site. In some embodiments, the engineered polypeptide comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 131-140.In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 131-140. In some embodiments, the engineered polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 131-140. In some embodiments, the natriuretic peptide comprises C-type natriuretic peptide (CNP) or a fragment thereof. In some embodiments, the CNP or a fragment thereof comprises 22 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises the C-terminal 22 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 36 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises the C-terminal 36 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 53 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises the C-terminal 53 contiguous bases of SEQ ID NO: 1.

[0007]

[0007] Described herein, in some aspects, is a cell comprising an engineered polynucleotide as described herein. In some embodiments, at least one fragment of the engineered polynucleotide is integrated into the genome of the cell. Also described herein, in some aspects, is a cell comprising an engineered polypeptide as described herein.

[0008]

[0008] Described herein, in some aspects, is a viral particle comprising an engineered polynucleotide described herein. In some embodiments, the viral particle comprises an AAV capsid. In some embodiments, the AAV capsid comprises a modified AAV capsid. In some embodiments, the modified AAV capsid comprises a modified AAV2 capsid.

[0009]

[0009] In some aspects, described herein are pharmaceutical compositions comprising an engineered polynucleotide described herein, an engineered polypeptide described herein, a cell described herein (e.g., a cell transduced with an engineered polynucleotide described herein), or a viral particle described herein. In some embodiments, the pharmaceutical composition is formulated for administration intrathecally, intraocularly, intravitreally, retinal, intravenously, intramuscularly, intraventricularly, intracerebral, intracerebellar, intraventricularly, intraparenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonary, intratracheal, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, inhalation spray form, intraluminal-GI route, or a combination thereof, to a subject in need of administration of the pharmaceutical composition. In some embodiments, the pharmaceutical composition is formulated for administration intravitreally, subretinally, or suprachoroidally. In some embodiments, the pharmaceutical composition is for treating an ocular disease or condition. In some embodiments, the pharmaceutical composition increases natriuretic peptide receptor-B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof in a subject in need of the pharmaceutical composition.

[0010]

[0010] Described herein, in some aspects, is a method of treating a disease or condition in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide described herein, an engineered polypeptide described herein, a cell described herein (e.g., a cell transduced with an engineered polynucleotide described herein), a viral particle described herein, a pharmaceutical composition described herein, or a combination thereof. Described herein, in some aspects, is a method of treating a disease or condition in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide described herein, an engineered polypeptide described herein, a cell described herein (e.g., a cell transduced with an engineered polynucleotide described herein), a viral particle described herein, a pharmaceutical composition described herein, or a combination thereof, wherein one administration step cures the disease or condition. Described herein, in some aspects, is a method of treating a disease or condition in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide described herein, an engineered polypeptide described herein, a cell described herein (e.g., a cell transduced with an engineered polynucleotide described herein), a viral particle described herein, a pharmaceutical composition described herein, or a combination thereof, wherein the administering step does not include daily administration.Described herein in some aspects is a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide described herein, an engineered polypeptide described herein, a cell described herein (e.g., a cell transduced with an engineered polynucleotide described herein), a viral particle described herein, a pharmaceutical composition described herein, or a combination thereof, wherein administering comprises weekly administration, biweekly administration, monthly administration, bimonthly administration, semi-annual administration, yearly administration, or a combination thereof. In some embodiments, the disease or condition comprises an eye disease. In some embodiments, the ocular disease is selected from the group consisting of ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, color vision disorders, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), Bardet-Biedl syndrome, Best disease, choroideremia, Leber's congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-vertebral dystrophy, vertebral-rod dystrophy, Oguchi disease, autosomal dominant radial drusen (Malattia leventinese (familial autosomal dominant drusen), blue-coloured monochromacy, or a combination thereof. In some embodiments, an engineered polynucleotide described herein, an engineered polypeptide described herein, a cell described herein (e.g., a cell transduced with an engineered polynucleotide described herein), a viral particle described herein, a pharmaceutical composition described herein, or a combination thereof increases natriuretic peptide receptor-B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof in a subject, thereby treating a disease or condition.In some embodiments, an engineered polynucleotide described herein, an engineered polypeptide described herein, a cell described herein (e.g., a cell transduced with an engineered polynucleotide described herein), a viral particle described herein, a pharmaceutical composition described herein, or a combination thereof increases the half-life of an agonist of natriuretic peptide receptor-B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof in a subject, thereby treating a disease or condition. In some embodiments, the half-life is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more, compared to the half-life of an endogenous agonist of natriuretic peptide receptor-B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof in the subject.

[0011]

[0011] Described herein in some aspects is a method of treating a disease or condition in a subject, comprising administering to the subject an engineered polynucleotide, wherein the engineered polynucleotide comprises a viral vector comprising an expression cassette for expressing in a cell of the subject an engineered polypeptide comprising an antibody or fragment thereof operably linked to a natriuretic peptide, wherein the cell expresses the engineered polypeptide, thereby treating the disease or condition. In some embodiments, the natriuretic peptide comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 4. In some embodiments, the natriuretic peptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 4. In some embodiments, the natriuretic peptide comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or fragment thereof comprises a fragment crystallizable (Fc) region. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 6-8. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 6-8. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence of ... natriuretic peptide is covalently attached to the N-terminus of the antibody or fragment thereof. In some embodiments, the natriuretic peptide is covalently attached to the C-terminus of the antibody or fragment thereof. In some embodiments, the natriuretic peptide is covalently attached to the antibody or fragment thereof by a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence that comprises (GGGGS)n, where n is an integer between 0 and 20. In some embodiments, n is an integer of 4.In some embodiments, the engineered polypeptide comprises a protease cleavage site. In some embodiments, the protease cleavage site comprises a furin protease site. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 131-140. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 131-140. In some embodiments, the engineered polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 131-140. In some embodiments, the natriuretic peptide comprises C-type natriuretic peptide (CNP) or a fragment thereof. In some embodiments, the CNP or a fragment thereof comprises 22 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises the C-terminal 22 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 36 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises the C-terminal 36 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 53 contiguous bases of SEQ ID NO: 1. In some embodiments, the CNP or fragment thereof comprises the C-terminal 53 contiguous bases of SEQ ID NO:1. In some embodiments, the engineered polynucleotide comprises an AAV vector. In some embodiments, the AAV vector comprises an AAV serotype comprising AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV serotype comprises AAV2. In some embodiments, the AAV vector encodes a modified AAV capsid. In some embodiments, the engineered polynucleotide is encapsulated in a viral particle. In some embodiments, the viral particle comprises an AAV capsid. In some embodiments, the AAV capsid is a modified AAV capsid. In some embodiments, the viral vector comprises a second expression cassette. In some embodiments, the second expression cassette encodes a therapeutic agent. In some embodiments, the therapeutic agent comprises a hormone. In some embodiments, the therapeutic agent comprises an agonist of the natriuretic peptide receptor (NPR).In some embodiments, the therapeutic agent comprises an agonist of the cyclic GMP (cGMP) signaling pathway. In some embodiments, the therapeutic agent comprises a VEGF inhibitor. The engineered polynucleotide is administered to the subject intrathecally, intraocularly, intravitreally, retina, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymal, subcutaneously, subretina, suprachoroidally, intratumorally, pulmonary, intratracheal, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, in an inhalation spray form, intraluminal-GI route, or a combination thereof. In some embodiments, the disease or condition comprises an eye disease. In some embodiments, the ocular disease is ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color vision disorders, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, traumatic glaucoma, Bardet-Biedl syndrome, Beth-Barr syndrome, glaucoma ... In some cases, the term "cataract surgery" refers to a condition in which the retina is in a drowning state, such as glaucoma, glaucoma, glaucoma, glaucoma, glaucoma syndrome, glaucoma ...

[0012]

[0012] Described herein in some aspects is a method of treating glaucoma in a subject, comprising administering to the subject an AAV2 vector, wherein the AAV2 vector encodes a natriuretic peptide covalently linked to an antibody or fragment thereof. In some embodiments, the natriuretic peptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:4. In some embodiments, the natriuretic peptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:4. In some embodiments, the natriuretic peptide comprises an amino acid sequence that is the amino acid sequence of SEQ ID NO:4. In some embodiments, the antibody or fragment thereof comprises a fragment crystallizable (Fc) region. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs:6-8. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs:6-8. In some embodiments, the antibody or fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs:6-8. In some embodiments, the antibody or fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs:6-8. In some embodiments, the natriuretic peptide is covalently linked to the N-terminus of the antibody or fragment thereof. In some embodiments, the natriuretic peptide is covalently attached to the C-terminus of the antibody or fragment thereof. In some embodiments, the natriuretic peptide is covalently attached to the antibody or fragment thereof by a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence comprising (GGGGS)n, where n is an integer between 0 and 20. In some embodiments, n is an integer equal to 4. In some embodiments, a protease cleavage site is adjacent to the natriuretic peptide and the antibody or fragment thereof. In some embodiments, the protease cleavage site comprises a furin protease site. In some embodiments, the natriuretic peptide comprises C-type natriuretic peptide (CNP) or a fragment thereof. In some embodiments, the CNP or a fragment thereof comprises 22 contiguous bases of SEQ ID NO:1. In some embodiments, the CNP or a fragment thereof comprises the C-terminal 22 contiguous bases of SEQ ID NO:1. In some embodiments, the CNP or a fragment thereof comprises 36 contiguous bases of SEQ ID NO:1.In some embodiments, the CNP or fragment thereof comprises 36 contiguous bases at the C-terminus of SEQ ID NO:1. In some embodiments, the CNP or fragment thereof comprises 53 contiguous bases at the C-terminus of SEQ ID NO:1. In some embodiments, the CNP or fragment thereof comprises 53 contiguous bases at the C-terminus of SEQ ID NO:1. In some embodiments, the AAV2 vector encodes a modified AAV capsid. In some embodiments, the AAV2 vector comprises a second expression cassette. In some embodiments, the second expression cassette encodes a therapeutic agent. In some embodiments, the therapeutic agent comprises a hormone. In some embodiments, the therapeutic agent comprises an agonist of the natriuretic peptide receptor (NPR). In some embodiments, the therapeutic agent comprises an agonist of the cyclic GMP (cGMP) signaling pathway. In some embodiments, the therapeutic agent comprises a VEGF inhibitor. In some embodiments, the AAV2 vector is encapsulated in an AAV viral particle prior to the administering step. In some embodiments, the AAV2 vector is administered to a subject in need of administration of the AAV2 vector intrathecally, intraocularly, intravitreally, retina, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymal, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonary, intratracheal, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, inhalation, inhalation spray form, intraluminal-GI route, or combinations thereof. In some embodiments, the AAV2 vector is administered intravitreally, subretinaly, or suprachoroidally. In some embodiments, administering the AAV2 vector increases natriuretic peptide receptor-B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or combinations thereof in the subject. In some embodiments, a single administration step of the AAV2 vector cures glaucoma. In some embodiments, administering the AAV2 vector does not include daily administration. In some embodiments, administering the AAV2 vector comprises weekly administration, biweekly administration, monthly administration, bimonthly administration, semi-annual administration, yearly administration, or a combination thereof.In some embodiments, administering the AAV2 vector increases the half-life of an agonist of natriuretic peptide receptor-B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof in the subject. In some embodiments, the half-life is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more than the half-life of an endogenous agonist of natriuretic peptide receptor-B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof in the subject. In some embodiments, the glaucoma comprises neovascular glaucoma (NG), glaucoma, traumatic glaucoma, or a combination thereof.

[0013]

[0013] Described herein, in some aspects, is an engineered polynucleotide comprising an AAV vector comprising one or more expression cassettes, the one or more expression cassettes encoding a peptide. Described herein, in some aspects, is an engineered polynucleotide comprising an AAV vector comprising one or more expression cassettes, the one or more expression cassettes encoding an engineered polypeptide comprising an antibody or fragment thereof operably linked to a peptide. In some embodiments, the AAV vector comprises an AAV serotype comprising AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV serotype comprises AAV2. In some embodiments, the peptide comprises CNP. In some embodiments, the CNP comprises at least 22 amino acid residues. In some embodiments, the CNP comprises at least 36 amino acid residues. In some embodiments, the CNP comprises at least 53 amino acid residues. In some embodiments, the CNP comprises an amino acid sequence that is at least 80% identical to SEQ ID NOs: 1-5. In some embodiments, the peptide is covalently attached to the N-terminus of the antibody or fragment thereof. In some embodiments, the peptide is covalently attached to the C-terminus of the antibody or fragment thereof. In some embodiments, the peptide is operably linked to the antibody or fragment thereof by a peptide linker. In some embodiments, the peptide linker is (GGGGS), where n is an integer between 0 and 10. n In some embodiments, the AAV vector encodes an engineered AAV capsid.

[0014]

[0014] Described herein in some aspects is an engineered polypeptide comprising an antibody or fragment thereof operably linked to a peptide, wherein the antibody or fragment thereof comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 6-8. In some embodiments, the peptide comprises CNP. In some embodiments, the CNP comprises at least 22 amino acid residues. In some embodiments, the CNP comprises at least 36 amino acid residues. In some embodiments, the CNP comprises at least 53 amino acid residues. In some embodiments, the CNP comprises an amino acid sequence at least 80% identical to SEQ ID NOs: 1-5. In some embodiments, the peptide is covalently attached to the N-terminus of the antibody or fragment thereof. In some embodiments, the peptide is covalently attached to the C-terminus of the antibody or fragment thereof. In some embodiments, the peptide is operably linked to the antibody or fragment thereof by a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence comprising (GGGGS)n, where n is an integer between 0 and 10.

[0015]

[0015] Described herein, in some aspects, is an engineered polynucleotide encoding an engineered polypeptide described herein. In some embodiments, the engineered polynucleotide is a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the AAV vector comprises an AAV serotype comprising AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV serotype comprises AAV2. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the viral vector comprises one or more expression cassettes. In some embodiments, the one or more expression cassettes encode a contiguous polypeptide comprising an engineered polypeptide described herein. In some embodiments, the contiguous polypeptide comprises a protease cleavable sequence. In some embodiments, the contiguous polypeptide comprises a furin cleavable sequence. In some embodiments, the contiguous polypeptide comprises a self-cleaving polypeptide sequence. In some embodiments, the one or more expression cassettes express at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent comprises a hormone. In some embodiments, the at least one additional therapeutic agent comprises an agonist of the natriuretic peptide receptor (NPR). In some embodiments, the at least one additional therapeutic agent comprises an agonist of the cyclic GMP (cGMP) signaling pathway. In some embodiments, the at least one additional therapeutic agent comprises a VEGF inhibitor. In some embodiments, the VEGF inhibitor binds to and inhibits VEGF-A, VEGF-B, VEGF-C, VEGF-D, or a combination thereof. In some embodiments, the VEGF inhibitor comprises an antibody.In some embodiments, the VEGF inhibitor comprises a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), a single domain antibody (sdAb), an Ig NAR, a camelid antibody, or a combination thereof, a binding fragment thereof, or a chemically modified derivative thereof. In some embodiments, the VEGF inhibitor comprises a non-antibody VEGF inhibitor. In some embodiments, the non-antibody VEGF inhibitor is VEGF receptor 1 (VEGFR1), VEGF receptor 2 (VEGFR2), VEGF receptor 3 (VEGFR3), a fragment thereof, or a combination thereof. In some embodiments, the non-antibody VEGF inhibitor comprises a soluble VEGFR1, a soluble VEGFR2, a soluble VEGFR3, a soluble fragment thereof, or a combination thereof. In some embodiments, the non-antibody VEGF inhibitor comprises VEGF-Trap or a modified form thereof.

[0016]

[0016] Described herein, in some aspects, are cells comprising an engineered polynucleotide described herein.

[0017] Described herein, in some aspects, are cells comprising an engineered polypeptide described herein.

[0017]

[0018] Herein, in some aspects, a pharmaceutical composition comprising an engineered polynucleotide described herein, an engineered polypeptide described herein, or a cell described herein is described. In some embodiments, the pharmaceutical composition is formulated for administration intrathecally, intraocularly, intravitreally, retina, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymal, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonary, intratracheal, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, inhalation spray form, intraluminal-GI route, or a combination thereof, to a subject in need of administration of the pharmaceutical composition. In some embodiments, the pharmaceutical composition is formulated for administration intravitreally, subretinaly, or suprachoroidally. In some embodiments, the pharmaceutical composition is for treating an ocular disease or condition. In some embodiments, the pharmaceutical composition increases natriuretic peptide receptor-B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof, in a subject in need of administration of the pharmaceutical composition.

[0018]

[0019] Described herein, in some aspects, is a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide described herein, an engineered polypeptide described herein, a cell described herein, or a pharmaceutical composition described herein. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises an ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color vision deficiencies, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, traumatic glaucoma, Bardet-Biedl syndrome, Beth-Delta syndrome, or combination thereof. In some cases, the term "cataract surgery" refers to a condition in which the retina is in a drowning state, such as glaucoma, glaucoma, glaucoma, glaucoma, glaucoma syndrome, glaucoma ...

[0019]

[0020] Described herein, in some aspects, is a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide described herein, an engineered polypeptide described herein, a cell described herein, or a pharmaceutical composition described herein, wherein the single administration step cures the disease or condition. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color vision deficiencies, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, traumatic glaucoma, Bardet-Biedl syndrome, Beth-Delta syndrome, or combination thereof. In some cases, the term "cataract surgery" refers to a condition in which the retina is in a drowning state, such as glaucoma, glaucoma, glaucoma, glaucoma, glaucoma syndrome, glaucoma ...

[0020]

[0021] Described herein, in some aspects, is a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide described herein, an engineered polypeptide described herein, a cell described herein, or a pharmaceutical composition described herein, wherein the administering does not include daily administration. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises an ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color vision deficiencies, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, traumatic glaucoma, Bardet-Biedl syndrome, Beth-Delta syndrome, or combination thereof. In some cases, the term "cataract surgery" refers to a condition in which the retina is in a drowning state, such as glaucoma, glaucoma, glaucoma, glaucoma, glaucoma syndrome, glaucoma ...

[0021]

[0022] This patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0022] [Figure 1A]

[0023] 1A-F show exemplary constructs for encoding and expressing the natriuretic peptides (eg, CNP) described herein. [Figure 1B] 1A-F show exemplary constructs for encoding and expressing the natriuretic peptides (eg, CNP) described herein. [Figure 1C]1A-F show exemplary constructs for encoding and expressing the natriuretic peptides (eg, CNP) described herein. [Figure 1D] 1A-F show exemplary constructs for encoding and expressing the natriuretic peptides (eg, CNP) described herein. [Figure 1E] 1A-F show exemplary constructs for encoding and expressing the natriuretic peptides (eg, CNP) described herein. [Figure 1F] 1A-F show exemplary constructs for encoding and expressing the natriuretic peptides (eg, CNP) described herein. [Figure 2A]

[0024] FIG. 2A shows an exemplary AAV vector encoding a natriuretic peptide described herein. [Figure 2B]

[0025] 2B shows an exemplary arrangement of CNP fused with a fusion partner described herein. The left panel shows unfused CNP. The right panel shows CNP fused with a fusion partner to form a CNP fusion protein (FP). [Diagram 3]

[0026] 3A-C show exemplary arrangements of a natriuretic peptide operably linked (eg, covalently attached) to an antibody or fragment thereof (eg, the fragment crystallizable region or Fc of an antibody). [Figure 4A]

[0027] FIG. 4A shows an exemplary anti-human CNP antibody titration standard curve. [Figure 4B]

[0028] FIG. 4B shows an exemplary comparison of detection of antibody dilution. [Figure 4C]

[0029] FIG. 4C shows an exemplary standard curve of the abundance of CNP-Fc encoded by AMI061, AMI087, or AMI088. [Diagram 5]

[0030] FIG. 5 shows exemplary AAV vector expression profiles in HEK293, HCH, or SkMC cells transduced with an AAV vector described herein (AMI061, AMI087, or AMI088). [Figure 6A]

[0031] 6A-B show exemplary SDS-PAGE and peptide sequencing confirming CNP or CNP-Fc expression by AAV vector-transduced cells. [Figure 6B] 6A-B show exemplary SDS-PAGE and peptide sequencing confirming CNP or CNP-Fc expression by AAV vector-transduced cells. [Figure 7A]

[0032] Figure 7A shows that AAV CNP-Fc36 encoded and expressed from an AAV vector was functional in vitro (as determined by cyclic guanosine monophosphate, cGMP production stimulated by expressed CNP36). AAV vector-encoded CNP or Fc1-CNP stimulated cyclic GMP production. The highest levels of cGMP were produced by CNP-Fc36 encoded by AAV vector AMI088. [Figure 7B]

[0033] FIG. 7B shows the affinity binding between the CNP fusions described herein and the NPR-B protein as measured by BiaCore assay. [Figure 7C]

[0034] FIG. 7C shows a single cycle kinetics (SCK) assay of binding between CNP and NPR-B. [Figure 7D]

[0035] FIG. 7D shows a Biacore assay of AMI263 (Fc4-CNP36) against NPR-B. [Figure 7E]

[0036] FIG. 7E shows a Biacore assay of CNP-Fc encoded by AMI088 against NPR-B. [Figure 7F]

[0037] FIG. 7F shows a Biacore assay of ANP against NPR-B. [Figure 7G]

[0038] FIG. 7G shows the Biacore assay of BNP against NPR-B. [Figure 7H]

[0039] FIG. 7H shows a Biacore assay of CNP-Fc encoded by AMI263 against NPR-B. [Figure 7I]

[0040] FIG. 7I shows a Biacore assay of CNP-Fc encoded by AMI087 against NPR-B. [Figure 8A]

[0041] Figures 8A-B show that AAV CNP-Fc36 encoded and expressed from an AAV vector was functional in vitro in a time course measurement (as determined by cGMP production stimulated by expressed CNP36). [Figure 8B] Figures 8A-B show that AAV CNP-Fc36 encoded and expressed from an AAV vector was functional in vitro in a time course measurement (as determined by cGMP production stimulated by expressed CNP36). [Figure 9A]

[0042] Figure 9A shows body weight measurements of mice before (day 0) or 7 days after administration of AAV vectors for in vivo functional studies. On the day of intravitreal (IVT) injection, all animals weighed 20-22 g, and all animals generally maintained their weight throughout the study. [Figure 9B]

[0043] Figure 9B shows a representative image from the central retina with hematoxylin and eosin (H&E) staining. [Figure 9C]

[0044] Figure 9C shows the number of retinal ganglion cells (RGCs) stained with H&E. The average number of RGCs in the right eye (OD; experimental eye) was lower than that in the left eye (OS; control eye), and there was no difference in the average number of RGCs in the OD and OS between Group 1 and Group 2. [Figure 10A]

[0045] Figure 10A shows flat mount analysis of RGC cell numbers in group 1 and group 2 mice from the in vivo functional study. Groups 1 and 2 were stained with 4',6-diamidino-2-phenylindole (DAPI) and neuron-specific class III beta-tubulin antibody (TUJ-1) and images were captured at 20x magnification. RGCs were counted in quadrants and cells / mm2 were averaged across each group. [Figure 10B]

[0046] FIG. 10B shows retinal ganglion cell (RGC) counts from the flat mount analysis of FIG. 10A (Groups 1 and 2). When RGC counts were quantified across groups, the control group (Group 1 OS; PBS, no NMDA) had a mean count of 11,000±700 cells / mm2. The NMDA control group (Group 1 OD; PBS+NMDA) had a reduced RGC count of 8,000±1,000 cells / mm2. Group 2 OD had a slightly increased RGC count over Group 1 OD at 9,000±500 cells / mm2. [Figure 10C]

[0047] Figure 10C shows flat mount analysis of RGC cell numbers in groups 3-8 of mice from the in vivo functional study. Groups 3-8 were stained with DAPI and four images were taken of each retina approximately 600 μm from the optic nerve head at 20x magnification. RGCs were counted in a central 248.1 × 325.7 μm area and cells / mm2 were averaged across each group. [Figure 10D]

[0048] Figure 10D shows retinal ganglion cell (RGC) counts from the flat mount analysis (groups 3-8) in Figure 10C. When RGC counts were calculated, the control group (group 7 OS; PBS, no NMDA) had a mean count of 12,000 ± 700 cells / mm2. The NMDA control group (group 7 OD; PBS + NMDA) had a reduced RGC count of 9,000 ± 300 cells / mm2. Group 8 OD had a similar RGC count to group 7 OS at 12,000 ± 300 cells / mm2. Group 3 OD had a similar RGC count to group 7 OD at 9,000 ± 1,000 cells / mm2. Group 4 OD had an RGC count of 10,000 ± 1,400 cells / mm2. Group 5 OD had an RGC count of 10,000 ± 600 cells / mm2. Group 6 OD had an RGC count of 10,000 ± 1,500 cells / mm2. [Figure 11]

[0049] Figure 11A-B shows the statistical analysis of groups 3-8 (one-way ANOVA followed by multiple comparisons using Dunnett statistical hypothesis comparison). Treatment of PBS-injected eyes with NMDA caused a statistically significant decrease in RGC numbers (P<0.0001). Increasing doses of MK-801 up to 100 μM restored group 8 RGC numbers to group 7 OS control levels, and the difference between NMDA-treated (group 7 OD) and NMDA+MK-801-treated (group 8) eyes was statistically significant (P<0.0001). When comparing groups 4-6 to group 3 NMDA / sham vector control, both AMI182 (group 5) and AMI088 (group 6) reached statistical significance (Figure 11A; P=0.0332 for group 5 vs. group 3; P=0.0021 for group 6 vs. group 3). When comparing RGC numbers in AAV-treated groups with NMDA / PBS controls (group 7 OD), only the AMI088 construct maintained statistical significance (P=0.002; FIG. 11B). [Figure 12A]

[0050] Figure 12A shows immunohistochemistry images obtained from retinal flat mounts. Groups 3 to 6 were stained for CNP36 in addition to DAPI to evaluate the location and level of AAV vector expression, and images (4x and 20x) were captured. [Figure 12B]

[0051] Figure 12B shows phase 1 immunohistochemistry (groups 1 and 2). Qualitatively, there was no difference in the number of TUJ-1+ and cone arrestin+ cells between the groups. All groups showed clear cone arrestin staining with some TUJ-1+ cells observed. [Figure 13]

[0052] Figure 13 shows the quantification of CNP-Fc or CNP. CNP36-Fc quantification in ocular and serum samples: Animals from groups 3 (AMI189) and 6 (AMI088) were analyzed using CNP36-Fc ELISA. Group 3 (sham vector) animals served as a negative control (Figure 13A). Animals in G6 were injected with AMI088, an AAV2 vector with N-terminal CNP36 fused to the C-terminus of a human IgG1-Fc fragment (CNP36-Fc). Data were analyzed with GraphPad Prism software using one-way ANOVA followed by Dunnett's multiple comparisons (****=<0.0001). Statistically significant differences were observed between groups 3 and 6. In samples without Fc fusion, CNP36 levels were lower due to the short half-life of CNP36. Ocular samples from groups 3, 4 and 5 were analyzed using the above-mentioned commercial ELISA kit (Figure 13B, left graph). Groups 4 and 5 showed slightly higher levels of CNP36 compared to group 3 (sham vector; negative control), but the observed difference was not significant. The low levels observed in ELISA indicated that only a small fraction of the total expressed CNP36 was quantified, since most of the protein was proteolytically degraded before quantification. As the test articles in groups 4 and 5 showed efficacy, CNP36 was expressed but not protected from proteolytic degradation. Neither CNP36-Fc nor CNP36 was expressed in serum samples. The right graph in Figure 13B shows an exemplary in vivo expression of CNP fusions detected by ELISA. [Figure 14]

[0053] Figure 14 shows RGC protection by AAV vector encoding CNP. Mice eyes were injected with 1μl IVT AAV construct at 4E+8vg / eye 28 days before NMDA injection. Images show that NMDA induced RGC# reduction, which was restored by MK-801. Sham vector showed reduction of RGC in NMDA only treatment group, while AMI182 and AMI088 groups showed higher RGC number than sham vector and NMDA treatment group. [Figure 15]

[0054] Figure 15 shows an exemplary SDS-PAGE showing the patterns associated with expression of VP1, VP2, or VP3. All AAV samples were: produced in Sf-9 cells; purified by two rounds of CsCl2 ultracentrifugation, buffer exchange; and sterile filtered. Approximately 1 ell vg / lane was loaded onto the gel. [Figure 16]

[0055] Figure 16 shows the effect of Fc4-CNP36 (FP) on intraocular pressure (IOP) changes in a rat partial optic nerve transection (pONT) model. Figure 16A shows the animal IOP monitored during a series of tests. Figure 16B shows the animal IOP changes after intravitreal administration (IVT) of Fc4-CNP36. Figure 16C shows the effect of Fc4-CNP36 concentration on IOP changes after intravitreal administration. Vehicle: 10 mM phosphate, pH 7.3, 180 mM NaCl, 0.001% Pluronic F68; FP(L)=2 μg / eye IVT; FP(M)=20 μg / eye IVT; and FP(H)=80 μg / eye IVT. [Figure 17]

[0056] Figure 17 shows the effect of Fc4-CNP36 on RGC protection in a rat pONT model. Figure 17A shows the effect of Fc4-CNP36 concentration on the reduction of detection of apoptotic retinal cells (DARC). Figure 17B shows the effect of Fc4-CNP36 concentration on RGC number. Vehicle: 10 mM phosphate, pH 7.3, 180 mM NaCl, 0.001% Pluronic F68; FP(L)=2 μg / eye IVT; FP(M)=20 μg / eye IVT; and FP(H)=80 μg / eye IVT. [Figure 18]

[0057] Figure 18 shows the protein concentration in the final sample of the rat pONT model. Only OS (left eye) was injected, but not OD (right eye). [Figure 19]

[0058] Figures 19A-B show the effect of AAV2.N54-Fc4-CNP36 and AAV2.N54-CPNP36 on retinal DARC reduction in a rat pONT model. DARC values ​​were subtracted from baseline. Final DARC numbers were plotted. Figure 19A: AMI273 was a vector of AAV2.N54-Fc4-CNP36 and expressed Fc4-CNP36 protein. Figure 19B: AMI302 was a vector of AAV2.N54-CNP36 and expressed CNP36 peptide. [Figure 20]

[0059] Figures 20A-B show the effect of AAV2.N54-Fc4-CNP36 (AAV-FP) and AAV2.N54-CPNP36 (AAV-P) on RGC protection in a rat pONT model. Figure 20A: AMI273 was a vector of AAV2.N54-Fc4-CNP36 and expressed the Fc4-CNP36 protein. Figure 20B: AMI302 was a vector of AAV2.N54-CNP36 and expressed the CNP36 peptide. [Figure 21]

[0060] FIG. 21 shows the number of RGCs in treated eyes of animals administered Fc4-CNP36 protein (FP) and AAV vector in a rat pONT model. [Figure 22]

[0061] Figure 22 shows Fc4-CNP36 concentrations in ocular and serum samples after administration of AAV vector (AAV-FP) IVT. The Fc4-CNP36 GOI was expressed in AMI273-treated eyes, but not in untreated eyes. [Diagram 23]

[0062] FIG. 23 shows CNP36 concentrations in ocular and serum samples following administration of AAV vector (AAV-P) IVT. [Figure 24]

[0063] FIG. 24 shows a comparison of DARC numbers and RGC local density. [Diagram 25]

[0064] Figures 25A-B show the determination of the EC50 for CNP-Fc described herein. Figure 25A shows the standard curve for cGMP. Figure 25B shows the data fitting and calculation of the EC50 for various CNP fusions (e.g., CNP-Fc) and comparable natriuretic peptides described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023]

[0065] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments. overview

[0066] In some aspects, engineered polynucleotides are described herein, including AAV vectors comprising one or more expression cassettes encoding a peptide. Also described herein are engineered polynucleotides comprising AAV vectors comprising one or more expression cassettes for encoding an engineered polypeptide comprising an antibody or fragment thereof operably linked to a peptide. For example, the engineered polypeptide comprises an antibody or fragment thereof operably linked to a CNP described herein. FIG. 2A illustrates an exemplary AAV vector encoding a peptide (e.g., CNP) or a fusion protein (e.g., a CNP-Fc fusion protein). FIG. 2B illustrates an exemplary arrangement of CNP fused to a fusion partner described herein. In some embodiments, the peptide encoded by the engineered polynucleotide is a natriuretic peptide. In some embodiments, the peptide is CNP. In some embodiments, the CNP is full-length CNP. In some embodiments, the CNP is truncated. In some embodiments, the CNP comprises at least 22 amino acid residues, at least 36 amino acid residues, or at least 53 amino acid residues. In some embodiments, the CNP is CNP-22 (SEQ ID NO:5). In some embodiments, the CNP is CNP-36 (SEQ ID NO: 4). In some embodiments, the CNP is CNP-53 (SEQ ID NO: 3). In some embodiments, the CNP comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 1-5. In some embodiments, the CNP comprises an amino acid sequence that is 100% identical to any one of SEQ ID NOs: 1-5.

[0024]

[0067] In some embodiments, the peptide is operably linked to an antibody or fragment thereof (e.g., an Fc fragment). In some embodiments, the antibody or fragment thereof comprises a signaling peptide (SP). For example, the engineered polypeptide may comprise amino acids that are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, the antibody or fragment thereof comprises a variable region (e.g., an immunoglobulin heavy chain variable or Vh). For example, the engineered polypeptide may comprise amino acids that are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:11 or SEQ ID NO:12.

[0025]

[0068] In some embodiments, the peptide is covalently attached to the N-terminus of the antibody or fragment thereof. In some embodiments, the peptide is covalently attached to the C-terminus of the antibody or fragment thereof. In some aspects, operably linking the peptide to the antibody or fragment thereof increases the stability of the peptide. In some aspects, operably linking the peptide to the antibody or fragment thereof increases the half-life of the peptide. In some aspects, operably linking the peptide to the antibody or fragment thereof increases the half-life of the peptide in vivo. In some aspects, operably linking the peptide to the antibody or fragment thereof increases the half-life of the peptide in circulation. In some aspects, operably linking the peptide to the antibody or fragment thereof increases the half-life of the peptide in cells. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 6-8. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to any one of SEQ ID NOs: 6-8. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 131-140. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to any one of SEQ ID NOs: 131-140. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 9-12. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to any one of SEQ ID NOs: 9-12. Figures 3A-C illustrate exemplary depictions of CNP-Fc fusion proteins described herein.

[0026]

[0069] In some embodiments, the engineered polynucleotide described herein comprises a vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV vector. In some embodiments, the AAV vector comprises an AAV serotype comprising AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV vector comprises an AAV serotype comprising AAV2. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the viral vector or AAV vector comprises one or more expression cassettes. In some embodiments, the one or more expression cassettes may encode CNP, or CNP operably linked to an antibody or fragment thereof (e.g., a CNP fusion protein), and at least one additional therapeutic agent. For example, the at least one additional therapeutic agent may be a hormone or agonist for stimulating the natriuretic peptide receptor (NPR) or for activating the cyclic GMP (cGMP) signaling pathway. In some embodiments, at least one additional therapeutic agent comprises a VEGF inhibitor.For example, at least one additional therapeutic agent can be an antibody that targets VEGF or an shRNA that targets VEGF transcript.In some embodiments, at least one additional therapeutic agent comprises a cytokine inhibitor (e.g., a tumor necrosis factor inhibitor).

[0027]

[0070] In some embodiments, the engineered polynucleotide described herein is encapsulated in or part of a viral particle. In some embodiments, the viral particle is at least partially encoded by the engineered polynucleotide. In some embodiments, the viral particle is not encoded by the engineered polynucleotide. In some embodiments, the viral particle is an AAV particle. In some embodiments, the viral particle comprises an AAV capsid. In some embodiments, the AAV capsid comprises a modified AAV capsid. In some embodiments, the modified AAV capsid comprises a modified AAV2 capsid. In some embodiments, the viral particle can be administered to a subject to treat a disease or condition. In some embodiments, the viral particle can be formulated into a pharmaceutical composition described herein.

[0028]

[0071] Described herein in some aspects is a method of treating a disease or condition in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide as described herein, an engineered polypeptide encoded by an engineered polynucleotide as described herein, a cell transduced with an engineered polynucleotide particle as described herein, or a pharmaceutical composition as described herein. In some embodiments, the engineered polynucleotide encodes an engineered polypeptide as described herein. In some embodiments, the method cures the disease or condition or significantly reduces the severity associated with the disease or condition. In some embodiments, the method confers protection against the disease or condition. In some embodiments, the method cures the disease or condition or significantly reduces the severity associated with the disease or condition after one administration. In some embodiments, the method cures the disease or condition or significantly reduces the severity associated with the disease or condition without the need for daily administration. In some embodiments, the disease or condition is an ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color vision deficiency, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, traumatic glaucoma, Bardet-Biedl syndrome, Best's disease, Eye disorders including choroideremia, Leber's congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum's disease, Stargardt's disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-vertebral dystrophy, vertebral-rod dystrophy, Oguchi's disease, autosomal dominant radial drusen (familial autosomal dominant drusen), blue-vertebral monochromacy, or a combination thereof. Engineered Polynucleotides

[0072] In some embodiments, the engineered polynucleotides encoding peptides or fusion proteins are described herein. In some embodiments, the engineered polynucleotides encode natriuretic peptides, such as C-type natriuretic peptide (CNP) or fragments thereof. In some embodiments, the CNP or fragments thereof comprises 22 contiguous bases of SEQ ID NO:1. In some embodiments, the CNP or fragments thereof comprises 22 contiguous bases at the C-terminus of SEQ ID NO:1. In some embodiments, the CNP or fragments thereof comprises 36 contiguous bases of SEQ ID NO:1. In some embodiments, the CNP or fragments thereof comprises 36 contiguous bases at the C-terminus of SEQ ID NO:1. In some embodiments, the CNP or fragments thereof comprises 53 contiguous bases ... at the C-terminus of SEQ ID NO:1.

[0029]

[0073] In some embodiments, the engineered polynucleotide encodes CNP or a CNP fusion protein (e.g., a CNP-Fc fusion protein described herein). In some embodiments, the engineered polynucleotide comprises a viral vector, such as an AAV vector, comprising one or more expression cassettes encoding CNP or a CNP fusion protein. In some embodiments, the engineered polynucleotide comprises a vector. In some embodiments, the vector is a viral vector. In some embodiments, the engineered polynucleotide comprises an AAV vector. In some embodiments, the engineered polynucleotide comprises an AAV vector encoding an engineered AAV capsid. In some embodiments, the AAV vector comprises one or more expression cassettes encoding an engineered polypeptide comprising a peptide or a fusion protein comprising an antibody or fragment thereof operably linked to a peptide. In some embodiments, the one or more expression cassettes encode an engineered polypeptide comprising CNP. In some embodiments, the one or more expression cassettes encode an engineered polypeptide comprising an antibody or fragment thereof operably linked to CNP. In some embodiments, the CNP comprises at least 22 amino acid residues. In some embodiments, the CNP comprises at least 36 amino acid residues. In some embodiments, the CNP comprises at least 53 amino acid residues. In some embodiments, the CNP comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 1-5. In some embodiments, the CNP comprises an amino acid sequence that is 100% identical to any one of SEQ ID NOs: 1-5. In some embodiments, the engineered polynucleotide encodes a natriuretic peptide that comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 4. In some embodiments, the engineered polynucleotide encodes a natriuretic peptide that comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 4.In some embodiments, the engineered polynucleotide encodes a natriuretic peptide comprising the amino acid sequence of SEQ ID NO:4.

[0030]

[0074] In some embodiments, the CNP encoded by the engineered polynucleotide is operably linked to an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a fragment crystallizable (Fc) region. In some embodiments, the CNP encoded by the engineered polynucleotide is operably linked to the N-terminus of the antibody or fragment thereof. In some embodiments, the CNP encoded by the engineered polynucleotide is operably linked to the C-terminus of the antibody or fragment thereof. In some embodiments, the CNP encoded by the engineered polynucleotide is covalently linked to the antibody or fragment thereof. In some embodiments, the CNP encoded by the engineered polynucleotide is covalently linked to the N-terminus of the antibody or fragment thereof. In some embodiments, the CNP encoded by the engineered polynucleotide is covalently linked to the C-terminus of the antibody or fragment thereof. In some embodiments, the CNP is covalently linked to the antibody or fragment thereof by a peptide linker.

[0031]

[0075] In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 6-8. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to any one of SEQ ID NOs: 6-8. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 131-140. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to any one of SEQ ID NOs: 131-140. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 9-12. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to any one of SEQ ID NOs: 9-12.

[0032]

[0076] In some embodiments, the engineered polynucleotide is a vector. In some embodiments, the engineered polynucleotide is a viral vector, including an AAV vector. In some embodiments, the engineered polynucleotide is an AAV vector, including an AAV serotype, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the engineered polynucleotide is an AAV vector, including an AAV2 serotype. In some embodiments, the AAV vector encodes a modified AAV capsid.

[0033]

[0077] In some embodiments, the engineered polynucleotide encodes a contiguous polypeptide comprising an engineered polypeptide described herein. In some embodiments, the contiguous polypeptide comprises a protease-cleavable sequence. In some embodiments, the contiguous polypeptide comprises a furin-cleavable sequence. In some embodiments, the contiguous polypeptide comprises a self-cleaving polypeptide sequence.

[0034]

[0078] In some embodiments, the engineered polynucleotide comprises one or more expression cassettes encoding CNP or a CNP fusion protein and at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent comprises a hormone. In some embodiments, the at least one additional therapeutic agent comprises an agonist of the natriuretic peptide receptor (NPR). In some embodiments, the at least one additional therapeutic agent comprises an agonist of the cyclic GMP (cGMP) signaling pathway. In some embodiments, the at least one additional therapeutic agent comprises a cytokine inhibitor. In some embodiments, the at least one additional therapeutic agent comprises a VEGF inhibitor, which binds to and inhibits VEGF-A, VEGF-B, VEGF-C, VEGF-D, or a combination thereof. In some embodiments, the VEGF inhibitor is an antibody. In some embodiments, the VEGF inhibitor is not an antibody.

[0035]

[0079] In some examples, the engineered polynucleotide comprises additional features, which may include sequences such as tags, signal peptides, intron sequences, promoters, stuffer sequences, and the like.

[0036]

[0080] In some examples, the engineered polynucleotide encodes a signal peptide. A signal peptide, sometimes also called a signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide, is a short peptide present at the N-terminus of most newly synthesized proteins that are destined for the secretory pathway. These proteins include proteins that reside inside certain organelles (endoplasmic reticulum, Golgi apparatus, or endosomes), are secreted from the cell, or are inserted into most cell membranes. In some examples, the nucleic acids provided herein can include a signal peptide. A signal peptide can be a peptide of any length, but typically is 15-30 amino acids long. A signal peptide can be about 10-15, 10-20, 10-30, 15-20, 15-25, 15-30, 20-30, or 25-30 amino acids long. A variety of signal peptides may be utilized, including, but not limited to, human antibody heavy chain (Vh), human antibody light chain (Vl), and aflibercept.

[0037]

[0081] In some examples, the engineered polynucleotide comprises an intron sequence. An intron is any nucleotide sequence within a sequence that can be removed by RNA splicing during maturation of the final RNA product. In other words, an intron is a non-coding region of an RNA transcript, or the DNA that codes for it, that is removed by splicing before translation. Although introns do not code for protein products, they play a role in regulating gene expression. Some introns themselves code for functional RNA through further processing after splicing to generate non-coding RNA molecules. Alternative splicing is widely used to generate multiple proteins from a single gene. In addition, some introns play an essential role in a wide range of gene expression regulation functions, such as nonsense-mediated decay and mRNA export. In one embodiment, the intron sequence is included in the nucleic acid of the present disclosure and can be selected from hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, hamster EF-1 alpha gene intron 1, intervening sequence intron, human growth hormone intron, and / or human beta globin intron. Any number of intron sequences are contemplated. In one embodiment, the intron sequence is SV40. In some examples, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or up to 10 intron sequences may be included in the nucleic acid.

[0038]

[0082] In one embodiment, the engineered polynucleotide includes additional features including a promoter. A promoter is a sequence of DNA to which a protein binds that initiates transcription of a single RNA from the DNA downstream of it. This RNA may code for a protein or may have a function by itself or alone, such as a tRNA, mRNA, or rRNA. The promoter is located upstream of the DNA (towards the 5' region of the sense strand), near the transcription start site of the gene. The promoter may be about 100-1000 base pairs in length. A variety of promoters are contemplated and may be used in the engineered polynucleotides of the present disclosure. In one embodiment, the promoter is a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EF1α) promoter, a simian vacuolating virus (SV40) promoter, a phosphoglycerate kinase (PGK1) promoter, a ubiquitin C (Ubc) promoter, a human beta-actin promoter, a CAG promoter, a tetracycline response element (TRE) promoter, a UAS promoter, an actin 5c (Ac5) promoter, a polyhedron promoter, a Ca2+ / calmodulin-dependent protein kinase II (CaMKIIa) promoter, a GAL1 promoter, a GAL 10 promoter, a TEF1 promoter, a glyceraldehyde 3-phosphate dehydrogenase (GDS) promoter, an ADH1 promoter, a CaMV35S promoter, a Ubi promoter, a human polymerase III RNA (H1) promoter, a U6 promoter, a polyadenylation construct thereof, and any combination thereof. In some examples, the promoter is a CMV promoter.

[0039]

[0083] Any of the engineered polynucleotides provided may contain a viral vector sequence. The viral vector may be, but is not limited to, a lentivirus, a retrovirus, or an adeno-associated virus. The viral vector may be an adeno-associated virus (AAV) vector. In some examples, the viral vector is an adeno-associated virus vector. Many serotypes of AAV vectors are contemplated, and may include, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and / or AAV12. Based on these initial serotypes, the AAV capsid of each serotype may be engineered to make them better suitable for biological functions, tissues, or cell selection. In some embodiments, the AAV vector is AAV2 and variants AAV2.N53 and AAV2.N54. Chimeric AAV vectors that may contain at least two AAV serotypes are also contemplated. In some cases, at least 3, at least 4, at least 5, at least 6, at least 7, or up to 8 different serotypes are combined with a chimeric AAV vector. In some cases, only a small portion of AAV is chimeric. For example, suitable portions can include capsid, VP1, VP2, or VP3 domains and / or Rep. In some cases, at least one of VP1, VP2, and VP3 has at least one amino acid substitution compared to otherwise equivalent wild-type AAV capsid protein. In some cases, mutations can occur in VP1 and VP2, VP1 and VP3, VP2 and VP3, or VP1, VP2, and VP3. In some embodiments, at least one of VP1, VP2, and VP3 has 1 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3, such as about 1 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, or about 20 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3. In some examples, the VPs can be removed. For example, in some embodiments, the mutant AAV does not include at least one of VP1, VP2, or VP3.

[0040]

[0084] In some cases, AAV vector can be modified.For example, AAV vector can include modification such as insertion, deletion, chemical change or synthetic modification.In some cases, a single nucleotide is inserted into AAV vector.In other cases, multiple nucleotides are inserted into vector.

[0041] Codon Optimization

[0085] In one embodiment, the engineered polynucleotides described herein include modifications that confer enhanced expression of biologics, such as CNP or CNP fusion proteins described herein. For example, some biologics contain unmodified sequences that are derived from native gene sequences and are not optimized for introduction and expression in target cells. In one embodiment, the isolated engineered polynucleotide is codon-optimized. Codon optimization can be specific to cell type-specific codon usage. Various organisms and cell types show bias toward the use of certain codons over other amino acids for the same amino acid. Some species are known to almost completely avoid certain codons. Similarly, certain cell types have a bias toward the use of certain codons over other amino acids for the same amino acid. In one embodiment, the method of optimizing the codons of an engineered polynucleotide can include reassigning codon usage based on the frequency of each codon usage in the target cell. In some examples, the target cell can be a cell of a certain tissue or organ. In some examples, the modification is made to increase guanine and / or cytosine content.

[0042]

[0086] In one embodiment, the engineered polynucleotide sequence can be modified to replace at least one codon with another codon that codes for the same amino acid. In some cases, the codon is modified within the coding region of the sequence. In some cases, the codon is modified within the non-coding region of the sequence. In some cases, the codon is modified within about 100, about 50, about 25, about 15, or about 5 bases from the stop codon. E-CAI can be used to estimate the value of codon compatibility index.

[0043]

[0087] Various modifications are contemplated herein. In some cases, codons can be exchanged. For example, sequences can be modified to exchange AGA with AGG. In other cases, CCC is exchanged with CCT. In other cases, AGC is exchanged with TCC. In other cases, CCC is exchanged with CCG. Any of the non-limiting exchanges provided in Table 1 can be applied to modify nucleic acid. Any number of codons in nucleic acid can be exchanged. In some examples, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 42, at least 44, at least 46, at least 48, or up to 50 codons may be replaced. In one embodiment, the engineered polynucleotide comprises 3 codon modifications. In one embodiment, the engineered polynucleotide comprises 16 codon modifications. In one embodiment, the engineered polynucleotide comprises 3-5, 5-10, 5-15, 10-15, 10-20, 15-20, 1-20, 12-20, 12-25, 15-30, or 15-25 codon modifications. In one embodiment, the engineered polynucleotide comprises two codon modifications, AGA to AGG and at least one of CCT to CCC, AGC to TCC, or CCC to CCG. In one embodiment, the engineered polynucleotide comprises three codon modifications, AGA to AGG and at least two of CCT to CCC, AGC to TCC, or CCC to CCG. In one embodiment, the engineered polynucleotide comprises four codon modifications, AGA to AGG, CCT to CCC, AGC to TCC, and CCC to CCG.The additional modifications may include any of the codon modifications provided in Table 1 in combination with any of the codons above and / or any possible additional modifications from Table 1. In one embodiment, the nucleic acid is modified such that AGA is replaced with AGG and CCT is replaced with CCC. In one embodiment, the nucleic acid is modified such that AGA is replaced with AGG and AGC is replaced with TCC. In one embodiment, the nucleic acid is modified such that AGA is replaced with AGG and CCC is replaced with CCG.

[0044] [Table 1]

[0045]

[0088] In some embodiments, the engineered polynucleotide sequence may comprise a viral vector sequence. In some embodiments, the viral vector sequence may be a scAAV vector sequence. In some embodiments, the AAV vector sequence may be a sequence of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV vector sequence may be a sequence of AAV2 serotype. In some embodiments, the viral vector sequence may comprise a sequence of at least two AAV serotypes. In some embodiments, the at least two serotypes may be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV11, and AAV12.

[0046]

[0089] In some cases, modification can also include chemical modification. Modified nucleic acid can include modification of its backbone, sugar, or nucleic acid base, and even new base or base pair. Modified nucleic acid can improve chemical and / or biological stability. Modification with various chemical substituents (e.g., hydrophobic groups) can also result in improved properties and functionality, such as new structural motifs and enhanced target binding.

[0047]

[0090] Exemplary chemical modifications include, but are not limited to, 2'F, 2'-fluoro; 2'OMe, 2'-O-methyl; LNA, locked nucleic acid; FANA, 2'-fluoroarabinose nucleic acid; HNA, hexitol nucleic acid; 2'MOE, 2'-O-methoxyethyl; ribuloNA, (1'-3')-β-L-ribulo nucleic acid; TNA, α-L-threose nucleic acid; tPhoNA, 3'-2'phosphonomethyl-threosyl nucleic acid; dXNA, 2'-deoxyxylonucleic acid; PS, phosphorothioate; phNA, alkylphosphonate nucleic acid; PNA, and peptide nucleic acid.

[0048] Dual expression

[0091] In some aspects, engineered polynucleotides are described herein that include one or more expression cassettes for expressing a peptide (e.g., CNP), a fusion protein (e.g., a CNP fusion protein), or a therapeutic agent. In some embodiments, the one or more expression cassettes encode a contiguous polypeptide. In some embodiments, the contiguous polypeptide includes a protease peptide sequence. In some embodiments, the protease peptide sequence is cleavable by a protease endogenously expressed in the cell. Non-limiting examples of proteases may include serine endoproteases, aspartic acid endoproteases, cysteine ​​thiol endoproteases, metalloendoproteases, or glutamic acid and threonine endoproteases. In some embodiments, the protease peptide sequence is cleavable by a serine endoprotease. In some embodiments, the protease peptide sequence is cleavable by furin. In some embodiments, the contiguous polypeptide includes a protease cleavable sequence. In some embodiments, the protease-cleavable sequence may be cleaved by any one of the proteases described herein. In some embodiments, the protease-cleavable sequence may be cleavable by furin. In some embodiments, the continuous polypeptide comprises a self-cleaving polypeptide sequence. In some embodiments, the self-cleaving polypeptide sequence comprises a 2A self-cleaving peptide sequence. Non-limiting examples of 2A self-cleaving peptide sequences may include T2A, P2A, E2A, F2A, or a combination thereof. In some embodiments, the self-cleaving polypeptide sequence comprises a F2A peptide sequence. In some embodiments, the continuous polypeptide comprises a protease-cleavable sequence and a self-cleaving polypeptide sequence. For example, the continuous polypeptide described herein may comprise a furin-F2A fusion polypeptide sequence. In some embodiments, the engineered polynucleotide comprises a viral vector, such as an AAV vector.

[0049]

[0092] In some embodiments, the engineered polynucleotide comprises one or more promoters or IRES. In some embodiments, the expression cassette comprises one or more promoters or internal ribosome entry sites (IRES). In some embodiments, the expression cassette is under the expression control of a promoter. In some embodiments, the expression cassette is under the expression control of a promoter. In some embodiments, the expression cassette can further exert expression control via at least one IRES.

[0050]

[0093] In some embodiments, the engineered polynucleotide comprises at least two, at least three, at least four, at least five, or more expression cassettes. In some embodiments, the engineered polynucleotide comprises two expression cassettes. In some embodiments, the CNP or CNP fusion protein (e.g., a CNP-Fc fusion protein) and at least one additional therapeutic agent are each expressed from an expression cassette.

[0051]

[0094] In some embodiments, at least one additional therapeutic agent is an inhibitor that targets a cytokine (e.g., tumor necrosis factor). In some embodiments, at least one additional therapeutic agent can be an antibody that targets a cytokine or an inhibitory nucleic acid that targets a cytokine transcript. For example, at least one additional therapeutic agent can be an inhibitory RNA, such as small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterogeneous nuclear RNA (hnRNA), that targets a cytokine transcript for degradation, thereby reducing the expression of the cytokine in cells.

[0052]

[0095] In some embodiments, at least one additional therapeutic agent is a VEGF inhibitor. For example, Figure 25B illustrates the functional analysis of a dual-expressed CNP fusion (Fc4-CNP36) and a VEGF inhibitor (aflibercept). In some embodiments, the VEGF inhibitor comprises an inhibitory RNA for targeting and degrading VEGF transcripts. In some embodiments, the VEGF inhibitor comprises an antibody or a fragment thereof. In some embodiments, the VEGF antibody binds to VEGF and reduces angiogenic signaling, including the VEGF signaling pathway. In some embodiments, the VEGF antibody binds to VEGF-A, VEGF-B, VEGF-C, VEGF-D, or a combination thereof. In some embodiments, the VEGF antibody binds to one or more isoforms of VEGF-A, including VEGF121, VEGF145, VEGF148, VEGF162, VEGF165, VEGF165b, VEGF183, VEGF189, or VEGF206. In some embodiments, the antibody comprises a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), a single domain antibody (sdAb), an Ig NAR, a camelid antibody, or a combination thereof, a binding fragment thereof, or a chemically modified derivative thereof. Non-limiting examples of VEGF antibodies include ranibizumab or bevacizumab. In some embodiments, the VEGF antibody comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to any one of SEQ ID NOs: 21-27 (Table 2), or a combination thereof, or a fragment thereof.

[0053] [Table 2]

[0054]

[0096] In some embodiments, the VEGF inhibitor is not an antibody. For example, the VEGF inhibitor described herein may include a VEGF receptor, a combination of VEGF receptors, or a fragment thereof for binding to VEGF to inhibit or reduce the VEGF signaling pathway. The VEGF receptor may include VEGF receptor 1 (FLT1), VEGF receptor 2 (KDR / FLK1), VEGF receptor 3 (FLT4), a fragment thereof, or a combination thereof. In some embodiments, the VEGF receptor may be a soluble VEGF receptor. For example, the soluble VEGF receptor may include soluble VEGFR1, soluble VEGFR2, soluble VEGFR3, a soluble fragment thereof, or a combination thereof. In some embodiments, the non-antibody VEGF inhibitor includes at least one of FLT1, KDR / FLK1, FLT4, a fragment thereof, or a combination thereof. In some embodiments, the non-antibody VEGF inhibitor includes at least one of soluble FLT1, soluble KDR / FLK1, soluble FLT4, a fragment thereof, or a combination thereof. In some embodiments, the non-antibody VEGF inhibitor comprises a VEGF-Trap. In some embodiments, the non-antibody VEGF inhibitor comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to any one of SEQ ID NOs:31-34 (Table 3).

[0055] [Table 3-1]

[0056] [Table 3-2]

[0057] [Table 3-3]

[0058] [Table 3-4]

[0059] Modified Capsid

[0097] Provided herein is a modified adeno-associated virus (AAV) capsid-containing composition and its method of use.Modified AAV capsid can comprise exogenous sequence compared to otherwise equivalent unmodified AAV capsid.Exogenous sequence can refer to exogenous polypeptide sequence.AAV capsid can be modified to provide them and any composition and / or method that they are utilized with improved functionality, thereby resulting in better therapeutic agents, particularly for ophthalmic use.

[0060]

[0098] The AAV wild-type (WT) genome contains at least three genes: rep, cap, and X. The X gene is located at the 3' end of the genome (nucleotides 3929-4393 in AAV2) and appears to code for a protein with an auxiliary function in genome replication. Significantly more information is available for rep and cap. The rep gene is located in the first half of the AAV WT genome and encodes a family of nonstructural proteins (Rep proteins) required for viral transcription control and replication and packaging of the viral genome into newly produced preassembled capsids. The second half of the AAV genome contains the cap gene, which encodes the viral proteins (VPs) VP1, VP2, and VP3, as well as the assembly activating protein (AAP). Transcription of all VPs, the capsid monomers, is controlled by a single promoter (p40 in the case of AAV2), resulting in a single mRNA. Splicing (VP1) and an aberrant translation initiation codon (VP2) are responsible for the approximately 10-fold lower abundance of VP1 and VP2 compared to VP3. When encoded by a single gene, AAV VPs share most of their amino acids. Specifically, the entire VP3 sequence is also contained within VP2 and VP1 (the "common VP3 region"), and VP2 and VP1 also share approximately 65 amino acids (the "common VP1 / VP2 region"). Only VP1 contains a unique sequence at its N-terminus (approximately 138 amino acids, VP1 unique). AAP was identified in 2010 as a 23 kDa protein encoded in the alternative cap ORF. It is used to stabilize and transport newly produced VP proteins from the cytoplasm to the cell nucleus. AAV serotypes 1-3, 6-9, and rh10 were unable to produce capsids in the absence of AAP, while low but detectable capsid production was reported for AAV4 and AAV5.

[0061]

[0099] In one embodiment, the AAV may include modifications. The modifications may be modifications of the rep, cap, and / or X coding polypeptide sequences of the AAV. In some examples, the modifications may be modifications of the cap polypeptide. The cap polypeptide may be modified in any one of the VP domains, such as VP1, VP2, and / or VP3. In some examples, VP1 is modified. In some examples, VP2 is modified. In some examples, VP3 is modified. In some embodiments, two or all of the VP domains may be modified. In some examples, VP1 and VP2 are modified. In some examples, VP1 and VP3 are modified. In addition, VP2 and VP3 may be modified, or VP1, VP2, and VP3 are modified. Other combinations are contemplated, such as Rep and Cap, Cap and X, Rep and X, and / or Rep, Cap, and X modifications. Any combination of domains may be modified, such as any one of the above VP modifications, with Rep and / or X modifications. In some examples, Rep and VP1 and / or VP2 are modified. In some embodiments, the subject Rep is modified. The rep modifications can include those provided herein and can be present in at least one of Rep 78, Rep 68, Rep 52, or Rep 40. In some examples, the Rep is of a different AAV serotype than the subject capsid.

[0062]

[0100] In some cases, the modification is of the AAV capsid. The capsid of an AAV serotype is assembled from 60 VP monomers with approximately 50 copies of VP3, 5 copies of VP2, and 5 copies of VP1. The topologically prominent capsid surface structures are holes or "channel-like structures" at each 5-fold axis of symmetry, a recess at each 2-fold axis of symmetry, and three ridges around each 3-fold axis of symmetry. The holes allow exchange between the capsid interior and exterior. The recesses, or more precisely the floors of each 2-fold axis of symmetry, are the thinnest parts of the viral capsid. The ridges around the 3-fold axes of symmetry carry five of the nine so-called variable regions (VRs). Specifically, VR-IV, -V, and -VIII form loops (loops 1-4) at the top of the ridges, whereas VR-VI and -VII are found at their bases. The VRs differ among serotypes and are responsible for serotype-specific variation in antibody and receptor binding. Due to its exposed position and its function in receptor binding, the VR forming the protuberance loop is an ideal location for capsid modifications aimed at redirecting or increasing AAV tropism (cell surface targeting). Tropism redirection (vector retargeting) combines, for example, the removal of native receptor binding by site-directed mutagenesis with the insertion of a ligand that mediates transduction through a novel, non-native AAV receptor, such that the tropism-expanded AAV vector acquires the ability to transduce cells through an additional receptor while retaining its native receptor-binding ability.

[0063]

[0101] In some aspects, the modification of the AAV capsid may refer to the insertion of an exogenous polypeptide sequence. In other aspects, the modification may refer to the deletion of the polypeptide sequence. The modification may also refer to the modification of at least one standard or non-standard amino acid residue in the polypeptide sequence.

[0064]

[0102] The insertion can include inserting at least one exogenous amino acid residue into the sequence encoding the AAV capsid. The amino acid can refer to a standard amino acid or a non-standard amino acid. Any number of amino acid residues can be inserted. In some examples, the insertion site can be in the GH loop or loop IV of the AAV capsid protein, for example, in the solvent-exposed part of the GH loop or loop IV of the AAV capsid protein.

[0065]

[0103] In some examples, the modification includes the insertion of an exogenous polypeptide sequence comprising the sequence of formula 1: X0-X1-X2-X1-X3-X1-X1-X4 (SEQ ID NO: 41). In some examples, X0 is valine (V), isoleucine (I), leucine (L), phenylalanine (F), tryptophan (W), tyrosine (Y) or methionine (M). In some examples, X1 is alanine (A), asparagine (N), glutamine (Q), serine (S), threonine (T), glutamic acid (E), aspartic acid (D), lysine (K), arginine (R), or histidine (H). In some examples, X2 is V, I, L, or M, and X3 is E, S, or Q. In some examples, X4 is K, R, E, or A. In some examples, formula 1 further comprises X5. X5 can be proline (P) or R.

[0066]

[0104] In some examples, formula 1 comprises LALG-X3-X1-X1-X4 (SEQ ID NO: 42), LKLG-X3-X1-X1-X4 (SEQ ID NO: 43), or VKLG-X3-X1-X1-X4 (SEQ ID NO: 44). In some examples, formula 1 comprises VKLG-X3-X1-X1-X4 (SEQ ID NO: 45). In some examples, the exogenous polypeptide comprises VKLG-X3-X1-T-X4 (SEQ ID NO: 46) and / or VKLG-X3-X1-X1-K (SEQ ID NO: 47). In some examples, the exogenous polypeptide comprises LALG-X3-X1-X1-X4 (SEQ ID NO: 48). In some examples, the exogenous polypeptide comprises LALG-X3-X1-T-X4 (SEQ ID NO: 49) and / or LALG-X3-X1-S-X4 (SEQ ID NO: 50). In some examples, the exogenous polypeptide comprises LALG-X3-X1-TR (SEQ ID NO: 51), LALG-X3-X1-TK (SEQ ID NO: 52), LALG-X3-X1-TE (SEQ ID NO: 53), and / or LALG-X3-X1-TA (SEQ ID NO: 54). In some examples, the exogenous polypeptide comprises LALG-X3-X1-SK (SEQ ID NO: 56). In some examples, the exogenous polypeptide comprises LKLG-X3-X1-X1-X4 (SEQ ID NO: 57). In some examples, the exogenous polypeptide comprises LKLG-X3-X1-T-X4 (SEQ ID NO: 58). In some examples, the exogenous polypeptide comprises LKLG-X3-X1-TK (SEQ ID NO: 59).

[0067]

[0105] In some examples, the exogenous polypeptide comprises a sequence of Formula 1. In some examples, the sequence of Formula I comprises a polypeptide sequence having at least 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or up to about 100% identity to a sequence of Table 4. In some examples, the exogenous polypeptide is a polypeptide of Table 4 having 0-2 modifications to the residues.

[0068]

[0106] In some examples, at least two of the exogenous polypeptides, such as the polypeptides described in formula 1, are inserted into the capsid sequence of the AAV provided herein. The at least two exogenous polypeptides can be inserted at the same position or at different positions. In one embodiment, any one of the exogenous polypeptide sequences provided in Table 4 can be inserted into a non-modified AAV capsid sequence, such as the wild-type sequence provided in Table 5, to generate a modified AAV capsid.

[0069] [Table 4-1]

[0070] [Table 4-2]

[0071] [Table 4-3]

[0072] [Table 4-4]

[0073] [Table 4-5]

[0074] [Table 4-6]

[0075] [Table 4-7]

[0076] [Table 4-8]

[0077] [Table 4-9]

[0078]

Table 4-10

[0079]

Table 4-11

[0080]

Table 4-12

[0081]

Table 4-13

[0082]

Table 4-14

[0083]

Table 5-1

[0084]

Table 5-2

[0085]

Table 5-3

[0086]

[0107] Similarly, deletion can comprise deleting at least one amino acid residue in the sequence encoding AAV capsid.Can delete any number of amino acids.In some examples, at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or up to about 50 exogenous amino acid residues can be inserted and / or deleted in the polypeptide sequence encoding AAV capsid. In some examples, at least or at most 1-5, 5-10, 10-15, 15-20, or a combination thereof, exogenous amino acid residues may be inserted and / or deleted in the polypeptide sequence encoding the AAV capsid. In some examples, about or at most about 5 amino acids to about 11 amino acids are inserted into an insertion site in the GH loop or loop IV of the capsid protein compared to the corresponding unmodified AAV capsid protein. For example, the insertion site may be between amino acids 587 and 588 of AAV2, or the corresponding position of the capsid subunit of another AAV serotype. It should be noted that the insertion site 587-588 is based on the AAV2 capsid protein. About 5 amino acids to about 11 amino acids may be inserted into the corresponding position in an AAV serotype other than AAV2 (e.g., AAV5, AAV6, AAV8, AAV9, etc.).

[0087]

[0108] In some embodiments, the insertion site is a single insertion site between two adjacent amino acids located between amino acids 570-614 of VP1 of any AAV serotype, e.g., the insertion site is between two adjacent amino acids located at amino acids 570-610, amino acids 580-600, amino acids 570-575, amino acids 575-580, amino acids 580-585, amino acids 585-590, amino acids 590-600, or amino acids 600-614 of VP1 of any AAV serotype or variant. For example, the insertion site can be between amino acids 580 and 581, amino acids 581 and 582, amino acids 583 and 584, amino acids 584 and 585, amino acids 585 and 586, amino acids 586 and 587, amino acids 587 and 588, amino acids 588 and 589, or amino acids 589 and 590. The site of insertion can be between amino acids 575 and 576, amino acids 576 and 577, amino acids 577 and 578, amino acids 578 and 579, or amino acids 579 and 580. The site of insertion can be between amino acids 590 and 591, amino acids 591 and 592, amino acids 592 and 593, amino acids 593 and 594, amino acids 594 and 595, amino acids 595 and 596, amino acids 596 and 597, amino acids 597 and 598, amino acids 598 and 599, or amino acids 599 and 600.

[0088]

[0109] In some embodiments, the insertion site may be between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 588 and 589 of AAV10.

[0089]

[0110] As another example, the insertion site can be between amino acids 450 and 460 of the AAV capsid protein shown in Table 5. For example, the insertion site can be at (e.g., immediately N-terminal to) amino acid 453 of AAV2, amino acid 454 of AAV1, amino acid 454 of AAV6, amino acid 456 of AAV7, amino acid 456 of AAV8, amino acid 454 of AAV9, or amino acid 456 of AAV10.

[0090]

[0111] In some embodiments, a capsid protein of interest comprises a GH loop that comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an amino acid sequence set forth in Table 5. One of skill in the art will know where an insertion site "corresponding to amino acids 587-588 of AAV2" is located in the capsid protein of any given AAV serotype based on a comparison of the amino acid sequences of the capsid proteins of various AAV serotypes.

[0091]

[0112] In some examples, the exogenous polypeptide may have 0 to 4 spacer amino acids (Y1-Y4) at the amino and / or carboxyl terminus of any one of the exemplary polypeptides of Table 4 or Formula 1. Suitable spacer amino acids include, but are not limited to, leucine, alanine, glycine, and / or serine.

[0092]

[0113] The modification of AAV capsid can include the modification of at least one amino acid residue in polypeptide sequence.In some cases, the modification can be made at any AAV capsid position described herein and can include any number of modifications.In some cases, the modification can include mutation.The mutation can include point mutation, missense mutation, nonsense mutation, deletion, duplication, frameshift, and / or repeat expansion.

[0093]

[0114] In one aspect, the amino acid may be a non-polar aliphatic residue, such as glycine, alanine, valine, leucine, isoleucine, or proline. In one aspect, the amino acid residue is aromatic and is phenylalanine, tyrosine, or tryptophan. In one aspect, the amino acid residue is polar and uncharged and is serine, threonine, cysteine, methionine, asparagine, or glutamine. In one aspect, the amino acid is positively charged and is lysine, arginine, or histidine. In one aspect, the amino acid is negatively charged and is aspartic acid or glutamic acid.

[0094]

[0115] In some cases, the mutation is a point mutation. The point mutation includes a change from a charged amino acid residue to a polar or non-polar amino acid residue. In some cases, the charged amino acid has a positive charge. In some cases, the charged amino acid has a negative charge.

[0095]

[0116] The point mutation may be a conservative mutation. Non-limiting examples of conservative mutations include a change from a non-polar aliphatic amino acid to a non-polar aliphatic amino acid, a change from a polar amino acid to a polar amino acid, a change from a positively charged amino acid to a positively charged amino acid, a change from a negatively charged amino acid to a negatively charged amino acid, and a change from an aromatic amino acid to an aromatic amino acid. For example, the 20 naturally occurring amino acids may share similar characteristics. The aliphatic amino acid may be glycine, alanine, valine, leucine, or isoleucine. The hydroxyl or sulfur / selenium-containing amino acid may be serine, cysteine, selenocysteine, threonine, or methionine. The cyclic amino acid may be proline. The aromatic amino acid may be phenylalanine, tyrosine, or tryptophan. The basic amino acid may be histidine, lysine, and arginine. The acidic amino acid may be aspartic acid, glutamic acid, asparagine, or glutamine. Conservative mutations can be serine to glycine, serine to alanine, serine to serine, serine to threonine, and serine to proline. Conservative mutations can be arginine to asparagine, arginine to lysine, arginine to glutamine, arginine to arginine, and arginine to histidine. Conservative mutations can be leucine to phenylalanine, leucine to isoleucine, leucine to valine, leucine to leucine, and leucine to methionine. Conservative mutations can be proline to glycine, proline to alanine, proline to serine, proline to threonine, and proline to proline. Conservative mutations can be threonine to glycine, threonine to alanine, threonine to serine, threonine to threonine, and threonine to proline. Conservative mutations can be alanine to glycine, alanine to threonine, alanine to proline, alanine to alanine, and alanine to serine. Conservative mutations can be valine to methionine, valine to phenylalanine, valine to isoleucine, valine to leucine, valine to valine.Conservative mutations can be glycine to alanine, glycine to threonine, glycine to proline, glycine to serine, glycine to glycine. Conservative mutations can be isoleucine to phenylalanine, isoleucine to isoleucine, isoleucine to valine, isoleucine to leucine, isoleucine to methionine. Conservative mutations can be phenylalanine to tryptophan, phenylalanine to phenylalanine, phenylalanine to tyrosine. Conservative mutations can be tyrosine to tryptophan, tyrosine to phenylalanine, tyrosine to tyrosine. Conservative mutations can be cysteine ​​to serine, cysteine ​​to threonine, cysteine ​​to cysteine. Conservative mutations can be histidine to asparagine, histidine to lysine, histidine to glutamine, histidine to arginine, histidine to histidine. Conservative mutations can be glutamine to glutamic acid, glutamine to asparagine, glutamine to aspartic acid, glutamine to glutamine. Conservative mutations can be asparagine to glutamic acid, asparagine to asparagine, asparagine to aspartic acid, asparagine to glutamine. Conservative mutations can be lysine to asparagine, lysine to lysine, lysine to glutamine, lysine to arginine, lysine to histidine. Conservative mutations can be aspartic acid to glutamic acid, aspartic acid to asparagine, aspartic acid to aspartic acid, aspartic acid to glutamine. Conservative mutations can be glutamine to glutamine, glutamine to asparagine, glutamine to aspartic acid, glutamine to glutamine. Conservative mutations can be methionine to phenylalanine, methionine to isoleucine, methionine to valine, methionine to leucine, methionine to methionine. Conservative mutations can be tryptophan to tryptophan, tryptophan to phenylalanine, tryptophan to tyrosine.

[0096]

[0117] Non-limiting examples of additional amino acid mutations include A to R, A to N, A to D, A to C, A to Q, A to E, A to G, A to H, A to I, A to L, A to K, A to M, A to F, A to P, A to S, A to T, A to W, A to Y, A to V, R to N, R to D, R to C, R to Q, R to E, R to G, R to H, R to I, R to L, R to K, R to M, R to F, R to P, R to S, R to T, R to W, R to Y, R to V, N to D, N to C, N to Q, N to E, N to G, N to H, N to I, N to L, N to K ,N to M,N to F,N to P,N to S,N to T,N to W,N to Y,N to V,D to C,D to Q,D to E,D to G,D to H,D to I,D to L,D to K,D to M,D to F,D to P,D to S,D to T,D to W,D to Y,D to V,C to Q,C to E,C to G,C to H,C to I,C to L,C to K,C to M,C to F,C to P,C to S,C to T,C to W,C to Y,C to V,Q to E,Q to G,Q to H,Q to I,Q to L,Q to K,Q to M,Q to F,Q to P,Q to S,Q to V T, Q to W, Q to Y, Q to V, E to G, E to H, E to I, E to L, E to K, E to M, E to F, E to P, E to S, E to T, E to W, E to Y, E to V, G to H, G to I, G to L, G to K, G to M, G to F, G to P, G to S, G to T, G to W, G to Y, G to V, H to I, H to L, H to K, H to M, H to F, H to P, H to S, H to T, H to W, H to Y, H to V, I to L, I to K, I to M, I to F, I to P, I to S, I to T, I to W, I to Y, I to V, L to to K, L to M, L to F, L to P, L to S, L to T, L to W, L to Y, L to V, K to M, K to F, K to P, K to S, K to T, K to W, K to Y, K to V, M to F, M to P, M to S, M to T, M to W, M to Y, M to V, F to P, F to S, F to T, F to W, F to Y, F to V, P to S, P to T, P to W, P to Y, P to V, S to T, S to W, S to Y, S to V, T to W, T to Y, T to V, W to Y, W to V, Y to V, and the reverse of any of the mutations already described.

[0097]

[0118] Any one of the above modifications, insertions, deletions, and / or mutations can be made at any residue in the AAV sequence. The sequence can be a capsid sequence. In other examples, the sequence can be a Rep and / or X sequence, rather than a capsid sequence. The sequence can be present in VP1, VP2, and / or VP3 as previously described. In some examples, the sequence modification is a modification of a loop of the capsid sequence, such as loop 3 and / or loop 4. In some examples, the modification is a modification of a residue of a sequence in Table 5.

[0098]

[0119] In some examples, the modification, e.g., insertion, deletion, and / or mutation, is a modification of a residue of a capsid polypeptide sequence of Table 5. In some examples, the modification is at a residue at positions 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, or a combination thereof. In some examples, the modification is at a residue at positions 200-300, 300-400, 400-500, 500-600, or a combination thereof. In some examples, the modification is at a residue at positions 300-500, or a combination thereof. In one embodiment, the insertion site is in the GH loop or loop IV of the AAV capsid protein, e.g., in a solvent exposed portion of the GH loop or loop IV of the AAV capsid protein. For example, the insertion site is within amino acids 570-611 of AAV2, within amino acids 571-612 of AAV1, within amino acids 560-601 of AAV5, within amino acids 571-612 of AAV6, within amino acids 572-613 of AAV7, within amino acids 573-614 of AAV8, within amino acids 571-612 of AAV9, or within amino acids 573-614 of AAV10.

[0099]

[0120] For example, the insertion site can be between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 589 and 590 of AAV10. In some examples, the modification is at positions 452, 453, 466, 467, 468, 471, 585, 586, 587, and / or 588 of AAV2. In some examples, the modification is at positions 452 or 453 of AAV2. In some examples, the modification is at positions 587 or 588 of AAV2. In some examples, the modification is an insertion at positions 452, 453, 466, 467, 468, 471, 585, 586, 587, and / or 588 of any one of SEQ ID NOs: 121-126. In some examples, the modification is an insertion at positions 452, 453, 466, 467, 468, 471, 585, 586, 587, and / or 588 of SEQ ID NO: 121. In some examples, the modification is a mutation, and the mutation is R585A or R588A of any one of SEQ ID NOs: 121-126. In some examples, the modification is a mutation, and the mutation is R585A or R588A of SEQ ID NO: 121.

[0100]

[0121] In some embodiments, a subject modified AAV capsid does not contain any other amino acid modification, mutation, substitution, insertion, or deletion other than an insertion of about 5 amino acids to about 11 amino acids in the loops (loops 3 and / or 4) compared to the corresponding unmodified AAV capsid protein. In other embodiments, a subject variant AAV capsid contains an insertion of about 5 amino acids to about 11 amino acids in loops 3 and / or 4 compared to the unmodified AAV capsid protein, as well as an insertion, deletion, or substitution of 1 to about 25 amino acids compared to the unmodified AAV capsid protein. In one embodiment, a subject AAV virion capsid does not contain any other amino acid substitution, insertion, or deletion other than an insertion of about 7 amino acids to about 10 amino acids in the GH loop or loop IV compared to the corresponding parent AAV capsid protein. In other embodiments, the subject AAV virion capsid comprises an insertion, deletion, or substitution of 1 to about 25 amino acids compared to the parent AAV capsid protein, in addition to an insertion of about 7 to about 10 amino acids in the GH loop or loop IV compared to the corresponding parent AAV capsid protein. For example, in some embodiments, the subject AAV virion capsid comprises an insertion, deletion, or substitution of 1 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, or about 20 to about 25 amino acids compared to the parent AAV capsid protein, in addition to an insertion of about 7 to about 10 amino acids in the GH loop or loop IV compared to the corresponding parent AAV capsid protein.

[0101]

[0122] In some examples, chimeric AAV capsids are provided herein. The chimeric capsid comprises polypeptide sequences from at least two AAV serotypes. The chimeric capsid can comprise a mixture of sequences selected from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and / or AAV12. In some examples, the chimeric serotypes differ between VP1, VP2, and / or VP3. In some examples, the chimeric capsid comprises sequences from at least two serotypes selected from AAV4 and AAV6, AAV5 and AAV6, AAV11 and AAV6, AAV12 and AAV6, and any combination thereof. In some examples, the first AAV serotype can be AAV4, and the second serotype can be AAV6. In some examples, the first AAV serotype and the second AAV serotype of the chimeric AAV vector can be AAV11 and AAV6. In some examples, the first AAV serotype and the second AAV serotype of the chimeric AAV vector can be AAV12 and AAV6. In some examples, the chimeric capsid comprises sequences from AAV2 and AAV5 or AAV2 and AAV6. In some examples, the chimeric capsid comprises sequences from AAV2 and AAV5, AAV2 and AAV6, AAV2 and AAV8, AAV2 and AAV9, AAV2 and AAV1, and AAV2 and AAV12.

[0102]

[0123] The modifications to the AAV provided herein can confer enhanced activity to the modified AAV compared to an otherwise unmodified or wild-type AAV. The modifications provided herein can improve cell transduction, tropism, and / or reduce immunogenicity associated with the capsid.

[0103]

[0124] In some examples, the modifications provided herein enhance cell transduction. Cell transduction can refer to the ability of AAV to infect cells (in vivo or in vitro) and / or deliver a transgene to a cell.

[0104]

[0125] In some examples, the modifications provided herein enhance tropism. Enhanced tropism refers to acquiring the ability to transduce cells through additional receptors compared to otherwise unmodified AAV. In some embodiments, enhanced tropism can improve infection of ocular cells by utilizing modified AAV, thereby improving gene therapy. In some examples, the modifications provided herein can improve tropism for ocular cells selected from bipolar cells, retinal ganglion cells, horizontal cells, amacrine cells, epithelial cells, retinal pigment cells, photoreceptor cells, or any combination thereof. In some examples, the modifications improve tropism for retinal cells,

[0126] Also provided herein is an AAV vector. The AAV vector comprises an inverted terminal repeat (ITR), Rep, Cap, AAP and X sequence. Typically, the AAV viral genome is flanked by ITRs that act as packaging signals and origins of replication. The rep gene encodes a family of multifunctional proteins (Rep proteins) that are involved in the control of viral transcription, replication, packaging and integration in AAVS1. For AAV2, four Rep proteins have been described. The expression of Rep78 and Rep68 is controlled by the AAV2-specific p5 promoter, while p19 controls the expression of smaller Rep proteins (Rep52 and Rep40). Rep68 and Rep40 are splice variants of Rep78 and Rep52, respectively. The numbers indicate molecular weight. Expression of the AAP and viral capsid proteins VP1 (90 kDa), VP2 (72 kDa), and VP3 (60 kDa), all encoded in the cap gene, is controlled by the p40 promoter. The X gene is located at the 3' end of the genome in a region shared with the cap gene and carries its own promoter (p81). The X protein appears to enhance viral replication, whereas the AAP is essential for capsid assembly. The three different VPs contribute to the polyhedral AAV2 capsid in a ratio of 1 (VP1):1 (VP2):10 (VP3).

[0105]

[0127] The modified capsid protein disclosed herein can be isolated, e.g., purified. In some embodiments, the modified capsid disclosed herein is contained in an AAV vector or AAV virion (e.g., recombinant AAV virion, rAAV, or AAV virus particle). In other embodiments, such modified AAV vector and / or AAV variant virion is used in an in vivo or ex vivo method of treating eye disease in primate retina, e.g., human retina.

[0106]

[0128] Also provided herein is a vector that comprises modified AAV capsid.Any one of the modifications previously described can be included in the vector provided herein.In some examples, the AAV vector comprises a modified capsid that comprises exogenous sequence in at least two loops of VP domain, compared to the otherwise equivalent AAV capsid sequence that lacks exogenous sequence.In some aspects, the vector provided herein can further comprise transgene sequence.

[0107] Engineered Polypeptides

[0129] Described herein is an engineered polypeptide comprising a peptide operably linked to an antibody or fragment thereof. In some embodiments, the engineered polypeptide is encoded from an engineered polynucleotide described herein. In some embodiments, the engineered polypeptide comprises a natriuretic peptide. In some embodiments, the engineered polypeptide comprises CNP. In some embodiments, the CNP comprises at least 20 amino acid residues, at least 22 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 35 amino acid residues, at least 36 amino acid residues, at least 40 amino acid residues, at least 45 amino acid residues, at least 50 amino acid residues, at least 53 amino acid residues, at least 55 amino acid residues, at least 60 amino acid residues, at least 61 amino acid residues, at least 65 amino acid residues, at least 70 amino acid residues, at least 75 amino acid residues, at least 80 amino acid residues, at least 85 amino acid residues, at least 90 amino acid residues, at least 95 amino acid residues, at least 100 amino acid residues, at least 105 amino acid residues, at least 110 amino acid residues, at least 115 amino acid residues, at least 120 amino acid residues, at least 125 amino acid residues, or at least 126 amino acid residues. In some embodiments, the CNP comprises 20 amino acid residues, 23 amino acid residues, 25 amino acid residues, 30 amino acid residues, 35 amino acid residues, 36 amino acid residues, 40 amino acid residues, 45 amino acid residues, 50 amino acid residues, 53 amino acid residues, 55 amino acid residues, 60 amino acid residues, 61 amino acid residues, 65 amino acid residues, 70 amino acid residues, 75 amino acid residues, 80 amino acid residues, 85 amino acid residues, 90 amino acid residues, 95 amino acid residues, 100 amino acid residues, 105 amino acid residues, 110 amino acid residues, 115 amino acid residues, 120 amino acid residues, 125 amino acid residues, or 126 amino acid residues. In some embodiments, the CNP comprises 22 amino acid residues. In some embodiments, the CNP comprises 36 amino acid residues. In some embodiments, the CNP comprises 53 amino acid residues. In some embodiments, the CNP comprises 61 amino acid residues. In some embodiments, the CNP comprises 126 amino acid residues.In some embodiments, the CNP comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 1-5. In some embodiments, the CNP comprises an amino acid sequence that is 100% identical to SEQ ID NOs: 1-5. In some embodiments, the CNP comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 1. In some embodiments, the CNP comprises an amino acid sequence that is 100% identical to SEQ ID NO: 1. In some embodiments, the CNP comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 2. In some embodiments, the CNP comprises an amino acid sequence that is 100% identical to SEQ ID NO: 2. In some embodiments, the CNP comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:3. In some embodiments, the CNP comprises an amino acid sequence that is 100% identical to SEQ ID NO:3. In some embodiments, the CNP comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:4. In some embodiments, the CNP comprises an amino acid sequence that is 100% identical to SEQ ID NO:4. In some embodiments, the CNP comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:5. In some embodiments, the CNP comprises an amino acid sequence that is 100% identical to SEQ ID NO:5.

[0108]

[0130] In some embodiments, the engineered polypeptide comprises an antibody or fragment thereof operably linked to a peptide or CNP. In some embodiments, the antibody or fragment thereof comprises an Fc region of an antibody. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 6-8. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 6. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 6. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 7. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 7. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 8. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO:8.

[0109]

[0131] In some embodiments, the engineered polypeptide comprises a peptide covalently attached to the N-terminus of an antibody or fragment thereof. In some embodiments, the engineered polypeptide comprises a peptide covalently attached to the C-terminus of an antibody or fragment thereof. In some embodiments, the engineered polypeptide comprises a peptide covalently attached or operably linked to an antibody or fragment thereof by a peptide linker. In some embodiments, the peptide linker comprises at least one glycine followed by a serine. In some embodiments, the peptide linker comprises a subunit with at least one glycine followed by a serine. For example, the subunit is (GS) n is shown as n is an integer between 0 and 20. In such a scenario, n is equal to 2 is equivalent to a peptide linker having an amino acid sequence of (GS)2 or GSGS. In some embodiments, the subunits may include one glycine followed by a serine (e.g., GS), two glycines followed by a serine (e.g., GGS), three glycines followed by a serine (e.g., GGGS), four glycines followed by a serine (e.g., GGGGS), five glycines followed by a serine (e.g., GGGGGS), or six glycines followed by a serine (e.g., GGGGGGS). In some embodiments, the peptide linker includes subunits with at least one glycine followed by a serine, at least two glycines followed by a serine, at least three glycines followed by a serine, at least four glycines followed by a serine, or at least six glycines followed by a serine. In some embodiments, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 17, 18, 19, or 20. n is an integer greater than 20. In some embodiments, n is an integer equal to 4. In some embodiments, n is the integer number 5.

[0110]

[0132] In some embodiments, the engineered polypeptide comprises a peptide covalently linked to an antibody or fragment thereof by a peptide linker, wherein n is an integer between 0 and 20 (GGGGS). n In some embodiments, the engineered polypeptide comprises an amino acid sequence comprising: n In some embodiments, the engineered polypeptide comprises an amino acid sequence comprising: n In some embodiments, the engineered polypeptide comprises an amino acid sequence comprising: n The amino acid sequence comprises:

[0111]

[0133] In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 131-140. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to any one of SEQ ID NOs: 131-140. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 131. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 131. In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 132. In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 132. In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 133. In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 133. In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 134. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO:134.In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 135. In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 135. In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 136. In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 136. In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 137. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 137. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 138. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 138. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 139. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 139.In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 140. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 140.

[0112]

[0134] In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 9-12. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to any one of SEQ ID NOs: 9-12. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 9. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 9. In some embodiments, the peptide operably linked to the antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 10. In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 10. In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 11. In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 11. In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 12. In some embodiments, a peptide operably linked to an antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 12.

[0113]

[0135] In some embodiments, an engineered polypeptide comprising a CNP operably linked to an antibody or fragment thereof increases the half-life of the linked CNP compared to the unlinked CNP. In some embodiments, a CNP operably linked to an antibody or fragment thereof increases the half-life by at least 0.1-fold, at least 0.2-fold, at least 0.3-fold, at least 0.4-fold, at least 0.5-fold, at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, at least 0.9-fold, at least 1.0-fold, at least 2.0-fold, at least 5.0-fold, at least 10.0-fold, at least 20.0-fold, at least 50.0-fold, or at least 100.0-fold compared to the half-life of the unlinked CNP. In some embodiments, a CNP operably linked to an antibody or fragment thereof increases the half-life by at least 1 minute, at least 2 minutes, at least 3 minutes, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 60 minutes, at least 120 minutes, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days, or at least 30 days compared to the half-life of unlinked CNP. In some embodiments, a CNP operably linked to an antibody or fragment thereof increases the in vivo half-life by at least 1 minute, at least 2 minutes, at least 3 minutes, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 60 minutes, at least 120 minutes, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days, or at least 30 days compared to the in vivo half-life of unlinked CNP.

[0114]

[0136] In some embodiments, an engineered polypeptide comprising a CNP operably linked to an antibody or fragment thereof increases protection against degradation of the linked CNP compared to unlinked CNP. In some embodiments, a CNP operably linked to an antibody or fragment thereof increases protection against degradation by at least 0.1-fold, at least 0.2-fold, at least 0.3-fold, at least 0.4-fold, at least 0.5-fold, at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, at least 0.9-fold, at least 1.0-fold, at least 2.0-fold, at least 5.0-fold, at least 10.0-fold, at least 20.0-fold, at least 50.0-fold, or at least 100.0-fold compared to unlinked CNP.

[0115]

[0137] In some embodiments, the engineered polypeptide may be administered to a subject to treat a disease or condition. In some embodiments, the engineered polypeptide may be formulated into a pharmaceutical composition that is administered to a subject to treat a disease or condition. In some embodiments, the engineered polypeptide comprising CNP and an antibody or fragment thereof may be administered to a subject to treat a disease or condition. In some embodiments, the engineered polypeptide comprising CNP and an antibody or fragment thereof. may be formulated into a pharmaceutical composition that is administered to a subject to treat a disease or condition.

[0116]

[0138] In some embodiments, the engineered polypeptide can increase an activity or signal cascade associated with the natriuretic peptide receptor (NPR). In some embodiments, the engineered polypeptide can increase an activity or signal cascade associated with the cyclic GMP (cGMP) signaling pathway. In some embodiments, the engineered polypeptide comprising CNP and an antibody or fragment thereof can increase an activity or signal cascade associated with the natriuretic peptide receptor (NPR). In some embodiments, the engineered polypeptide comprising CNP and an antibody or fragment thereof can increase an activity or signal cascade associated with the cyclic GMP (cGMP) signaling pathway.

[0117]

[0139] In some embodiments, an engineered polypeptide may be administered to a subject to treat a disease or condition by increasing activity or signal cascade associated with the natriuretic peptide receptor (NPR). In some embodiments, an engineered polypeptide may be administered to a subject to treat a disease or condition by increasing activity or signal cascade associated with the cGMP signaling pathway. In some embodiments, an engineered polypeptide comprising CNP and an antibody or fragment thereof may be administered to a subject to treat a disease or condition by increasing activity or signal cascade associated with the natriuretic peptide receptor (NPR). In some embodiments, an engineered polypeptide comprising CNP and an antibody or fragment thereof may be administered to a subject to treat a disease or condition by increasing activity or signal cascade associated with the cGMP signaling pathway.

[0118] Pharmaceutical Compositions

[0140] Described herein is a pharmaceutical composition comprising an engineered polynucleotide, an AAV vector comprising an engineered polynucleotide, an engineered polypeptide, a cell transduced with an AAV vector comprising an engineered polynucleotide, a viral particle comprising an engineered polynucleotide, or a combination thereof. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition comprises two or more active agents disclosed herein. In some embodiments, the pharmaceutical composition comprising an engineered polynucleotide, an AAV vector comprising an engineered polynucleotide, or an AAV vector comprising an engineered polynucleotide treats a disease or condition described herein. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the disease or condition comprises ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color vision deficiency, age-related macular degeneration (nAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, traumatic glaucoma, Bardet-Biedl syndrome, Best disease, choroideremia, Leber's congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-vertebral dystrophy, vertebral-rod dystrophy, Oguchi disease, autosomal dominant radial drusen (familial autosomal dominant drusen), blue vertebral monochromacy, or a combination thereof.

[0119]

[0141] For in vivo delivery, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the engineered polypeptide, the cell transduced with the AAV vector comprising the engineered polynucleotide, or combinations thereof, can be formulated into a pharmaceutical composition and generally administered intravitreally or parenterally (e.g., administered via routes of administration such as intramuscular, subcutaneous, intratumoral, transdermal, intrathecal, etc.). In some embodiments, the pharmaceutical composition is formulated for administration intrathecally, intraocular, intravitreal, retinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, subretinal, suprachoroidal, intratumoral, pulmonary, intratracheal, intraperitoneal, intravesical, intravaginal, rectal, oral, sublingual, transdermal, inhalation, inhalation spray form, intraluminal-GI route, or combinations thereof to a subject in need of administration of the pharmaceutical composition.

[0120]

[0142] In some embodiments, the pharmaceutical composition can be used to treat a subject, such as a human or mammal, that requires the pharmaceutical composition. In some cases, the subject can be diagnosed with a disease, such as an eye disease. In some embodiments, the subject's pharmaceutical composition is co-administered with a second-line treatment. The second-line treatment can include any treatment for ophthalmic use. In some cases, the second-line treatment includes nutritional therapy, vitamins, laser treatment, such as laser photocoagulation, photodynamic therapy, visudyne, anti-VEGF therapy, eyewear, eye drops, numbing drugs, binocular vision disorder treatment, behavioral / visual cognitive therapy, and the like. In some embodiments, any of the biologics previously described can be considered as a second-line treatment.

[0121]

[0143] In some embodiments, an effective amount of the pharmaceutical composition results in a reduction in the rate of loss of retinal function, anatomical integrity, or retinal health, and thus the rate of disease progression, for example, a reduction in the rate of loss of disease, and thus the rate of disease progression, by 2-fold, 3-fold, 4-fold, or 5-fold or more, for example a reduction in the rate of loss of disease, and thus the rate of disease progression, by 10-fold or more.

[0122]

[0144] In some embodiments, an effective amount of the pharmaceutical composition reduces angiogenic signaling in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more, compared to angiogenic signaling in a cell that was not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition reduces angiogenesis in a subject in need of treatment with the pharmaceutical composition by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more, compared to angiogenesis in a subject when the subject is not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition reduces vascular leakage in a subject in need of treatment with the pharmaceutical composition by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more compared to vascular leakage in the subject when the subject is not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition reduces inflammation in a subject in need of treatment with the pharmaceutical composition by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more compared to inflammation in the subject when the subject is not treated with the pharmaceutical composition.

[0123]

[0145] In some embodiments, an effective amount of a subject rAAV virion results in visual function, increased retinal function, improved retinal anatomy or health, and / or improved eye movement, and / or improved neurological function, e.g., a 2-fold, 3-fold, 4-fold, or 5-fold or greater improvement in retinal function, retinal anatomy or health, and / or eye movement, e.g., a 10-fold or greater improvement in retinal function, retinal anatomy or health, and / or eye movement. As will be readily appreciated by those of skill in the art, the dosage required to achieve the desired therapeutic effect is typically within the range of 1×10 8 ~Approx. 1×10 15 recombinant virions, typically in the range of 1×10 8 ~Approx. 1×10 15 This is called the "vector genome."

[0124]

[0146] In some aspects, the compositions, e.g., pharmaceutical compositions, provided herein are administered to a subject in need of the pharmaceutical composition. In some examples, administration is at least about 0.5×10 of the AAV vector. 9 vg, 1.0×109vg, 1.0×10 10 , 1.0×10 11 vg, 3.0×10 11 vg, 6×10 11 vg, 8.0×10 11 vg, 1.0×10 12 vg, 1.0×10 13 vg, 1.0×10 14 vg, 1.0×10 15 vg, 1.5×10 15 For example, for in vivo injection, e.g., direct injection into the eye, a therapeutically effective dose includes delivering a dose of about 10 of the AAV virions to the subject. 6 ~about 10 15 order of magnitude, for example about 10 8 ~10 12 For in vitro transduction, the effective amount of engineered AAV virions delivered to cells can be on the order of about 10 engineered AAV virions. 8 ~about 10 13Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose-response curves.

[0125]

[0147] Administration can be repeated for any period of time. In some embodiments, administration is performed twice daily, once every other day, twice weekly, bimonthly, once every three months, monthly, every other month, semi-annually, annually, or biennially.

[0126]

[0148] Dosage treatment can be a single dose schedule or a multiple dose schedule.Moreover, subject can be administered as many doses as necessary.Those skilled in the art can easily determine the appropriate number of doses.In some embodiments, pharmaceutical composition is administered via intravitreal injection, subretinal injection, microinjection or suprachoroidal injection.

[0127]

[0149] In the practice of the treatment or method of use provided herein, a therapeutically effective amount of the pharmaceutical composition described herein is administered to a mammal having a disease, disorder, or condition, such as cancer, to be treated.In some embodiments, the mammal is a human.The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used, and other factors.The therapeutic agent, and in some cases the composition described herein, can be used alone or in combination with one or more therapeutic agents as a component of a mixture.

[0128]

[0150] The pharmaceutical compositions described herein can be administered to a subject by suitable routes of administration, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal routes of administration.The compositions described herein can include but are not limited to aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, sustained release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsed release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[0129]

[0151] The pharmaceutical compositions may be manufactured in a conventional manner, such as by means of conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping or compressing processes, by way of example only.

[0152] In certain embodiments, the pharmaceutical compositions provided herein contain one or more preservatives to inhibit microbial activity.Suitable preservatives include mercury-containing substances, such as merfen and thimerosal; stabilized chlorine dioxide; and quaternary ammonium compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0130]

[0153] In some embodiments, the pharmaceutical compositions described herein are formulated into any suitable dosage form, including, but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, sustained release formulations, pulsed release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations. In one aspect, the therapeutic agents disclosed herein, e.g., therapeutic agents, are formulated into pharmaceutical compositions suitable for intramuscular, subcutaneous, or intravenous injection. In one aspect, formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for rehydration into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, cremophor, and the like), suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial growth can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some cases, it is desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be brought about by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0131]

[0154] In another aspect, the dosage form comprises microencapsulated formulation.In some embodiments, one or more other compatible materials are present in the microencapsulated material.Non-limiting examples of materials include pH adjuster, disintegration promoter, antifoaming agent, antioxidant, flavoring agent, and carrier material, such as binder, suspending agent, disintegrating agent, filler, surfactant, dissolving agent, stabilizer, lubricant, wetting agent, and diluent.

[0132]

[0155] The dosage form of the liquid formulation for oral administration is optionally an aqueous suspension selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersion, emulsion, solution, elixir, gel, and syrup.In addition to the therapeutic agent, the liquid dosage form optionally includes additives such as (a) disintegrant; (b) dispersant; (c) wetting agent; (d) at least one preservative, (e) thickener, (f) at least one sweetener, and (g) at least one flavoring agent.In some embodiments, the aqueous dispersion further includes a crystal formation inhibitor.

[0133]

[0156] In some embodiments, the pharmaceutical compositions described herein are self-emulsifying drug delivery systems (SEDDS). Emulsions are dispersions of one immiscible phase in another immiscible phase, usually in the form of droplets. Generally, emulsions are made by vigorous mechanical dispersion. In contrast to emulsions or microemulsions, SEDDS spontaneously form emulsions upon addition of excess water without any external mechanical dispersion or agitation. The advantage of SEDDS is that only light mixing is required to distribute the droplets throughout the solution. In addition, water or aqueous phase is added as needed just before administration, thereby ensuring the stability of unstable or hydrophobic active ingredients. Thus, SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provides improved bioavailability of hydrophobic active ingredients.

[0134]

[0157] In addition, the pharmaceutical compositions optionally contain one or more pH adjusting or buffering agents, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trishydroxymethylaminomethane, and buffers such as citric acid / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0135]

[0158] In addition, the pharmaceutical composition may optionally contain one or more salts in an amount necessary to make the osmolality of the composition acceptable.Such salts include salts with sodium, potassium, or ammonium cations, and salts with chloride, citrate, ascorbic acid, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0136] kit

[0159] In some embodiments, disclosed herein is a kit for use, comprising the engineered polynucleotide described herein, the AAV comprising the engineered polynucleotide, the engineered polypeptide, the cell transduced with the AAV vector comprising the engineered polynucleotide, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof. In some embodiments, the kit disclosed herein can be used to treat a disease or condition in a subject. In some embodiments, the kit comprises a collection of materials or components apart from comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the engineered polypeptide, the cell transduced with the AAV vector comprising the engineered polynucleotide, or the pharmaceutical composition.

[0137]

[0160] In some embodiments, the kits described herein include components for selecting for a homogenous AAV population containing the engineered polynucleotides described herein. In some embodiments, the kits include components for assaying the number of units of a biomolecule (e.g., AAV) synthesized and / or released by or expressed on the surface of a host cell. In some embodiments, the kits include components for performing an assay such as an enzyme-linked immunosorbent assay (ELISA). The exact nature of the components configured in the kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating a disease or condition (e.g., cancer) disclosed herein in a subject. In some embodiments, the kits are configured specifically for the purpose of treating a mammalian subject. In some embodiments, the kits are configured specifically for the purpose of treating a human subject.

[0138]

[0161] Instructions for use may be included in the kit. In some embodiments, the kit includes instructions for administering the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the engineered polypeptide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the pharmaceutical composition, or a combination thereof to a subject in need of administration of the kit. In some embodiments, the kit includes instructions for further engineering a cell to express a biological molecule (e.g., the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the engineered polypeptide, the AAV comprising the engineered polynucleotide, or the cell transduced with the AAV vector). In some embodiments, the kit includes instructions for thawing or otherwise restoring biological activity of the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, or the AAV comprising the engineered polynucleotide, which may be cryopreserved or lyophilized during storage or transport. In some embodiments, the kit comprises instructions for measuring the viability of the restored engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, or the AAV comprising the engineered polynucleotide to ensure efficacy for its intended purpose (e.g., therapeutic efficacy when used to treat a subject).

[0139]

[0162] If necessary, the kit may also contain other useful components, such as diluents, buffers, pharma- ceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, dressings, or other useful tools. The materials or components assembled in the kit may be stored and provided to the physician in any convenient and suitable manner that preserves their operability and usefulness. For example, the components may be in dissolved, dehydrated, or lyophilized form; they may be provided at room temperature, refrigerated, or frozen temperature. The components are typically contained in a suitable packaging material(s).

[0140] Delivery method

[0163] The engineered polynucleotide can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the engineered polynucleotide can be transferred into host cells by physical, chemical, or biological means. In some embodiments, the engineered polynucleotide can be delivered to host cells by encapsulating the engineered polynucleotide in a viral particle, such as an AAV particle. In some embodiments, the engineered polynucleotide can be delivered to cells via physical methods, such as calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, electroporation, and the like.

[0141]

[0164] Physical methods of introducing the encoding engineered polynucleotide into cells can include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene guns, electroporation, and the like. One method of introducing an engineered polynucleotide into a host cell is calcium phosphate transfection.

[0142]

[0165] Chemical means of introducing engineered polynucleotides encoding non-naturally occurring nucleic acids into cells can include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, spherical nucleic acids (SNAs), liposomes, or lipid nanoparticles. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle). Other state-of-the-art targeted delivery methods of nucleic acids are available, such as targeted nanoparticle delivery of engineered polynucleotides or vectors encoding engineered polynucleotides.

[0143]

[0166] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for introducing the engineered polynucleotide or a vector encoding the engineered polynucleotide into cells (in vitro, ex vivo, or in vivo). In another aspect, the vector can be associated with a lipid. The lipid-associated vector can be encapsulated in the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule associated with both the liposome and the engineered polynucleotide, entrapped in a liposome and complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in or complexed with a micelle, or otherwise associated with a lipid. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, in some embodiments, they exist in a bilayer structure as micelles or as a "collapsed" structure. Alternatively, they simply disperse in the solution, possibly forming aggregates that are not uniform in size or shape.Lipids are fatty substances that, in some embodiments, are naturally occurring or synthetic lipids.For example, lipids include the lipid droplets that naturally occur in cytoplasm, as well as the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0144]

[0167] Lipids suitable for use are obtained from commercial sources, and stock solutions of lipids in chloroform or chloroform / methanol are often stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes are often characterized as having a vesicular structure with a phospholipid bilayer and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers. However, compositions that have structures in solution that differ from normal vesicular structures are also encompassed. For example, lipids in some embodiments adopt micellar structures or simply exist as heterogeneous aggregates of lipid molecules. Similarly, lipofectamine-nucleic acid complexes are contemplated.

[0145]

[0168] In some examples, non-viral delivery methods include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, exosomes, polycation or lipid: cargo conjugates (or aggregates), naked polypeptides (e.g., recombinant polypeptides), naked DNA, artificial virions, and agent-enhanced uptake of polypeptides or DNA. In some embodiments, the delivery method includes conjugating or encapsulating the compositions or engineered polynucleotides described herein with at least one polymer, such as a natural polymer or a synthetic material. The polymer may be biocompatible or biodegradable. Non-limiting examples of suitable biocompatible, biodegradable synthetic polymers include aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylene oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamide esters, polyoxaesters containing amine groups, and poly(anhydrides). Such synthetic polymers can be homopolymers or copolymers (e.g., random, block, segmented, graft) of two or more of multiple different monomers, such as lactic acid, lactide, glycolic acid, glycolide, epsilon-caprolactone, trimethylene carbonate, p-dioxanone, etc. In one example, the scaffold can be composed of a polymer that includes glycolic acid and lactic acid, such as a polymer with a 90 / 10 or 5 / 95 ratio of glycolic acid to lactic acid. Non-limiting examples of naturally occurring biocompatible, biodegradable polymers can include glycoproteins, proteoglycans, polysaccharides, glycosaminoglycans (GAGs) and fragment(s) derived from these components, elastin, laminin, dechlorin, fibrinogen / fibrin, fibronectin, osteopontin, tenascin, hyaluronic acid, collagen, chondroitin sulfate, heparin, heparan sulfate, ORC, carboxymethylcellulose, and chitin.

[0146]

[0169] In some examples, the engineered polynucleotides described herein can be packaged and delivered to cells via extracellular vesicles. The extracellular vesicles can be any membrane-bound protein. In some embodiments, the extracellular vesicles can be any membrane-bound particle secreted by at least one cell. In some examples, the extracellular vesicles can be any membrane-bound particle synthesized in vitro. In some examples, the extracellular vesicles can be any membrane-bound particle synthesized without cells. In some examples, the extracellular vesicles can be exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptosomes, oncosomes, exospheres, enveloped viruses, exomers, or other very large extracellular vesicles.

[0147]

[0170] In some embodiments, engineered polynucleotides can be delivered to cells via biological methods, such as the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors, in some embodiments, are derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, and adeno-associated viruses, and the like. Exemplary viral vectors include retroviral vectors, adenoviral vectors, adeno-associated virus vectors (AAV vectors), pox vectors, parvovirus vectors, baculovirus vectors, measles virus vectors, or herpes simplex virus vectors (HSV). In some examples, retroviral vectors include gamma retroviral vectors, such as vectors derived from Moloney murine leukemia (Keukemia) virus (MoMLV, MMLV, MuLV, or MLV), or murine stem cell virus (MSCV) genomes. In some examples, retroviral vectors also include lentiviral vectors, such as vectors derived from the human immunodeficiency virus (HIV) genome. In some examples, the AAV comprises serotypes including AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or combinations thereof. Based on these initial serotypes, the AAV capsid of each serotype can be engineered to be more suitable for biological function, tissue, or cell selection. In some embodiments, the AAV is AAV2, and variants AAV2.N53 and AAV2.N54, which are used in the examples of the present disclosure. Chimeric AAVs that can contain at least two AAV serotypes are also contemplated. In some examples, at least 3, at least 4, at least 5, at least 6, at least 7, or up to 8 different serotypes are combined in a chimeric AAV. In some examples, only a small portion of the AAV is chimeric. For example, the suitable portion can include capsid, VP1, VP2, or VP3 domains, and / or Rep.In some examples, at least one of VP1, VP2, and VP3 has at least one amino acid substitution compared to an otherwise equivalent wild-type AAV capsid protein. In some examples, the mutations can occur in VP1 and VP2, VP1 and VP3, VP2 and VP3, or VP1, VP2, and VP3. In some embodiments, at least one of VP1, VP2, and VP3 has 1 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3, such as about 1 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, or about 20 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3. In some examples, the VPs can be removed. For example, in some embodiments, the mutant AAV does not include at least one of VP1, VP2, or VP3.

[0148] Methods for modifying cells

[0171] In one aspect, the present invention also provides a method for modifying cells to generate engineered cells. The cell may refer to a primary cell, a recombinant cell, or a cell line. In some examples, the cell is a packaging cell. The packaging cell may be, for example, any one of HEK293 cells, HeLa cells, and Vero cells. The engineered cell may be a primary cell. In some examples, the engineered cell may be an ocular cell. Suitable ocular cells include, but are not limited to, photoreceptors, ganglion cells, RPE cells, amacrine cells, horizontal cells, Müller cells, and the like.

[0149]

[0172] In some cases, the cell is a packaging cell that is used to generate viral particles.To generate AAV virions or viral particles, AAV vectors are introduced into suitable host cells using known techniques, such as by transfection.In some cases, transfection techniques are used, such as CaPO4 transfection or electroporation, and / or hybrid adenovirus / AAV vectors are used to infect cell lines, such as human embryonic kidney cell line HEK293 (a human kidney cell line that contains a functional adenovirus E1 gene that provides transactivation E1 protein).Suitable transfection methods include calcium phosphate co-precipitation, direct microinjection, electroporation, liposome-mediated gene transfer, and high-velocity microprojectile-based nucleic acid delivery, which are known in the art.

[0150]

[0173] To engineer cells, a plurality of cells may be contacted with the isolated engineered polynucleotide. The contacting step may include any duration, including from about 5 minutes to about 5 days. The contacting step may last for about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, or about 60 minutes. In some examples, the contacting step may last for 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, or up to about 5 days.

[0151]

[0174] In some examples, the packaging cell line supernatant is treated by PEG precipitation to concentrate the virus. In other examples, a centrifugation step can be used to concentrate the virus. For example, a column can be used to concentrate the virus during centrifugation. In some embodiments, the precipitation is performed at about 4° C. or lower (e.g., about 3° C., about 2° C., about 1° C., or about 1° C.) for at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours, or at least about 24 hours. In some embodiments, the recombinant AAV is isolated from the PEG precipitated supernatant by low-speed centrifugation followed by CsCl gradient separation. The low-speed centrifugation can be at about 4000 rpm, about 4500 rpm, about 5000 rpm, or about 6000 rpm for about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes. In some examples, the recombinant AAV is isolated from the PEG precipitated supernatant by centrifugation at about 5000 rpm for about 30 minutes followed by CsCl gradient separation. In some cases, CsCl purification can be replaced by IDX gradient ultracentrifugation. Supernatant can be collected at about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours after transfection, or any time between these two time points. Supernatant can also be purified, concentrated, or combined. For example, concentration or viral titer can be determined by qPCR or silver staining.

[0152]

[0175] In one embodiment, a plurality of AAV particles (containing the engineered polynucleotides described herein) isolated from the engineered cells is also provided. The viral titer is about 10 2 vp / mL, approx. 10 3 vp / mL, approx. 10 4 vp / mL, approx. 10 5 vp / mL, approx. 10 6 vp / mL, approx. 10 7 vp / mL, approx. 10 8 vp / mL, or up to about 10 9 The viral titer can be approximately 10 2GC / mL, approx. 10 3 GC / mL, approx. 10 4 GC / mL, approx. 10 5 GC / mL, approx. 10 6 GC / mL, approx. 10 7 GC / mL, approx. 10 8 GC / mL, or up to about 10 9 In some examples, the viral titer may be about 10 2 TU / mL, approximately 10 3 TU / mL, approximately 10 4 TU / mL, approximately 10 5 TU / mL, approximately 10 6 TU / mL, approximately 10 7 TU / mL, approximately 10 8 TU / mL, or up to about 10 9 The optimal viral titer may be in TU / mL. Optimal viral titers may vary depending on the cell type being transduced. The viral range may be about 1000 MOI to about 2000 MOI, about 1500 MOI to about 2500 MOI, about 2000 MOI to about 3000 MOI, about 3000 MOI to about 4000 MOI, about 4000 MOI to about 5000 MOI, about 5000 MOI to about 6000 MOI, about 6000 MOI to about 7000 MOI, about 7000 MOI to about 8000 MOI, about 8000 MOI to about 9000 MOI, about 9000 MOI to about 10,000 MOI. For example, to infect 1 million cells using an MOI of 10,000, 10,000 x 1,000,000 = 10 10 GC is required.

[0153]

[0176] In some examples, multiple AAV particles can be formulated into a unit dosage form. Various formulations are contemplated for delivery to adults or children, including, but not limited to, 0.5×10 9 vg, 1.0×10 9 vg, 1.0×10 10 , 1.0×10 11 vg, 3.0×10 11 vg, 6×10 11 vg, 8.0×10 11 vg, 1.0×10 12 vg, 1.0×10 13 vg, 1.0×1014 vg, 1.0×10 15 vg, or up to 1.5×10 15 The composition of viral particles may be stored frozen or in a suitable container.

[0154]

[0177] The compositions and methods provided herein may be sufficient to enhance the delivery and / or expression of a subject biologic by at least about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to 100% more than an otherwise equivalent unmodified nucleic acid. In some examples, the otherwise equivalent unmodified nucleic acid is a nucleic acid encoding VEGF-Trap. In some examples, the modification is at least about 1 fold, about 6 fold, about 11 fold, about 16 fold, about 21 fold, about 26 fold, about 31 fold, about 36 fold, about 41 fold, about 46 fold, about 51 fold, about 56 fold, about 61 fold, about 66 fold, about 71 fold, about 76 fold, about 81 fold, about 86 fold, about 91 fold, about 96 fold, about 101 fold, about 106 fold, about 111 fold, about 116 fold, about 121 fold, about 126 fold, about 131 fold, about 136 fold, about 141 fold, about 146 fold, about 151 fold, about 156 fold, about 161 fold, about 166 fold, about 171 fold, about 176 fold, about 181 fold, about 186 fold, It may be sufficient to enhance delivery and / or expression of a subject biologic by about 191-fold, about 196-fold, about 201-fold, about 206-fold, about 211-fold, about 216-fold, about 221-fold, about 226-fold, about 231-fold, about 236-fold, about 241-fold, about 246-fold, about 251-fold, about 256-fold, about 261-fold, about 266-fold, about 271-fold, about 276-fold, about 281-fold, about 286-fold, about 291-fold, about 296-fold, about 301-fold, about 306-fold, about 311-fold, about 316-fold, about 321-fold, about 326-fold, about 331-fold, about 336-fold, about 341-fold, about 346-fold, or about 350-fold more. In one embodiment, the increase in expression comprises at least a 5-fold, at least a 10-fold, at least a 20-fold, at least a 50-fold, at least a 100-fold, at least a 200-fold, or at least a 500-fold increase as determined by in vitro assays. Suitable in vitro assays include ELISA, Western blot, Luminex, microscopy, imaging, and / or flow cytometry.

[0155]

[0178] The subject AAV virions may exhibit an increase in infectivity of retinal cells of at least 1-fold, at least 6-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or greater than 50-fold, as compared to the infectivity of retinal cells (photoreceptors, ganglion cells, RPE cells, amacrine cells, horizontal cells, Muller cells, and the like) by AAV virions containing otherwise equivalent WT AAV capsid proteins.

[0156] Treatment

[0179] Provided herein are methods of treating the diseases or conditions described herein. In some aspects, the methods confer protection against the disease or condition. The methods of treatment may include introducing an engineered polynucleotide, an AAV vector comprising the engineered polynucleotide, an AAV comprising the engineered polynucleotide, a cell transduced with an AAV vector, a viral particle comprising a viral particle comprising an engineered polynucleotide, a pharmaceutical composition, or a combination thereof, into a subject in need of treatment. Also provided are methods of treating a disease or condition, comprising administering a pharmaceutical composition to a subject in need of treatment of the disease or condition. The pharmaceutical composition may include a sequence encoding a biologic comprising an engineered polynucleotide, an AAV vector comprising the engineered polynucleotide, an AAV vector comprising the engineered polynucleotide, a viral particle comprising an engineered polynucleotide, or a combination thereof. In some embodiments, administration is by any suitable administration, including systemic administration (e.g., intravenous, intravitreal, subretinal, etc.). In some embodiments, the subject is a human.

[0157]

[0180] In some embodiments, the method comprises treating a disease or condition in a subject in need of such treatment by administering to the subject a therapeutically effective amount of an engineered polynucleotide, engineered polypeptide, engineered polynucleotide-transduced cell, or pharmaceutical composition described herein. In some embodiments, the method treats the disease or condition, and a single administration of the engineered polynucleotide, engineered polypeptide, engineered polynucleotide-transduced cell, or pharmaceutical composition described herein cures the disease or condition. In some embodiments, the method treats the disease or condition, and the administration of the engineered polynucleotide, engineered polypeptide, engineered polynucleotide-transduced cell, or pharmaceutical composition described herein does not include daily administration. In some embodiments, the disease or condition comprises an eye disease. Non-limiting examples of eye diseases include ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color vision disorders, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, traumatic glaucoma, Bardet-Biedl syndrome, Best's disease, The disease or condition may include choroideremia, Leber's congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum's disease, Stargardt's disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-vertebral dystrophy, vertebral rod dystrophy, Oguchi's disease, autosomal dominant radial drusen (familial autosomal dominant drusen), blue-vertebral monochromacy, or a combination thereof. In some embodiments, the disease or condition is neovascular glaucoma (NG). In some embodiments, the disease or condition is glaucoma. In some embodiments, the disease or condition is traumatic glaucoma.

[0158]

[0181] In some embodiments, administering a therapeutically effective amount of an engineered polynucleotide, engineered polypeptide, engineered polynucleotide-transduced cell, or pharmaceutical composition described herein to a subject protects the subject from a disease or condition. For example, administering a therapeutically effective amount of an engineered polynucleotide, engineered polypeptide, engineered polynucleotide-transduced cell, or pharmaceutical composition can protect the subject from developing a disease or condition resulting from injury. As shown in Example 4, engineered polypeptides promoted protection of retinal ganglion cells of the eye after transection injury. In some embodiments, administering a therapeutically effective amount of an engineered polynucleotide, engineered polypeptide, engineered polynucleotide-transduced cell, or pharmaceutical composition protects or promotes cell survival in the subject. In some embodiments, administering a therapeutically effective amount of an engineered polynucleotide, engineered polypeptide, engineered polynucleotide-transduced cell, or pharmaceutical composition protects or promotes ocular cell survival in the subject. In some embodiments, administering a therapeutically effective amount of the engineered polynucleotide, engineered polypeptide, engineered polynucleotide-transduced cell, or pharmaceutical composition protects or promotes survival of retinal ganglion cells in the subject. In some embodiments, administering a therapeutically effective amount of the engineered polynucleotide, engineered polypeptide, engineered polynucleotide-transduced cell, or pharmaceutical composition reduces intraocular pressure in the subject.

[0159]

[0182] In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, or the pharmaceutical composition is administered at least once during a period of time (e.g., once every 2 days, twice a week, once a week, every week, three times a month, twice a month, once a month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, once a year). In some embodiments, the composition is administered two or more times during a period of time (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 times). In some embodiments, the administration described herein comprises a single administration. In some embodiments, the administration described herein does not include daily administration.

[0160]

[0183] In some embodiments, the method includes administering a therapeutically effective amount of an engineered polynucleotide, an AAV vector comprising an engineered polynucleotide, an AAV comprising an engineered polynucleotide, a cell transduced with an AAV vector, or a pharmaceutical composition in a variety of dosage forms and routes, including, for example, oral or topical administration. In some embodiments, the composition may be administered intravitreal, subretinal, suprachoroidal, parenteral, intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, subarachnoid, intraocular, intracisternal, ocular, endothelial, topical, intranasal, intrapulmonary, rectal, intraarterial, intrathecal, inhalation, intralesional, intradermal, intradural, intracapsular, subcapsular, intracardiac, transtracheal, subcuticular, subarachnoid, or intraspinal administration, such as by injection or infusion. In some embodiments, the composition may be administered by absorption through epithelial or mucocutaneous linings (e.g., oral mucosal rectal and intestinal mucosal administration). In some embodiments, the composition is delivered via multiple routes of administration.

[0161]

[0184] In some embodiments, the method comprises administering the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof by intravenous infusion. In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof is administered by slow continuous infusion over an extended period of time, for example, for a period of more than 24 hours. In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof is administered as an intravenous injection or short-term infusion. In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof, is administered via the vitreous route. In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof, may be administered locally, for example, via direct injection of the agent into an organ, optionally as a depot, or as a sustained release formulation or implant.

[0162]

[0185] In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof may be administered with other therapies, such as antiviral therapy, chemotherapy, antibiotics, cell therapy, cytokine therapy, or anti-inflammatory agents. In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof may be administered before, during, or after the onset of a disease or condition, and the timing of administering the composition containing the therapeutic agent may vary. In some examples, the composition may be used as a prophylactic agent and may be administered continuously to a subject (e.g., a subject for immunization or a subject for treatment) susceptible to coronavirus or prone to a coronavirus-related condition or disease. Prophylactic administration may attenuate the probability of the onset of an infection, disease, or condition, or may reduce the severity of an infection, disease, or condition.

[0163]

[0186] The engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof, may be administered to a subject prior to the onset of symptoms. In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof may be administered to a subject (e.g., a subject for immunization or a subject for treatment) following (e.g., as soon as possible) a test result, such as a test result providing a diagnosis, a test showing the presence of coronavirus in a subject (e.g., a subject for immunization or a subject for treatment), or a test showing disease progression, e.g., a decrease in blood oxygen levels. The therapeutic agent may be administered after the onset of a disease or condition is detected or suspected (e.g., as soon as practicable). The therapeutic agent can be administered after possible exposure to coronavirus (e.g., as soon as practicable), for example after a subject (e.g., a subject for immunization or a subject for treatment) has come into contact with an infected subject or is known to have come into contact with an infected subject who is contagious.

[0164]

[0187] Actual dosage levels of an agent (e.g., an engineered polynucleotide or pharmaceutical composition) of the present disclosure may be varied to obtain an amount of agent that achieves a desired therapeutic response for a particular subject, composition, and mode of administration without toxicity to the subject (e.g., a subject for immunization or a subject for treatment). The dosage level selected may depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present disclosure used, the route of administration, the time of administration, the rate of excretion, the duration of treatment, other drugs, compounds, and / or materials used in conjunction with the particular composition used, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors known in the medical arts.

[0165]

[0188] Dosage regimes may be adjusted to provide the optimum desired response (e.g., therapeutic and / or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased depending on the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions into unit dosage forms. Unit dosage form as used herein refers to a physically discrete unit suitable as a unitary dose for a subject (e.g., a subject for immunization or a subject for treatment); each unit contains a predetermined amount of active agent calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of the unit dosage forms of the present disclosure are determined by and may depend directly on (a) the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of synthesizing such active agents for the treatment of susceptibility in an individual. The dose may be determined with reference to the plasma or local concentration of the cyclic polyribonucleotide or antibody or antigen-binding fragment thereof. Dosage can be determined with reference to plasma or local concentrations of the linear polyribonucleotide or antibody or antigen-binding fragment thereof.

[0166]

[0189] The engineered polynucleotides, AAV vectors comprising engineered polynucleotides, AAVs comprising engineered polynucleotides, cells transduced with AAV vectors, viral particles comprising engineered polynucleotides, pharmaceutical compositions, or combinations thereof described herein may be in unit dosage forms suitable for single administration of precise dosage amounts. In unit dosage forms, the formulation may be divided into unit doses containing appropriate amounts of the composition. In unit dosage forms, the formulation may be divided into unit doses containing appropriate amounts of one or more linear polyribonucleotides, antibodies or antigen-binding fragments thereof, and / or therapeutic agents. The unit dosages may be in the form of packages containing discrete amounts of the formulations. Non-limiting examples are packaged injectables, vials, and ampoules. The aqueous suspension compositions disclosed herein may be packaged in single-dose non-reopenable containers. Multi-dose reopenable containers may be used, for example, with or without preservatives. The injectables disclosed herein may be present in unit dosage forms, for example, ampoules, or multi-dose containers containing preservatives.

[0167]

[0190] In some examples, the increase in the level of a biologic in a subject is at least a 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, or 500-fold increase as determined by a diagnostic assay.

[0168]

[0191] Suitable diagnostic assays may include ophthalmic diagnostic assays. Ophthalmic diagnostic assays may include ophthalmic examinations, such as refraction, eye scans, optical coherence tomography, Farnworth Munsell 100 Hue test, computerized imaging of the optic disc, and nerve fiber layer analysis (GDX, HRT, OCT), corneal topography, electroretinogram (ERG), electronystagmogram (EOG), visual evoked potentials (VEP), visual evoked responses (VER), fluorescein angiography, optical coherence tomography (OCT), retinal photography, fundus photography, specular microscopy, Goldmann perimeter, Humphrey perimeter, FDT, Octopus perimeter, biometry / IOL calculation, A-scan, B-scan, and combinations thereof.

[0169]

[0192] In some cases, retinal examination can be used.Non-limiting methods for evaluating retinal function and its changes include evaluating visual acuity (e.g., best-corrected visual acuity [BCVA], walking, navigation, object detection and identification), evaluating visual field (e.g., static and kinetic perimetry), performing clinical examination (e.g., anterior and posterior slit lamp examination), evaluating electrophysiological response to all wavelengths of light and dark (e.g., all forms of electroretinogram (ERG) [full field, multifocal and pattern], all forms of visual evoked potential (VEP), electronystagmogram (EOG), color vision, dark adaptation and / or contrast sensitivity). Non-limiting methods for assessing anatomy and retinal health and changes therein include optical coherence tomography (OCT), fundus photography, adaptive optics-applied scanning laser ophthalmoscopy (AO-SLO), fluorescence and / or autofluorescence; ocular motility and eye movement measurements (e.g., nystagmus, fixation preference, and stability), reported outcome measures (patient-reported changes in visually guided and non-visually guided behaviors and activities, patient-reported outcomes [PROs], questionnaire-based quality of life assessments, daily activities, and neurofunctional measures (e.g., magnetic resonance imaging (MRI)).

[0170]

[0193] In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof, exhibits 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more potency compared to a comparable cell not contacted with the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, or the pharmaceutical composition.

[0171]

[0194] In some embodiments, the method of treatment described herein can treat eye diseases.Relevant eye diseases and conditions can include, but are not limited to, blindness, color vision disorder, age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma, Bardet-Biedl syndrome, Best disease, choroideremia, Leber's congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-vertebral dystrophy, vertebral rod dystrophy, Oguchi disease, autosomal dominant radial drusen (familial autosomal dominant drusen), blue-vertebral monochromacy.In one embodiment, the eye disease or condition is AMD.AMD can be wet AMD or dry AMD.

[0172]

[0195] In some cases, administration of the pharmaceutical composition is sufficient to reduce at least one symptom of a disease or condition, treat the disease or condition, and / or eliminate the disease or condition. In some cases, the improvement of the disease or condition can be confirmed by any of the diagnostic assays provided. In other words, the improvement can be obtained through interviewing the treated subject. For example, the subject can communicate to the attending physician that their vision has improved compared to their vision before administration of the subject's drug. In other cases, an in vivo animal model can be used to confirm the reduction of the disease or condition after treatment. Suitable animal models include mouse models, primate models, rat models, dog models, and the like.

[0173]

[0196] The use of absolute or sequential terms, such as "will," "will not," "shall," "shall not," "must," "must not," "first," "firstly," "next," "then," "before," "after," "lastly," and "finally" are not meant to limit the scope of the embodiments disclosed herein, but are exemplary.

[0174]

[0197] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "together," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be included in a similar manner to the term "comprising."

[0175]

[0198] As used herein, the terms "at least one," "one or more," and "and / or" are open-ended expressions whose operation is both conjunctive and disjunctive. For example, the terms "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" each mean A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together, respectively.

[0176]

[0199] As used herein, "or" can refer to "and," "or," and "and / or," and can be used both exclusively and inclusively. For example, the term "A or B" can refer to "A or B," "A but not B," "B but not A," and "A and B." In some cases, the context may dictate a particular meaning.

[0177]

[0200] Any systems, methods, software, and platforms described herein are modular, and thus terms such as "first" and "second" do not necessarily imply a priority, importance, or order of implementation.

[0178]

[0201] The term "about" when referring to a number or numerical range means that the number or numerical range being referred to is an approximation within experimental variation (or within statistical experimental error) and that the number or numerical range may vary, for example, by 1% to 15% from the stated number or numerical range. In examples, the term "about" refers to ±10% of the stated number or value.

[0179]

[0202] The terms "increased", "increasing" or "increase" are used herein to generally mean an increase of a statistically significant amount. In some embodiments, the term "increased" or "increase" means an increase of at least 10% compared to a reference level, such as at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase up to and including 100%, or any increase between 10-100% compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, compared to a reference level.

[0180]

[0203] The terms "reduced", "reducing", or "reduced" as used herein generally refer to a statistically significant amount of reduction. In some embodiments, the terms "reduced" or "reduced" refer to a reduction of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% (e.g., nonexistent levels, or undetectable levels compared to a reference level), or any reduction between 10-100% compared to a reference level. In the context of a marker or condition, these terms refer to a statistically significant reduction in such levels. The reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or greater, preferably to a level that is accepted as within the normal range for individuals without a given disease.

[0181]

[0204] "AAV", "AAV construct", or "recombinant AAV", or "AAV" refers to adeno-associated viruses of any known serotype, including AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, or scAAV, rh10, chimeric or hybrid AAV, or any combination, derivative, or variant thereof. AAVs are small, non-enveloped, single-stranded DNA viruses. They are non-pathogenic parvoviruses and may require helper viruses, such as adenovirus, herpes simplex virus, vaccinia virus, and CMV, for replication. Wild-type AAV is common in the general population and is not associated with any known pathogenicity. Hybrid AAVs are AAVs that contain capsid proteins from one AAV serotype and genomic material from another AAV serotype. Chimeric AAV can comprise gene sequences and / or protein sequences from two or more AAV serotypes, and can comprise mutations made to the gene sequences of the two or more AAV serotypes.Exemplary chimeric AAV can comprise a chimeric AAV capsid, for example, a capsid protein with one or more regions of amino acids from two or more AAV serotypes.AAV variant is an AAV that comprises one or more amino acid mutations in its genome or protein compared to its parent AAV, one or more amino acid mutations in its capsid protein compared to its parent AAV.As used herein, AAV includes avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, where primate AAV refers to the AAV that infects non-primates, and non-primate AAV refers to the AAV that infects non-primate animals, for example, the avian AAV that infects avian animals. In some instances, wild-type AAV contains the rep and cap genes, where the rep gene is required for viral replication and the cap gene is required for the synthesis of capsid proteins. As used herein, the terms "recombinant AAV" and "rAAV" are interchangeable.

[0182]

[0205] The term "recombinant AAV vector" or "AAV vector" or "AAV vector" refers to a vector derived from any of the AAV serotypes mentioned above. In some examples, an AAV vector may contain one or more of the AAV wild-type genes, such as the rep and / or cap genes, deleted in whole or in part, but containing the functional elements required for packaging and using the AAV virus for gene therapy. For example, functional inverted terminal repeat or ITR sequences adjacent to an open reading frame or cloned exogenous sequence are known to be important for the replication and packaging of AAV virions, but the ITR sequences can be modified from the wild-type nucleotide sequence, including nucleotide insertion, deletion, or substitution, so that the AAV is suitable for use in the embodiments described herein, such as gene therapy or gene delivery systems. In some aspects, a self-complementary vector (sc) can be used, such as a self-complementary AAV vector, which can avoid the need for viral double-stranded DNA synthesis and can result in higher expression of transgene proteins. In some embodiments, AAV vectors can be generated to allow for the selection of optimal serotypes, promoters, and transgenes. In some examples, the vectors can be targeted or modified vectors that selectively bind to or infect immune cells.

[0183]

[0206] The term "AAV virion" or "AAV virion" refers to a viral particle that comprises a capsid that includes at least one AAV capsid protein that encapsidates an AAV vector described herein, which in some embodiments may further comprise a heterologous polynucleotide sequence or transgene. The virion may be an engineered virion.

[0184]

[0207] The terms "control," "host," "individual," and "patient" are used interchangeably herein and refer to an animal, typically a mammal. Any suitable mammal may be administered the compositions (e.g., engineered guide RNA) described herein or treated by the methods described herein. The subject may be a vertebrate or an invertebrate. The subject may be an experimental animal. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), livestock (e.g., horses, cows, goats, sheep, pigs), and experimental animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal may be of any age or at any stage of development (e.g., adult, teenager, child, pediatric, or fetus). The mammal may be male or female. In some embodiments, the subject is a human. The subject may be a patient. The subject may be afflicted with a disease. The subject may exhibit symptoms of the disease. The subject may not exhibit symptoms of the disease but still have the disease. The subject may be receiving medical care from a caregiver (e.g., the subject is hospitalized and being treated by a physician).

[0185]

[0208] The terms "protein", "peptide" and "polypeptide" are used interchangeably in their broadest sense and refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The terms also encompass amino acid polymers that have been modified, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" refers to any of the natural and / or unnatural or synthetic amino acids, including glycine and D or L optical isomers, and amino acid analogs and peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. As used herein, the term "amino acid" refers to any of the natural and / or unnatural or synthetic amino acids, including glycine and D and L engineered isomers, amino acid analogs, and peptidomimetics. As used herein, the term "fusion protein" refers to a protein consisting of more than one naturally occurring or recombinantly produced protein domain, where each domain typically performs a different function. In this regard, the term "linker" refers to a protein fragment used to link these domains together, optionally preserving the conformation of the fusion protein domains and / or preventing undesirable interactions between the fusion protein domains that may comprise their respective functions.

[0186]

[0209] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, which means that when aligned, the percentage of bases or amino acids are the same when comparing the two sequences.Sequence similarity can be determined in several different ways.To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, available on the world wide web at ncbi.nlm.nih.gov / BLAST / .Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package.

[0187]

[0210] Although preferred embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention is limited by the specific examples provided herein. Although the present invention has been described with reference to the above specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Multiple variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. Furthermore, it is understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions described herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in the practice of the present invention. It is therefore contemplated that the present invention includes within its scope any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents are covered thereby.

[0188] Embodiment

[0211] Embodiment 1. An engineered polynucleotide comprising an AAV vector comprising one or more expression cassettes, wherein the one or more expression cassettes encode a peptide.

[0189]

[0212] Embodiment 2. An engineered polynucleotide comprising an AAV vector comprising one or more expression cassettes, wherein the one or more expression cassettes encode an engineered polypeptide comprising an antibody or fragment thereof operably linked to a peptide.

[0190]

[0213] Embodiment 3. The engineered polynucleotide of embodiment 1 or 2, wherein the AAV vector comprises an AAV serotype comprising AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof.

[0191]

[0214] Embodiment 4. The engineered polynucleotide of embodiment 3, wherein the AAV serotype comprises AAV2.

[0215] Embodiment 5. The engineered polynucleotide of any one of embodiments 1 to 4, wherein the peptide comprises CNP.

[0192]

[0216] Embodiment 6 The engineered polynucleotide of embodiment 5, wherein the CNP comprises at least 22 amino acid residues.

[0217] Embodiment 7 The engineered polynucleotide of embodiment 5, wherein the CNP comprises at least 36 amino acid residues.

[0193]

[0218] Embodiment 8 The engineered polynucleotide of embodiment 5, wherein the CNP comprises at least 53 amino acid residues.

[0219] Embodiment 9. The engineered polynucleotide of any one of embodiments 6-8, wherein the CNP comprises an amino acid sequence that is at least 80% identical to SEQ ID NOs: 1-5.

[0194]

[0220] Embodiment 10. The engineered polynucleotide of embodiment 2, wherein the peptide is covalently attached to the N-terminus of the antibody or fragment thereof.

[0221] Embodiment 11. The engineered polynucleotide of embodiment 2, wherein the peptide is covalently attached to the C-terminus of the antibody or fragment thereof.

[0195]

[0222] Embodiment 12. The engineered polynucleotide of embodiment 2, wherein the peptide is operably linked to the antibody or fragment thereof by a peptide linker.

[0223] Embodiment 13. The peptide linker is n is an integer between 0 and 10 (GGGGS) n 13. The engineered polynucleotide of embodiment 12, comprising an amino acid sequence comprising:

[0196]

[0224] Embodiment 14. The engineered polynucleotide of any one of embodiments 1 to 4, wherein the AAV vector encodes an engineered AAV capsid.

[0225] Embodiment 15. An engineered polypeptide comprising an antibody or fragment thereof operably linked to a peptide, wherein the antibody or fragment thereof comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs:6-8.

[0197]

[0226] Embodiment 16 The engineered polypeptide of embodiment 15, wherein the peptide comprises CNP.

[0227] Embodiment 17 The engineered polypeptide of embodiment 16, wherein the CNP comprises at least 22 amino acid residues.

[0198]

[0228] Embodiment 18 The engineered polypeptide of embodiment 16, wherein the CNP comprises at least 36 amino acid residues.

[0229] Embodiment 19. The engineered polypeptide of embodiment 16, wherein the CNP comprises at least 53 amino acid residues.

[0199]

[0230] Embodiment 20. The engineered polypeptide of any one of embodiments 17-19, wherein the CNP comprises an amino acid sequence that is at least 80% identical to SEQ ID NOs: 1-5.

[0200]

[0231] Embodiment 21. The engineered polypeptide of any one of embodiments 15 to 20, wherein the peptide is covalently attached to the N-terminus of the antibody or fragment thereof.

[0201]

[0232] Embodiment 22. The engineered polypeptide of any one of embodiments 15 to 20, wherein the peptide is covalently attached to the C-terminus of the antibody or fragment thereof.

[0202]

[0233] Embodiment 23. The engineered polypeptide of any one of embodiments 15 to 22, wherein the peptide is operably linked to the antibody or fragment thereof by a peptide linker.

[0203]

[0234] Embodiment 24. The engineered polypeptide of embodiment 23, wherein the peptide linker comprises an amino acid sequence comprising (GGGGS)n, where n is an integer between 0 and 10.

[0204]

[0235] Embodiment 25. An engineered polynucleotide encoding an engineered polypeptide according to any one of embodiments 15 to 24.

[0236] Embodiment 26 The engineered polynucleotide of embodiment 25, which is a vector.

[0205]

[0237] Embodiment 27 The engineered polynucleotide of embodiment 26, wherein the vector is a viral vector.

[0238] Embodiment 28 The engineered polynucleotide of embodiment 27, wherein the viral vector comprises an AAV vector.

[0206]

[0239] Embodiment 29. The engineered polynucleotide of embodiment 28, wherein the AAV vector comprises an AAV serotype including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof.

[0207]

[0240] Embodiment 30 The engineered polynucleotide of embodiment 29, wherein the AAV serotype comprises AAV2.

[0241] Embodiment 31. The engineered polynucleotide of any one of embodiments 28 to 30, wherein the AAV vector encodes an engineered AAV capsid.

[0208]

[0242] Embodiment 32. The engineered polynucleotide of any one of embodiments 27 to 31, wherein the viral vector comprises one or more expression cassettes.

[0243] Embodiment 33. The engineered polynucleotide of embodiment 2 or embodiment 32, wherein the one or more expression cassettes encode a contiguous polypeptide, the contiguous polypeptide comprising an engineered polypeptide of any one of embodiments 2 to 33.

[0209]

[0244] Embodiment 34 The engineered polynucleotide of embodiment 33, wherein the contiguous polypeptide comprises a protease-cleavable sequence.

[0245] Embodiment 35 The engineered polynucleotide of embodiment 33, wherein the consecutive polypeptide comprises a furin-cleavable sequence.

[0210]

[0246] Embodiment 36 The engineered polynucleotide of embodiment 33, wherein the contiguous polypeptide comprises a self-cleaving polypeptide sequence.

[0247] Embodiment 37. The engineered polynucleotide of embodiment 1, 2, or 32, wherein the one or more expression cassettes express at least one additional therapeutic agent.

[0211]

[0248] Embodiment 38 The engineered polynucleotide of embodiment 37, wherein the at least one additional therapeutic agent comprises a hormone.

[0249] Embodiment 39. The engineered polynucleotide of embodiment 38, wherein the at least one additional therapeutic agent comprises an agonist of a natriuretic peptide receptor (NPR).

[0212]

[0250] Embodiment 40 The engineered polynucleotide of embodiment 38, wherein the at least one additional therapeutic agent comprises an agonist of the cyclic GMP (cGMP) signaling pathway.

[0213]

[0251] Embodiment 41 The engineered polynucleotide of embodiment 37, wherein the at least one additional therapeutic agent comprises a VEGF inhibitor.

[0252] Embodiment 42 The engineered polynucleotide of embodiment 41, wherein the VEGF inhibitor binds to and inhibits VEGF-A, VEGF-B, VEGF-C, VEGF-D, or a combination thereof.

[0214]

[0253] Embodiment 43 The engineered polynucleotide of embodiment 41 or embodiment 42, wherein the VEGF inhibitor comprises an antibody.

[0254] Embodiment 44. The engineered polynucleotide of embodiment 43, wherein the VEGF inhibitor comprises a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, an (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), a single domain antibody (sdAb), an Ig NAR, a camelid antibody, or a combination thereof, a binding fragment thereof, or a chemically modified derivative thereof.

[0215]

[0255] Embodiment 45 The engineered polynucleotide of embodiment 41 or embodiment 42, wherein the VEGF inhibitor comprises a non-antibody VEGF inhibitor.

[0256] Embodiment 46. The engineered polynucleotide of embodiment 45, wherein the non-antibody VEGF inhibitor is VEGF receptor 1 (VEGFR1), VEGF receptor 2 (VEGFR2), VEGF receptor 3 (VEGFR3), a fragment thereof, or a combination thereof.

[0216]

[0257] Embodiment 47. The engineered polynucleotide of embodiment 45, wherein the non-antibody VEGF inhibitor comprises a soluble VEGFR1, a soluble VEGFR2, a soluble VEGFR3, a soluble fragment thereof, or a combination thereof.

[0217]

[0258] Embodiment 48 The engineered polynucleotide of embodiment 45, wherein the non-antibody VEGF inhibitor comprises VEGF-Trap or a modified form thereof.

[0259] Embodiment 49. A cell comprising an engineered polynucleotide according to any one of embodiments 1 to 14 or 25 to 48.

[0218]

[0260] Embodiment 50. A cell comprising an engineered polypeptide according to any one of embodiments 15 to 24.

[0261] Embodiment 51. A pharmaceutical composition comprising an engineered polynucleotide according to any one of embodiments 1 to 14 or 25 to 48, an engineered polypeptide according to any one of embodiments 15 to 24, or a cell according to embodiment 49 or embodiment 50.

[0219]

[0262] Embodiment 52. The pharmaceutical composition of embodiment 51, which is formulated for administration intrathecally, intraocularly, intravitreally, retinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, subretinal, suprachoroidal, intratumoral, pulmonary, intratracheal, intraperitoneal, intravesical, intravaginal, rectal, oral, sublingual, transdermal, inhalation, inhalation spray form, intraluminal-GI route, or a combination thereof, to a subject in need of administration of the pharmaceutical composition.

[0220]

[0263] Embodiment 53. The pharmaceutical composition of embodiment 52, which is formulated for intravitreal, subretinal, or suprachoroidal administration.

[0264] Embodiment 54. The pharmaceutical composition of embodiment 52, for treating an eye disease or condition.

[0221]

[0265] Embodiment 55. The pharmaceutical composition of embodiment 52, which increases natriuretic peptide receptor-B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof, in a subject in need of the pharmaceutical composition.

[0222]

[0266] Embodiment 56. A method for treating a disease or condition in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide according to any one of embodiments 1-14 or 25-48, an engineered polypeptide according to any one of embodiments 15-24, a cell according to embodiment 47 or embodiment 48, or a pharmaceutical composition according to embodiments 49-52.

[0223]

[0267] Embodiment 57. A method of treating a disease or condition in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide according to any one of embodiments 1-13 or 24-46, an engineered polypeptide according to any one of embodiments 14-23, a cell according to embodiment 49 or embodiment 50, or a pharmaceutical composition according to embodiments 49-52, wherein a single administration cures the disease or condition.

[0224]

[0268] Embodiment 58. A method of treating a disease or condition in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide according to any one of embodiments 1-13 or 24-46, an engineered polypeptide according to any one of embodiments 14-23, a cell according to embodiment 47 or embodiment 48, or a pharmaceutical composition according to embodiments 51-55, wherein the administering step does not include daily administration.

[0225]

[0269] Embodiment 59. The method of any one of embodiments 56 to 58, wherein the disease or condition comprises an eye disease.

[0270] Embodiment 60. The eye disease is ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color vision disorders, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, traumatic glaucoma, Bardet-Biedl syndrome, Best disease, colloidal ophthalmopathy, glaucoma ... 60. The method of embodiment 59, comprising a condition that is associated with a protanopia, a glaucoma ... EXAMPLES

[0226]

[0271] The following illustrative examples are representations of embodiments of the stimuli, systems, and methods described herein and are not meant to be limiting in any way. Example 1. Designs and Experiments for Expressing CNP and CNP Fusion Proteins

[0272] A series of CNP and CNP fusion proteins (CNP fused to a human antibody heavy chain secretory signal peptide at the 5' end, IgG1Fc fragment or IgG4Fc fragment at the N-terminus or C-terminus of CNP as shown in Figure 3) were designed and tested. The designed CNP22 or CNP36 and CNP fusion proteins were reverse translated into DNA sequences with homo sapiens codon output. The DNA sequences of CNP and CNP fusion proteins were further manually modified to adjust the GC content for synthesis as three overlapping DNA fragments generating the AAV constructs described herein (e.g., any one of the AAV constructs in Figures 1-2).

[0227]

[0273] For example, to generate AMI061-pFB-scCMV-Vh-Leader-CNP36-Fc, AMI059 was first cut by StuI and SphI to remove the aflibercept coding sequence and polyA signal. Then, the codon-optimized Vh-Leader-CNP36-Fc fragment was PCR amplified with primers A056, A057, A025 and AMI014 as a template. Finally, the PCR fragment was assembled into the StuI and SphI sites of AMI059 to generate AMI061 by NEBuilder HiFi DNA Assembly kit. To generate AMI087-pFB-scCMV-Vh-Leader-CNP36-4xGGGGS-Fc-WPREmini, AMI061 was first cut by SnaBI and SphI to remove the partial CMV promoter and CNP36-4xGGGGS-Fc fragment. The partial CMV promoter and CNP36-4xGGGGS fragment were then amplified with primers A051 and A166 and AMI061 as template. The Fc fragment was amplified with primers A167 and A025 and AMI061 as template. These two fragments were joined by PCR with primers A051 and A025 and assembled into the SnaBI and SphI sites of AMI061 by NEBuilder HiFi DNA Assembly kit to generate AMI087. To generate AMI088-pFB-scCMV-Vh-Leader-Fc-4xGGGGS-CNP36-WPREmini, AMI060, AMI059 were first cut with AflII and XhoI to remove the CNP-VGGRK-Fc fragment. Then, the Fc fragment was amplified with primers A180 and A181, the 4xGGGGS-CNP36 fragment was amplified with primers A182 and A183 and AMI060 as template. These two PCR fragments were joined by primers A180 and A183 and finally assembled into the AflII and XhoI sites of AMI060 by NEBuilder HiFi DNA Assembly kit to generate AMI088.To generate AMI182-pFB-scCMV-Vh-Leader-CNP36-2xSTOP-4xGGGGS-Fc-WPREmini, the 4xGGGGS-Fc fragment was first PCR amplified with primers A618 and A619 and AMI087 as a template. The PCR fragment was then assembled into the XcmI and EcoNI sites of AMI087 by NEBuilder HiFi DNA Assembly kit to generate AMI182. To generate AMI183-pFB-scCMV-3xSTOP-Vh-Leader-CNP36-4xGGGGS-Fc-WPREmini, AMI087 was first cut with StuI and EcoNI to remove the CNP and partial Fc coding sequences. The CNP and partial Fc fragment with three built-in stop codons were then amplified using primers A620, A621, A622, and A619 and AMI087 as a template. Finally, the CNP and partial Fc fragment with three stop codons were assembled into the StuI and EcoNI sites of AMI087 using the NEBuilder HiFi DNA Assembly kit to generate AMI183. The identity of the AAV construct was confirmed by PCR amplification and sequencing analysis by utilizing the primers listed in Table 6.

[0228] [Table 6-1]

[0229] [Table 6-2]

[0230] Generation of recombinant baculoviruses for AAV vector production

[0274] Recombinant baculoviruses (rBVs) were generated using the Bac-to-Bac baculovirus expression system according to the manufacturer's instructions. Briefly, pFB shuttle plasmids containing the target genes were each diluted to 1 ng / μL in TE buffer, and 2 ng of each DNA was mixed with 20 μL of Δcath-DH10Bac competent bacteria containing the cathepsin gene-deleted bacmid DNA molecule, incubated on ice for 30 min, and then heat-shocked at 42°C for 30 s. After incubation on ice for 2 min, the bacteria were allowed to recover at 37°C for 4 h and then plated on agar plates containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 40 μg / mL IPTG, and 100 μg / mL X-gal. After 48 hours of incubation at 37°C, two white colonies containing recombinant bacmid DNA were picked and miniprep bacmid DNA was purified under sterile conditions. Approximately 5 μg of each bacmid DNA and 10 μL of GeneJet reagent (SignaGen Laboratories, Fredrick, MD) were each diluted in 100 μL of ESFAF medium and then mixed together for approximately 30 minutes to form the transfection mixture. Sf9 cells were plated in 6-well plates at 1.5e+6 cells / well in 2 mL of ESFAF medium for approximately 30 minutes at 28°C. After removing the old medium from the Sf9 cells, each transfection mixture was diluted in 800 μL of ESFAF medium and then added to the Sf9 cells. After overnight incubation at 28°C, an additional 1 mL ESFAF medium was added to each well. After a total incubation time of 4 days, the medium containing the rBV was harvested and amplified at a 1:200 ratio to generate sufficient quantities of rBV for use in the AAV production process.

[0231] AAV production and purification

[0275] rBVs carrying AAV2 Rep and mutant capsid genes and target expression cassettes, respectively, were used to co-infect Sf-9-V432AG cells for AAV production. Briefly, 10 moi of rBV-Cap-Rep and 5 moi of rBV-target cassette were used to co-infect Sf9 cell line at a density of ~5e+6 cells / mL in 50% fresh ESFAF medium for 3 days at 28°C in a shaking incubator with a shaking speed of 180 revolutions per minute (rpm). At the end of infection, cell pellets were collected by centrifugation at 3,000 rpm for 10 min. Cells were lysed in Sf9 lysis buffer containing 50 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 1% Sarkosyl, 1% Triton X-100, and 125 units / mL Benzonase with shaking at 350 rpm for 1 hour at 37°C after vigorous vortexing. At the end of the shaking, the salt concentration was increased to 500 mM by vortexing, and the lysate was cleared by centrifugation at 8,000 rpm for 20 minutes at 4°C. The cleared lysate was transferred to an ultracentrifuge tube in an SW28 swinging bucket rotor containing 5 mL of 1.50 g / cc and 10 mL of 1.30 g / cc cesium chloride solution. After centrifugation at 28,000 rpm for approximately 18 hours at 15°C, the AAV band was collected by syringe and transferred to an ultraclear centrifuge tube in a 70ti centrifuge rotor. Centrifuge tubes were filled with 1.38 g / cc cesium chloride solution and heat sealed. AAV samples were ultracentrifuged twice at 65,000 rpm for ~18 hours at 15°C, and AAV bands were collected by syringe. Purified AAV samples were buffer exchanged into PBS buffer containing 0.001% Pluronic F-68 and filter sterilized with a 0.22 μm syringe filter. Sterilized AAV samples were stored at 4°C for up to one month and then transferred to -80°C for longer storage. AAV titers were determined by real-time PCR.

[0232] Transient expression of constructs in mammalian cell culture systems

[0276] Human HEK293 cells were cultured in DMEM medium containing 10% FBS in a CO2 incubator at 37 °C. For maintenance passages, cells were split 1:10 twice a week. For transfection, cells were split at 2 × 10 in 10 mL medium overnight. 6 Cells / dish were seeded in a 10 cm cell culture dish. 14 μg of plasmid DNA and 22 μL of Lipofectamine 3000 were diluted in 0.5 mL of Opti medium, respectively, and mixed. After 5 min of incubation at room temperature, the mixture was added dropwise to the cells and incubated in a CO2 incubator at 37°C for 48 h. The medium was collected for further experiments.

[0233]

[0277] HEK293 cells were cultured at 2 × 10 6 Cells were seeded onto 10 cm tissue culture dishes at a density of 1000 x 1000 cells / well. Transfection of each CNP22 or CNP36 or CNP fusion variant plasmid was performed using 14 μg DNA / dish with Lipofectamine 3000 reagent according to the manufacturer's protocol. Cell culture supernatants were harvested and analyzed for protein expression by Western blot 48 hours after transfection. All transfections were performed in triplicate in at least three independent experiments.

[0234]

[0278] CNP fusion (either CNP-Fc or CNP-Fc variant) protein was determined by SDS-PAGE and Western blot analysis. HEK293 cell medium (supernatant) was harvested 48 or 72 hours after vector introduction. A total volume of 30 μL of cell supernatant was mixed with 10 μl of 4× loading buffer and loaded onto a NuPAGE 10% Tris-Glycine gel for electrophoresis. Proteins were subsequently transferred to a PVDF membrane. The membrane was treated with casein blocker in PBS at room temperature for at least 1 hour, probed with goat anti-human IgG1Fc antibody biotin conjugate, and then incubated with horseradish peroxidase-conjugated streptavidin.

[0235] Purification of CNP and CNP fusion proteins

[0279] All functional protein sequences were converted to DNA sequences, cloned into plasmids for expression, and cloned into baculovirus vectors for recombinant AAV packaging. Expressible plasmids were amplified, DNA preparations were made, and used for transient transfection into HEK293 cells. Cell culture harvest was used for purification of CNP36-Fc proteins by Protein A affinity column chromatography. These fusion proteins were purified to homogeneity and characterized for purity by SDS-PAGE.

[0236]

[0280] Expressed CNP, CNP-Fc, or CNP-Fc proteins were purified from HEK293 cell culture harvests by Protein A affinity column chromatography. Harvested serum-free medium was filtered through a 0.2 μm filter to remove particulates and loaded onto a Protein A column (1 mL size) at a flow rate of 1.5-2.0 mL / min. The column was washed with wash buffer (20 mM Tris-HCl, pH 7.3, 150 mM NaCl, 5 mM EDTA), eluted with elution buffer (0.1 M glycine, pH 2.5), and neutralized to pH 6.8-7.4 with 1 / 10 neutralization buffer (1.0 M Tris HCl, pH 10). The neutralized proteins were buffer exchanged into 1× PBS, filter-sterilized through a 0.2 μm syringe filter pre-wetted with PBS, and stored at -80°C. The column chromatogram showed a sharp peak of CNP fusion protein eluted from the column when the pH reached 3-4.0 (Figure 6A). Similar observations of FP-CNP expression were made based on the analysis of the chromatograms and SDS-PAGE (Figure 6B). The protein concentration of each preparation was determined by BCA protein assay (Figure 5). Table 7 shows the exact size of either CNP-Fc or CNP-Fc expressed by transduced HEK293 cells. The N-terminal sequence of the CNP-Fc protein (AMI088) was determined by Edman degradation (Figure 6A). The purified protein preparations were used for in vitro biological function assays. Protein AMI263 was also used in optic nerve crush (ONC) evaluation for its role in protecting retinal ganglion cells (RGC) from injury (Table 8).

[0237] [Table 7]

[0238] [Table 8]

[0239]

[0281] The biological function of these purified proteins was assayed for their stimulation of cGMP production using the NPR-B receptor positive cell line NIH3T3 and the NPR-B negative cell line HEK293 cells. Determination of cGMP was assayed by ELISA using commercially available CNP as a standard curve. Figure 7A and Figure 8A-B (time course) show the simulation of cGMP produced by CNP-Fc36. Figure 7A shows the cGMP release from the CNP fusions described herein that bind to NPR-B. Figure 7B shows the kinetic affinity binding between the CNP fusions described herein and the NPR-B protein as measured by BiaCore assay. Kinetic affinity binding analysis was performed at 25 °C in HBS running buffer (20 mM NaH2PO4-Na2HPO4·H2O, 150 mM NaCl, pH 7.4) supplemented with 0.005% (v / v) system surfactant P20 using a Biacore3000 optical biosensor with a research grade CM5 sensor chip. Surface preparation. NPR-B was immobilized at different levels in three flow cells by standard amine coupling, and Fc1 was left unmodified to serve as a reference. All surface plasmon resonance (SPR) analysis was performed on a BIAcore3000 system using a series of CM5 sensor chips. Data processing and analysis were performed using BIAevaluation software. All sensorgrams were double referenced by subtracting the response to a reference flow cell and a blank sample. Human NPR-B (R&D systems) was covalently coupled to the CM5 chip by amine coupling. A surface density of 6600 RU was used for measurements with natriuretic peptides. Successive injections of CNP (0.5-8 nM) were performed at a flow rate of 30 μl / min (300 s each) followed by a dissociation time of 900 s. Saturation of the binding sites was observed and the surface was regenerated by two injections (60 s each) of 0.5 M NaCl. Kinetic parameters were calculated assuming simple 1:1 (Langmuir) binding. Figure 7C shows the single cycle kinetics (SCK) assay of binding between CNP and NPR-B (KD=36.3Pm; ka=1.31e7; and kd=4.74E-4).Figure 7D shows the Biacore assay of AMI263(Fc4-CNP36) against NPR-B (KD=0.36pM; ka=1.38E6; and kd=4.92E-6). All SPR analyses were performed on a BIAcore3000 system using a series of CM5 sensor chips. Data processing and analysis were performed using BIAevaluation software. All sensorgrams were double-referenced by subtracting the response to a reference flow cell and a blank sample. Human NPR-B (R&D systems) was covalently coupled to a CM5 chip by amine coupling. A surface density of 3100RU was used for measurements with natriuretic peptides. Sequential injections of AMI263(FC4-CNP) (0.125-2nM) were performed at a flow rate of 30μl / min (each 300s) followed by a dissociation time of 900s. Saturation of the binding sites was observed. Kinetic parameters were calculated assuming simple 1:1 (Langmuir) binding. Figure 7E shows the Biacore assay of AMI088 for NPR-B (KD=34.1 pM; ka=1.31E7; and kb=4.47E-4). All SPR analyses were performed on a BIAcore3000 system using a series of CM5 sensor chips. Data processing and analysis were performed using BIAevaluation software. All sensorgrams were double-referenced by subtracting the response to a reference flow cell and a blank sample. Human NPR-B (R&D systems) was covalently coupled to a CM5 chip by amine coupling. A surface density of 3100 RU was used for measurements with natriuretic peptides. Sequential injections of AMI088(FC1-CNP) (0.31-5 nM) were performed at a flow rate of 30 μl / min (each 300 s) followed by a dissociation time of 900 s. Saturation of the binding sites was observed. Kinetic parameters were calculated assuming simple 1:1 (Langmuir) binding. Figure 7F shows the Biacore assay of ANP against NPR-B (KD=52.3nM; ka=2.36E3; and kd=1.24E-4). All SPR analyses were performed on a BIAcore3000 system using a series of CM5 sensor chips. Data processing and analysis were performed using BIAevaluation software.All sensorgrams were double-referenced by subtracting the response to a reference flow cell and a blank sample. Human NPR-B (R&D systems) was covalently coupled to a CM5 chip by amine coupling. A surface density of 3100 RU was used for measurements with natriuretic peptides. Sequential injections of AMI263 (FC4-CNP) (0.5-300 nM) were performed at a flow rate of 30 μl / min (300 s each) followed by a dissociation time of 900 s. Saturation of binding sites was observed. Kinetic parameters were calculated assuming simple 1:1 (Langmuir) binding. Figure 7G shows the Biacore assay of BNP against NPR-B (KD=664 pM; ka=9.4E5; and kb=6.24E-4). All SPR analyses were performed on a BIAcore3000 system using a series of CM5 sensor chips. Data processing and analysis were performed using BIAevaluation software. All sensorgrams were double-referenced by subtracting the response to a reference flow cell and a blank sample. Human NPR-B (R&D systems) was covalently coupled to a CM5 chip by amine coupling. A surface density of 3100 RU was used for measurements with natriuretic peptides. Sequential injections of AMI263 (FC4-CNP) (0.5-300 nM) were performed at a flow rate of 30 μl / min (300 s each) followed by a dissociation time of 900 s. Saturation of the binding sites was observed. Kinetic parameters were calculated assuming simple 1:1 (Langmuir) binding. Figure 7H shows the Biacore assay of AMI263 for NPR-B (KD=13.1 nM; ka=1.14E4; and kd=1.49E-3). All SPR analyses were performed on a BIAcore3000 system using a series of CM5 sensor chips. Data processing and analysis were performed using BIAevaluation software. All sensorgrams were double-referenced by subtracting the response to a reference flow cell and a blank sample. Human NPR-B (R&D system) was covalently coupled to a CM5 chip by amine coupling. A surface density of 3100 RU was used for measurements with natriuretic peptides.Sequential injections of AMI263 (FC4-CNP) (9.37-150 nM) were performed at a flow rate of 30 μl / min (each 300 s) followed by a dissociation time of 900 s. Saturation of the binding sites was observed. Kinetic parameters were calculated assuming simple 1:1 (Langmuir) binding. Figure 7I shows the Biacore assay of AMI087 against NPR-B (KD=41.9 nM; ka=3.65E3; and kd=1.53E-3). All SPR analyses were performed on a BIAcore3000 system using a series of CM5 sensor chips. Data processing and analysis were performed using BIAevaluation software. All sensorgrams were double-referenced by subtracting the response to a reference flow cell and a blank sample. Human NPR-B (R&D system) was covalently coupled to the CM5 chip by amine coupling. A surface density of 3100 RU was used for measurements with natriuretic peptides. Sequential injections of AMI087(FC4-CNP) (9.37-150 nM) were performed at a flow rate of 30 μl / min (each 300 s) followed by a dissociation time of 900 s. Saturation of the binding sites was observed. Kinetic parameters were calculated assuming simple 1:1 (Langmuir) binding. Table 32 shows a summary of the affinity between NPR-B and CNP or CNP fusions described herein. Table 33 shows the corrected concentrations of proteins for the Biacore assay.

[0240] [Table 9]

[0241] [Table 10]

[0242] Stimulation of cyclic guanynyl monophosphate (cGM) production by CNP

[0282] Purified CNP fusion proteins were assessed for biological function in cell-based assays with C-type natriuretic peptide receptor B (NPR-B) positive cell line NIH / 3T3 and negative cell line HEK293 cells. Tests were also performed in the presence of A-type natriuretic peptide (ANP) and CNP22 control (Figures 7A-B and 8A-B).

[0243] Adeno-associated virus (AAV) vector production and delivery

[0283] Several AAV2.N54-CNP and AAV2.N54-CNP-FC constructs have been produced to deliver various forms of CNP22, CNP36, or CNP fusions (CNP fused to either the N-terminus or C-terminus of the Fc fragment). The AAV2.N54-CNP and AAV2.N54-CNP-FC constructs could transduce airway epithelia cells by sinus, nasal, and / or pulmonary delivery methods. Other serotypes of AAV could also be used depending on the target tissue or cells to be delivered. For example, AAV6 tends to preferentially transduce lung cells.

[0244]

[0284] V432A cells, an Sf9-derived insect cell line, were cultured in ESF AF medium supplemented with 100 units / ml penicillin and 100 μg / ml streptomycin at 28°C in storage bottles. Cells were grown at a cell density of 7 × 10 6 Upon reaching cells / ml, they were split 1:4 for maintenance. Recombinant baculovirus (rBV) was generated according to the manufacturer's protocol. Briefly, the construct was used to transduce DH10Bac and recombinant bacmid DNA was isolated. The bacmid DNA was transduced into V432A cells to generate rBV. The rBV was quantified by qPCR. Table 9 lists exemplary AAV constructs encoding CNP or CNP fusion proteins. Table 10 lists the amino acid sequences of the CNP and CNP fusion proteins used in the experiments of Examples 1-3. Table 11 lists the nucleic acid sequences of the AAV constructs encoding CNP and CNP fusion proteins used in the experiments of Examples 1-3.

[0245]

[0285] AAV vector purity was determined by the SimplyBlue Staining assay. Briefly, 26 μl of AAV sample was mixed with 10 μL of 4× loading buffer + 4 μL of 10× reducing reagent and incubated at 95° C. for 2 min. Approximately 1E+11 vg of each AAV sample was loaded into each lane of a 10% SDS-PAGE gel and run at 100 volts until the dye reached the bottom of the gel. Gels were stained according to the manufacturer's protocol. SDS-PAGE gel patterns were obtained with the expected VP1, VP2 and VP3 component levels (FIG. 15).

[0246]

[0286] The AAV2.N54-CNP or AAV2.N54-CNP-FC constructs listed in Table 2 were further evaluated for production of each construct protein using suspension culture of HEK293 cells. V432A cells were cultured at 7 × 10 6The rBVs were cultured to 100 cells / ml and diluted 1:1 with fresh ESF AF medium. Approximately 200 viruses per cell of the rBVs containing the designed rep-cap gene and approximately 100 viruses per cell of the rBVs containing the DNA sequence encoding CNP-Fc or CNP-Fc protein were added separately and allowed to infect V432 cells for 3 days in a shaking incubator at 28°C. The infected V432A cells were harvested by centrifugation at 3,000 rpm for 10 minutes. The cell pellets were lysed in SF9 lysis buffer (50 mM Tris-HCl, pH 7.8, 50 mM NaCl, 2 mM MgCl2, 1% Sarkosyl, 1% Triton X-100, and 140 units / ml Benzonase®). Genomic DNA was digested by incubation at 37°C for 1 hour. At the end of the incubation, sodium chloride was added to adjust the salt concentration of the lysate to approximately 1M to further dissociate the AAV vector from the cell matrix. Cell debris was removed by centrifugation at 8000 rpm for 30 minutes. The cleared lysate was loaded onto a CsCl step gradient and subjected to ultracentrifugation at 28,000 rpm for 20 hours in a swinging bucket rotor. The virus band was pulled by a syringe with an 18-gauge needle, loaded onto a second CsCl gradient and subjected to linear-ultracentrifugation at 65,000 rpm for 20 hours. The virus band was then pulled and passed through two PD-10 desalting columns to remove CsCl and detergent, and simultaneously exchanged into buffer B (1×PBS, 0.1 M sodium citrate, and 0.001% Pluronic F-68). Quantitative real-time PCR (qPCR) was performed to determine the copy number of the AAV vector genome with ITR primers and probes (Table 12).

[0247] [Table 11]

[0248] [Table 12-1]

[0249]

Table 12-2

[0250]

Table 12-3

[0251]

Table 12-4

[0252]

Table 13-1

[0253]

Table 13-2

[0254]

Table 13-3

[0255]

Table 13-4

[0256]

Table 13-5

[0257]

Table 13-6

[0258]

Table 13-7

[0259]

Table 13-8

[0260]

Table 13-9

[0261]

Table 13-10

[0262]

Table 13-11

[0263]

Table 13-12

[0264]

Table 13-13

[0265]

Table 13-14

[0266]

Table 13-15

[0267]

Table 13-16

[0268]

Table 13-17

[0269]

Table 13-18

[0270]

Table 13-19

[0271]

Table 13-20

[0272]

Table 13-21

[0273]

Table 13-22

[0274]

Table 13-23

[0275]

Table 13-24

[0276]

Table 13-25

[0277]

Table 13-26

[0278]

Table 13-27

[0279]

Table 13-28

[0280]

Table 13-29

[0281]

Table 13-30

[0282]

Table 13-31

[0283]

Table 13-32

[0284]

Table 13-33

[0285]

Table 13-34

[0286]

Table 13-35

[0287]

Table 13-36

[0288]

Table 13-37

[0289]

Table 13-38

[0290]

Table 13-39

[0291]

Table 13-40

[0292]

Table 13-41

[0293]

Table 13-42

[0294]

Table 13-43

[0295]

Table 13-44

[0296]

Table 13-45

[0297]

Table 13-46

[0298]

Table 13-47

[0299]

Table 13-48

[0300]

Table 13-49

[0301]

Table 13-50

[0302]

Table 13-51

[0303]

Table 13-52

[0304]

Table 13-53

[0305]

Table 13-54

[0306]

Table 13-55

[0307]

Table 13-56

[0308]

Table 13-57

[0309]

Table 13-58

[0310]

Table 13-59

[0311]

Table 13-60

[0312]

Table 13-61

[0313] [Table 14]

[0314] Example 2. CNP-Fc ELISA development and standardization C-type natriuretic peptide (CNP) has two isoforms, CNP-22 and CNP-53. Example 2 shows that CNP-36 fused to Fc increased the half-life of CNP and also facilitated purification and ELISA detection when compared to native (unfused) CNP. Transduction and CNP-Fc expression experiments were performed with three AAV vectors (AMI061, AMI087, and AMI088). Figure 1A illustrates the three vectors. Figures 1B-F show additional vectors that can encode CNP or CNP fusion proteins. An enzyme-linked immunosorbent assay (ELISA) was developed to quantify CNP-Fc fusion protein in cell culture supernatants. An ELISA assay was used to quantify CNP-Fc expressed in HEK293 cells, human chondrocytes (HCH) cells, and human skeletal muscle cells (hSkMCs) upon transduction with one of the three AAV constructs described herein. Table 13 shows the materials and equipment for performing the ELISAs described herein.

[0315] [Table 15]

[0316] Buffers and Substrates. Coating buffer: 3.7 g sodium bicarbonate, 0.64 g sodium carbonate in 1 L MilliQ water, pH 9.6. Store at room temperature; Blocking buffer: commercial casein blocking buffer in PBS with 0.1% Tween20. Store at 4°C; Working diluent: same as blocking buffer; Washing buffer: 1x PBS with 0.1% tween20 (expiry date, 30 days from date of manufacture); Coating antibody (Ab): human CNP antibody (monoclonal), coding concentration was 2 μg / mL; Detection Ab: goat pAb anti-human Fc. Diluted in wells to a final dilution of 1:20,000. Store at 4°C; HRP: Streptavidin-HRP stock at 1 mg / mL, diluted to 1:10,000. Store at 4°C; Substrate: TMB. Store at 4°C; and Stop solution: 2N HCl. Store at ambient temperature.

[0317]

[0289] Procedure 1. Coat a 96-well plate with anti-CNP antibody at a final concentration of 2 μg / mL in coating buffer in a volume of 50 μL per well and cover the plate with a sealing cover. Place the plate at 4° C. overnight.

[0318] 2. The next day, remove the plate and wash 3 times with wash buffer, tapping the plate on a paper towel to remove excess solution. 3. Using a multichannel pipette, add 300 μL of Blocking Buffer to each well. Place the plate with sealing cover in a 37° C. incubator for 2 hours.

[0319] 4. After incubation, discard the blocking buffer and tap on a paper towel to remove excess buffer. 5. Prepare standard and sample dilutions in blocking buffer according to the experimental scheme and add 50 μL to each well. NOTE: The reaction volume for each step is equal to the initial coating buffer volume. For example, if the coating buffer is 50 µL, then the sample, capture, detection, TMB and stop solutions should all be 50 µL, except for the blocking and wash buffers (300 µL).

[0320] 6. Cover the plate with a sealing cover and return to the incubator for 1 hour. 7. After 1 hour, discard the solution and wash the plate 6 times with 300 μL of wash buffer. Remove excess solution by tapping into paper towels as above.

[0321] 8. Add detection antibody at a 1:20,000 dilution in blocking buffer and incubate for 1 hour at 37°C. 9. Discard the solution and repeat the washing procedure as described in step 7.

[0322] 10. Add streptavidin-HRP at a 1:10,000 dilution in blocking buffer for 45 minutes at 37°C. 11. Discard solution and wash plate as described in step 7.

[0323] 12. Add 50 µL of TMB and avoid placing the plate under a direct light source for 15-20 min (can be <15 min) or until saturation begins to be visible in the highest concentration wells. 13. Add 50 μL of stop solution and within 15 minutes read the plate at 450 nm with a reference at 600 nm.

[0324]

[0290] ELISA Development 1. Coating / Capture Antibody Titration: Anti-CNP antibodies at different concentrations (5, 2, 1 and 0.5 μg / mL) were coated onto wells and plated in a 96-well plate. AMI061, AMI087 and AMI088 (CNP constructs) were also titrated from 0-200 ng / mL by serial dilution. Detection Ab was diluted up to 1:4000 and streptavidin-HRP up to 1:10,000.

[0325] 2. Detection antibody titration: Results from the above experiments showed a coating Ab concentration of 2 μg / mL to be optimal for the assay. However, all constructs showed a plateau at 100 and 200 ng / mL concentrations. Therefore, along with the detection antibody, the standard concentration was reduced from 200 ng / mL to 50 ng / mL. Standards were now serially diluted from 50 ng / mL to 0.8 ng / mL.

[0326] Results. Coating Ab titration: Following the protocol described in the ELISA development section, wells were coated with different concentrations of anti-CNP antibodies and different CNP constructs were added to specific wells. The scheme of addition is shown in Table 14. The numbered wells indicate the concentration of CNP constructs added to the wells in ng / mL. Figure 4A shows the titration of coating Ab for each construct. The resulting standard curve was fitted using hyperbolic fitting in GraphPad Prism software. At coating concentrations of 0.5 and 1 μg / mL, the absorbance was below 1 O.D. The coating concentration of 5 μg / mL showed saturation and 2 μg / mL showed an OD higher than 1. Therefore, 2 μg / mL may be an appropriate concentration to use for the standard curve. Detection Ab was titrated to achieve a better fitting of the standard curve. Detection Ab titration: At 1:4000 dilution of detection Ab, the curve fitting was not optimal. This may result in inaccurate sample estimation in later experiments. Therefore, the detection Ab was diluted 1:5000, 1:10,000, 1:20,000 and 1:40,000. The coating Ab concentration was 2 μg / mL, and the titration of the CNP construct was reduced from 200 ng / mL to 50 ng / mL and serially diluted with the assay diluent blank to obtain concentrations of 50, 25, 12.5, 6.25, 3.125, 1.56, 0.76 ng / mL. Streptavidin-HRP was diluted 1:10,000 as used in previous experiments. Figure 4B shows the standard curves of all three CNP constructs with hyperbolic fitting. The detection Ab titration of 1:20,000 obtained a better fit compared to the other dilutions. Anti-CNP-Fc ELISA to quantify the expression of CNP in HEK293 cell supernatants: 1E+06 HEK293 cells were plated in 6-well plates. Cells were transduced with AAV2 (N54-AMI061, N54-AMI087 and N54-AMI088) constructs with CNP fused to Fc at 100,000 MOI. Constructs were designed and optimized for GC content to better promote expression. Viral vector titers were determined after purification using qPCR. Titers and other parameters are shown in Table 15.Five days after transduction, cell supernatants were harvested and CNP-Fc ELISA was performed at different sample dilutions (undiluted, 1:5, 1:10, 1:50, 1:100, 1:200, 1:500, 1:1000, 1:2000 and 1:4000 dilutions). Experiments were performed in duplicate. The resulting standard curves were plotted and fitted using a hyperbolic equation using GraphPad Prism software as described above (Figure 4C). The intra-assay precision of the CNP-Fc ELISA was calculated and is shown in Table 16. The percentage coefficient of variation (CV) values ​​of all standards are below 10%, indicating good precision of the assay. Undiluted samples showed saturating signals. For AMI061, dilutions 1:500, 1:1000 and 1:2000 showed readouts that were within the detectable range. For AMI087 and AMI088, OD values ​​were in the detectable range at 1:4000 dilution. AMI088 also showed the highest expression when compared to other CNP constructs. These results are summarized in Table 17. Cells not transduced with CNP constructs showed very little expression with 6.2, 2.5 and 6.4 ng / mL for AMI061, AMI087 and AMI088, respectively.

[0327] [Table 16]

[0328] [Table 17]

[0329] [Table 18]

[0330] [Table 19]

[0331] The standard curve of the CNP-Fc ELISA is hyperbolic and needs to be fitted using hyperbolic fitting. The ELISA method described herein may be required to quantify CNP-Fc in cell culture supernatants, preclinical and clinical samples. A brief summary of the conditions under which this ELISA was standardized includes: 2 μg / mL anti-CNP antibody coating concentration was used; CNP-Fc purified protein was serially diluted from 50 ng / mL to 0.78 ng / mL; and detection Ab was diluted to 1:20,000 and streptavidin-HRP was diluted to 1:10,000.

[0332] Example 3. Evaluating efficacy in an NMDA excitotoxicity mouse model of retinal degeneration This study evaluates the effect of adeno-associated virus (AAV) vectors on retinal ganglion cell protection after injury caused by N-methyl-D-aspartate (NMDA) administration to mice. Injection volumes and treatment options can be found in Table 18. Groups 7 and 8 were added to test high concentrations of MK-801. All tissues for bioanalysis were snap frozen instead of homogenized. A method for spiking IBMX into whole blood was established and defined. The study was performed in wild-type mice (Mus Musculus, C57BL / 6).

[0333] [Table 20]

[0334] NMDA preparations The molecular weight of NMDA is 147.3 g / mol. On the day of administration, 20 mg of NMDA was weighed out and added to 2.7 mL of PBS to make a 50 mM solution [Stock A]. The Stock A solution was diluted 1:9 in PBS (100 μL Stock A + 900 μL PBS) to make a final 5 mM NMDA solution. The final solution was syringe filtered through a 0.22 μm filter into a sterile vial. Solutions were made on the day of injection and kept protected from light and refrigerated until administration. MK-801 formulation The molecular weight of MK-801 is 337.37 g / mol. The received container of MK-801 contained 5 mg of MK-801. On the day of administration, 1.0 mL of PBS was added to the MK-801 to give a concentration of 14.82 mM solution [Stock A]. The stock was serially diluted as follows: 10 μL of Stock A + 731 μL of PBS: 0.2 mM (200 μM) MK801 [Stock B]. The final solution was syringe filtered through a 0.22 μm filter into a sterile vial. Solutions were made on the day of injection and kept protected from light and refrigerated until administration.

[0335] Group 1 administration

[0296] 300 μL of 5 mM NMDA stock was combined with 300 μL of sterile PBS and 1 μL was injected on day 0 for a final concentration of 2.5 mM NMDA.

[0336] Group 2 administration

[0297] 300 μL of 5 mM NMDA stock was combined with 300 μL of sterile filtered MK-801 and 1 μL was injected on day 0 for a final concentration of 2.5 mM NMDA / 100 μM MK-801.

[0337] Groups 3 to 6

[0313] 300 μL of 5 mM NMDA stock was combined with 300 μL of sterile PBS and 1 μL was injected on day 0 for a final concentration of 2.5 mM NMDA.

[0338] Intravitreal injection On day -28 or day 0 based on the experimental design, mice were given 0.01-0.05 mg / kg buprenorphine subcutaneously (SQ). Animals were then sedated for intravitreal injection with ketamine / xylazine or isoflurane inhalation, and one drop of 0.5% proparacaine HCl was applied to both eyes. The conjunctiva was gently grasped with Dumont #4 forceps, and the injection was performed using a 33G needle and Hamilton syringe. After dispensing the contents of the syringe, the syringe needle was slowly removed. According to the injection procedure, one drop of ofloxacin eye drop was applied topically to the cornea and conjunctiva with a lubricating eye drop.

[0339] During NMDA injection on day 0, animal 409 (group 4) had inflammation and adhesions in the OD, animal 519 (group 5) had cataracts in the OD, and animal 524 (group 5) had cataracts in the OU. In group 6, animals 625 and 626 had cataracts in the OS, and animals 629, 630, and 632 had cataracts in the OD. No other abnormalities were noted on study records.

[0340] Cageside Observation Morbidity and mortality were observed daily with cageside observations, with particular attention to both eyes. All animals were alert, alert, and responsive at all observation time points, with no further findings in the study records. On the day of IVT injection, all animals weighed 20-22 g, and all animals generally maintained their weight over the course of the study (Figure 9A). Euthanasia and terminal blood collection (Groups 3–6) Animals in groups 3-6 had terminal blood drawn on day 7. IBMX was used during blood collection to inhibit phosphodiesterase activity. Prior to necropsy on day 7, but after dosing on day -28, all remaining spare animals were utilized to ensure that the addition of IBMX to whole blood did not interfere with serum clotting and extraction. Briefly, each syringe for cardiac puncture was pre-rinsed with a 10 mM IBMX stock solution prior to blood draw. All mice were sedated to deep sedation under isoflurane and euthanized by exsanguination. After pre-rinsing each syringe with 10 mM IBMX, a 25G needle was inserted into the heart, the animals were exsanguinated and euthanized, and blood was collected. The volume of blood collected was documented, and the appropriate volume of IBMX was added to a 1.5 mL RNAse / DNAse-free microcentrifuge tube. For every 85 μL of whole blood collected, 15 μL of 10 mM IBMX was added for a final concentration of 1.5 mM IBMX in whole blood. Blood was then drawn into tubes, mixed gently with IBMX by inversion, and allowed to clot at room temperature for at least 20 minutes before serum processing. Samples were centrifuged at 4000×g for 10 minutes at room temperature in a benchtop microcentrifuge. After centrifugation, clear serum was transferred to pre-labeled polypropylene tubes, flash frozen in liquid nitrogen, and stored frozen at −80° C.

[0341] Ocular tissue collection and processing After euthanasia, the eyes of selected animals were processed for histological or immunological studies. The OD of animals 519 (group 5) and 629 (group 6) showed very small lenses, blood in the retina, and eyes filled with a gel-like substance. No other abnormalities at necropsy and tissue processing were noted in the study records. In groups 1 and 2, selected eyes were harvested into 10% neutral buffered formalin immediately after euthanasia. Eyes were placed in 70% ethanol at a later date. Eyes were then processed and blocked in paraffin for sectioning. Sagittal sections (5 μm thick) of each eye were prepared for all animals. At least three slides containing ribbons of approximately five sections were collected in sequence. The optic nerve was included in the sections. Slides were stained with hematoxylin and eosin (H&E) and examined using a light microscope. A representative image of the central retina from the H&E histopathology is shown in FIG. 9B. Retinal ganglion cells (RGCs) were counted (Table 19 and FIG. 9C). The mean RGC number in the right eye (OD; experimental eye) was lower than that in the left eye (OS; control eye), and there was no difference in the mean RGC number in the OD and OS between Group 1 and Group 2.

[0342] [Table 21]

[0343] Ocular tissue collection for retinal flat mount analysis (all groups) Eyes were enucleated and immediately fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and stored overnight at 4°C. At a later date, eyes were transferred to cold immunocytochemistry (ICC) buffer (PBS containing 0.5% BSA and 0.2% Tween 20) until processing. Using a dissecting microscope, eyes were carefully trimmed of extraneous tissue at the rim and the anterior chamber was removed using sharp curved scissors. The retina of the eyecup was rinsed with cold ICC buffer. Eyecups were placed in cold ICC buffer containing 1 / 100 rat anti-CNP36 (groups 3-6 only; resuspended at 0.5 mg / mL in PBS) for 3 hours at 4°C. Eyecups were then washed extensively and stained with 1 / 200 donkey anti-rat Cy3 and 1 / 1,000 DAPI for 2-3 hours at 4°C. Groups 1 and 2 were stained with 1 / 500 TUJ-1 and 1 / 1000 DAPI, whereas groups 7 and 8 were incubated with 1 / 1,000 DAPI only. Eyecups were thoroughly but gently washed with cold ICC buffer. Using sharp curved scissors and an eyelash knife, the retina was peeled from the optic disc and removed from the RPE / choroid. Orthotopic eyes were then prepared to the center and the retina was flat-mounted, covered, and sealed. 2D fluorescent microscopy images were acquired using an Olympus Bx63 upright fluorescent microscope. TUJ-1 signal was highly variable across groups 1 and 2, providing little additional information, and the Cy2 channel was not imaged. Images at 20x were acquired in quadrants and a central 248.1 x 325.7 μm region was analyzed for RGC number using Olympus cellSens and ImageJ software.

[0344] Groups 1 and 2 were stained with DAPI and TUJ-1 and images were captured at 20x (Figure 10A). RGC numbers were counted in quadrants and cells / mm2 were averaged across groups. When RGC numbers were quantified across groups (Figure 10B), the control group (Group 1 OS; PBS, no NMDA) had a mean number of 11,000±700 cells / mm2. The NMDA control group (Group 1 OD; PBS+NMDA) had a reduced RGC number of 8,000±1,000 cells / mm2. Group 2 OD had a slightly increased RGC number over Group 1 OD at 9,000±500 cells / mm2.

[0345] Groups 3-8 were stained with DAPI and four images were taken of each retina approximately 600 μm from the optic disc at 20x magnification. RGCs were counted in a central 248.1 × 325.7 μm area and cells / mm2 were averaged across each group (Figure 10C). When RGC numbers were calculated, the control group (Group 7 OS; PBS, no NMDA) had a mean number of 12,000 ± 700 cells / mm2 (Figure 10D). The NMDA control group (Group 7 OD; PBS + NMDA) had a reduced RGC number of 9,000 ± 300 cells / mm2. Group 8 OD had a similar RGC number to Group 7 OS at 12,000 ± 300 cells / mm2. Group 3 OD had a similar RGC number to Group 7 OD at 9,000 ± 1,000 cells / mm2. Group 4 OD was an RGC count of 10,000 ± 1,400 cells / mm2. Group 5 OD was an RGC count of 10,000 ± 600 cells / mm2. Group 6 OD was an RGC count of 10,000 ± 1,500 cells / mm2.

[0346] Statistical analysis was performed on the RGC data from groups 3-8 (one-way ANOVA followed by multiple comparisons using Dunnett statistical hypothesis comparison). Treatment of PBS-injected eyes with NMDA caused a statistically significant decrease in RGC numbers (P<0.0001). Increasing doses of MK-801 up to 100 μM restored group 8 RGC numbers to group 7 OS control levels, and the difference between NMDA-treated (group 7 OD) and NMDA+NK-801-treated (group 8) eyes was statistically significant (P<0.0001). When comparing groups 4-6 with group 3 NMDA / sham vector control, both AMI182 (group 5) and AMI088 (group 6) reached statistical significance (Figure 11A; P=0.0332 for group 5 vs. group 3; P=0.0021 for group 6 vs. group 3). When comparing RGC numbers in AAV-treated groups to NMDA / PBS controls (group 7 OD), only the AMI088 construct maintained statistical significance (P=0.002; Figure 11B). Groups 3-6 were stained with CNP36 in addition to DAPI to assess the location and levels of expression of AAV constructs, and images (4x and 20x) were captured (Figure 12A). Ocular tissue collection for immunohistochemistry (Groups 1 and 2 only) All eyes designated for immunohistochemistry (IHC) were enucleated, the approximate site of injection was marked, and the eyes were fixed in 4% paraformaldehyde in separately labeled vials overnight at 4°C. Eyes were then transferred to 0.1 M phosphate buffer (PB) and passed through a successive sucrose gradient (10-30%, 1 hour each), before being embedded in OCT medium and frozen on dry ice. Whole eyes were cryosectioned (14 μm sections) and stained with the following antibodies as shown in Table 20:

[0347] [Table 22]

[0348] Negative controls were performed by utilizing only the secondary antibody staining cocktail. Five slides across the retina of each eye were stained. Two pictures per retinal section - one at or near the injection site and one from the central region - were taken using an Olympus Bx63 upright fluorescent microscope and cellScan software. Qualitatively, no differences were found in the number of TUJ-1+ and cone arrestin+ cells between groups. All groups showed clear cone arrestin staining with some TUJ-1+ cells observed (Figure 12B). Further staining and analysis was not performed on sections as staining was not an informative endpoint.

[0349] Eye tissue collection (groups 3-6) Eyes assigned for assay were stored at ≦-70°C until removal, flash freezing, and shipping on dry ice. Tissues were placed into appropriate pre-weighed, labeled analytical vials, immediately re-weighed to determine sample weight, and placed on dry ice until transfer to the freezer. Samples were weighed on a scale capable of measuring to 4 decimal places. Samples collected included: serum (2mL polypropylene screw cap tubes); and whole eye including lens (2mL polypropylene screw cap tubes).

[0350] conclusion The purpose of this non-clinical study was to evaluate the effect of AAV constructs on retinal ganglion cell protection after damage caused by NMDA excitotoxicity in mice. Mice were pretreated with AAV constructs on day -28 at a dose of 4e+8vg / eye, and then retinal degeneration was induced with NMDA on day 0 in the right eye only. Groups 1, 2, 7, and 8 received co-injections of NMDA with either PBS (groups 1, 7) or MK-801 (groups 2, 8).

[0351]

[0312] Animals maintained normal body weight throughout the study. When RGC numbers in groups 1 and 2 were measured by histopathology, the experimental eyes (OD) showed lower RGC numbers than the control eyes (OS). When RGC numbers were measured by flat-mount immunohistochemistry, the NMDA-treated eyes (groups 1 and 7 OD) showed significantly lower RGC numbers than the control eyes (groups 1 and 7 OS). Pretreatment with 100 μM MK-801 (group 8) reduced the damage from NMDA administration. Among the AAV treatments, pretreatment with AMI088 resulted in statistically significantly higher RGC numbers compared to the NMDA control, with approximately 20% higher RGC numbers, but was not as effective as 100 μM MK-801. When AAV-treated groups were compared to the sham AAV+NMDA group, both AMI182 and AMI088 reached statistically significant higher RGC numbers.

[0352] Overall, 100 μM MK-801 was a sufficient positive control for retinal ganglion cell protection after damage caused by NMDA-induced excitotoxicity, as it completely restored the NMDA RGC deficit phenotype. Further studies can examine different dose levels of AMI182 and AMI088, as well as different time points after NMDA administration.

[0353] This study was designed to determine the efficacy of the proposed method to induce retinal degeneration similar to that seen in humans. One method of inducing this retinal degeneration is by intravitreal injection of NMDA. The number of animals, data collection time points, and parameters for measurement were selected based on the minimum required to meet the objectives of the study.

[0354] Biochemical studies and associated analyses were performed at the conclusion of the study. Ocular samples (e.g., whole eyes) from groups 3-6 were enucleated and frozen after isolation. All samples were received frozen upon receipt. Left eyes (OS) from all animals (n=8) in each group receiving different AAV constructs were analyzed. Table 21 shows documentation of tissue weight, volume of RIPA lysis and extraction buffer with protease inhibitors added to each sample prior to homogenization. Samples were placed on ice and homogenized using a sonicator as follows: 3 cycles of 20 sec + 20 sec rest. After sonication, samples were rested on ice.

[0355] [Table 23-1]

[0356] [Table 23-2]

[0357] CNP36 is a peptide produced by the vector AAV2.N54-CNP36 (AMI182). AMI182 is the same DNA transgene derived from AMI087 with multiple stop codons at the last cysteine ​​residue of CNP36. Thus, the Fc open reading frame was disrupted.

[0358] CNP36 and CNP-Fc36 expression in ocular (whole eye) and serum samples was quantified using a commercial CNP36 ELISA kit and an in-house CNP36-Fc ELISA (Example 2), respectively. The commercial ELISA was performed according to the step-by-step procedure described in the user manual. For the in-house ELISA, 2 μg / mL of anti-CNP36 antibody was coated onto a 96-well plate and incubated overnight at 4° C. The plate was washed with washing buffer and blocked with blocking buffer. Ocular and serum samples were added directly to specific wells without dilution. The samples were incubated for 1 hour. The plate was washed and detection antibody was added to each well at a 1:20,000 dilution. After 1 hour of incubation, the plate was washed and streptavidin-horseradish peroxidase (HRP) was added at a 1:10,000 dilution. After 45 min of incubation, the plates were washed and CNP36-Fc was detected by the addition of 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The reaction was stopped by the addition of stop solution and the plates were immediately read at 450 nm with a reference wavelength of 600 nm. CNP36-Fc quantification in ocular and serum samples Animals from groups 3 (AMI189) and 6 (AMI088) were analyzed using CNP36-Fc ELISA. Group 3 (sham vector) animals served as a negative control (Figure 13A). Animals in G6 were injected with AMI088, an AAV2 vector with N-terminal CNP36 fused to the C-terminus of a human IgG1-Fc fragment (CNP36-Fc). Data were analyzed with GraphPad Prism software using one-way ANOVA followed by Dunnett's multiple comparisons ( ****=<0.0001). Statistically significant differences were observed between groups 3 and 6. However, in samples without Fc fusion, CNP36 levels were extremely low, likely due to the very short half-life of CNP36. Ocular samples from groups 3, 4, and 5 were analyzed using the above-mentioned commercial ELISA kit (Figure 13B, left graph). Groups 4 and 5 showed slightly higher levels of CNP36 compared to group 3 (sham vector; negative control), but the observed differences were not significant. The low levels observed in the ELISA indicated that only a small fraction of the total expressed CNP36 was quantified, as most of the protein was proteolytically degraded prior to quantification. CNP36 was expressed, but optimal conditions for protection from proteolysis and subsequent detection were difficult, as test articles from groups 4 and especially 5 showed efficacy. Neither CNP36-Fc nor CNP36 were expressed in serum samples. Figure 14 shows RGC protection by AAV vectors encoding CNP. Mice eyes were injected with 1μl IVT AAV constructs at 4E+8vg / eye 28 days before NMDA injection. Images showed that NMDA induced RGC# reduction, which was rescued by MK-801. Sham vector showed reduction of RGCs in NMDA only treatment group, but AMI182 group and AMI088 group showed higher RGC number than sham vector and NMDA treatment group. Table 22 shows the expression summary of CNP36 and CNP36-Fc detection in ocular homogenate samples.

[0359] [Table 24]

[0360] The AAV2 constructs showed efficacy with retinal ganglion cell recovery in a pilot NMDA excitotoxicity study. CNP36-Fc is stable and therefore quantifiable in ocular samples. CNP36 without Fc fusion was difficult to quantitate due to its short half-life from rapid proteolysis. Expression of CNP36 and CNP36-Fc was not detected. For CNP36-Fc, this indicates that the protein did not leak systemically but acts locally, which may be a different therapeutic advantage. The sham vector showed no expression of CNP36, which acts as a good negative control for the study.

[0361] [Table 25-1]

[0362] [Table 25-2]

[0363] [Table 25-3]

[0364] Example 4. Treatment of a rat model of partial optic nerve transection (pONT) the purpose

[0320] Using an experimental rat model of partial optic nerve transection (pONT), we assessed the effects of adeno-associated virus (AAV) CNP peptide (AAV-P) and protein-Fc fusion (AAV-FP) on retinal microglial activity using detection of apoptotic retinal cells (DARC) imaging in vivo; retinal ganglion cell (RGC) apoptosis / stress using optical coherence tomography (OCT) imaging in vivo; intraocular inflammation using optical coherence tomography (OCT) imaging in vivo; intraocular pressure (IOP) using Tonolab tonometry in vivo; RGC survival using immunohistochemistry histologically on retinal whole-mounts; and immunohistochemistry histologically on retinal whole-mounts.

[0365] Materials and Methods: Test Article Information Table 24 shows the final calculation of the dose per eye: 4 μL / eye

[0366] [Table 26]

[0367] On days -21 or -28, AAV-P, AAV-FP, vehicle, negative and positive AAV controls: thaw one vial (50 μL) of each diluted vial above at ambient temperature for at least 24 hours, spin down condensed water on the vial walls, and centrifuge for 30 seconds in a minifuge / Eppendorf centrifuge to pellet aggregates. Pure Fc-peptide protein vials were thawed at ambient temperature for approximately 60 minutes, then spun briefly in an Eppendorg centrifuge to settle condensed water and remove aggregates prior to injection.

[0368] Ocular homogenization procedure A RIPA and protease inhibitor mixture was prepared by adding one tablet of protease inhibitor to 10 mL of RIPA buffer. Ocular tissue was measured for the amount of buffer needed. For every 1 mg of tissue, 10 μL of buffer was added. The tube containing the tissue was sonicated on ice for 20 seconds, then rested on ice for 20 seconds. Sonication and cooling on ice was repeated two more times for a total of three cycles. These cycles can be repeated as desired (if tissue is still not homogenized and clumps are visible).

[0369] After complete homogenization, the tubes were placed on an orbital shaker in a cold room for 2 hours. After 2 hours, the tubes were centrifuged at 13,000 rpm for 5 minutes. The supernatant was collected and stored at -80°C.

[0370] Randomized Experimental Design In total, 72 male Dark Agouti (DA) rats aged 8-10 weeks were used in this study. The 72 animals were randomly divided into three categories: Category 1 (Table 25): AAV-P (AMI302) and sham vector only (AMI189) (n=24); Category 2 (Table 26): AAV-FP (AMI273), buffer only (PBS-F68), and positive control AAV (AMI088) (n=30); and Category 3 (Table 27): pure fusion protein (FP-263) (n=18).

[0371] Each category contained 6 blocks with each block covering a different treatment, e.g., n=6 (see categories 1, 2, and 3). Rat IDs for each treatment are shown in the left column of each block. L: low dose; M: medium dose; and H: high dose.

[0372] Intravitreal injections (IVT, 4 μL) were given under general anesthesia (GA) depending on the category, administered once only in the left eye (pONT eye) using a 34-gauge Hamilton needle on day 0 after BL imaging. pONT was performed at week 3 in categories 1 and 2. In category 3, pONT was performed immediately after baseline imaging.

[0373] Partial optic nerve transection (pONT) surgery was performed under GA at week 3 in categories 1 and 2. In category 3, pONT surgery was performed on day 0 after BL imaging. Briefly, an incision was made in the superior conjunctiva to expose the optic nerve sheath. A vertical incision was made in the dura and a 0.2 mm horizontal incision was made in the dorsal optic nerve at a distance of 2 mm posterior to the eye. An ophthalmic scalpel with a 0.2 mm steel cutting guard was used in this procedure. Damage to major ocular blood vessels was avoided and was confirmed at the end of the surgery by fundus examination.

[0374]

[0329] Animals were culled at week 4 for categories 1 and 2 and at week 1 for category 3. Prior to culling, blood samples (at least 2 mL) were collected by cardiac puncture under terminal procedures and centrifuged to collect serum and stored at -80°C.

[0375] [Table 27]

[0376] [Table 28]

[0377] [Table 29]

[0378] In vivo assessment DARC / OCT. Under GA, DARC / OCT imaging was performed at baseline (BL) in categories 1 and 2 at weeks 1, 2, and 4. pONT was performed at week 3. In category 3, DARC / OCT was performed at baseline at week 1, before the final with pONT performed immediately after baseline imaging. Briefly, fluorescently labeled annexin 776 (6 mg / ml, 40 μL) was administered intranasally 2 hours prior to DARC imaging, which was then evaluated by confocal scanning laser ophthalmoscope (cSLO). OCT imaging was performed at the posterior pole of the center of the optic disc using a Spectralis cSLO.

[0379] IOP. Intraocular pressure (IOP) was measured under inhalation anesthesia using a Tonolab tonometer at BL three times per week (Monday, Wednesday, and Friday) at weeks 1, 2, and 4 in categories 1 and 2, and at week 1 in category 3. Ten IOP readings were collected from each eye of each animal at each time point.

[0380] Histological assessment Three blocks of animals (Blocks 1-3) from each category (36 rats in total) were used for immunohistochemistry studies. After sorting, both eyes of each animal were enucleated and retinal whole mounts were dissected. Immunostaining was performed with anti-RBPMS and anti-Iba-1 antibodies to evaluate RGC survival and microglial activity, respectively. The immunostained retinal whole mounts were then imaged under a fluorescent microscope.

[0381] Protein Analysis The remaining three blocks of animals from each category (Blocks 4–6) (36 rats in total) were used for protein analysis. After sorting, both eyes from each animal were enucleated, flash frozen in liquid nitrogen, cryogenically homogenized in a pestle and mortar, and stored at −80°C until protein analysis.

[0382] ELISA ELISA can be used for the concentration of CNP-Fc in process intermediates and drug substances using adeno-associated vectors for gene therapy, or for CNP-Fc determination in analytical samples (cell culture supernatants), preclinical samples (plasma, homogenized tissues, vitreous fluid, etc. from monkeys, pigs, and rodents) and clinical samples. CNP-Fc comprises a recombinant fusion protein consisting of CNP-36 fused with human IgG. CNP-Fc can be affinity purified from HEK293 cells transduced by AMI088, a rAAV2 carrying the CNP gene that can only make a 36 amino acid long peptide fused with Fc. Serial dilutions of CNP-Fc can be used in the assay. The detection range of CNP-Fc concentration in this assay can be 0.78-50 ng / mL.Materials and equipment may include: CNP-Fc stored at -80°C in 1x PBS (concentration may be determined by BCA); CNP coating / capture antibody: 100 μg / mL, antigen expressed and purified in HEK293 cells (GeneScript, Cat#Z03073); Goat anti-human IgG Fc(Biotin) pre-adsorbed (Abcam, Cat#ab98618); HRP-streptavidin conjugate (Abcam, Cat#ab7403); TMB substrate: 1-Step™ Ultra TMB-ELISA substrate solution (ThermoFisher, Cat#34028); 96-well microplate reader (Molecular Device: VERSAmax tunable Microplate reader); Coating buffer: 3.7 g sodium bicarbonate (NaHCO3), 0.64 g sodium carbonate (Na2CO3), and 1 L Milli Q water, pH 9.60, storage condition is room temperature for 1 month; 1x PBS (Phosphate Buffered Saline): 8.0 g sodium chloride, 1.3 g sodium phosphate dibasic, 0.2 g sodium phosphate monobasic, and 1.0 liter Milli-Q water, pH 7.4, storage condition 1 year at room temperature; Washing Buffer (PBST): 1X phosphate buffered saline and 0.1% Tween20 (v / v), storage condition 30 days from date of preparation at room temperature; Blocking Buffer (BB): 1X phosphate buffered saline (PBS) with 0.1% Tween20 (v / v) and 1% casein, storage condition 30 days from date of preparation at 4°C; Dilution Buffer (DB): same as blocking buffer; Stop Solution for TMB Substrate: 2N HCl, diluted in-house from stock HCl purchased from Millipore Sigma, Cat# 1003172510; and 96-well microplates.

[0383]

[0335] The ELISA procedure may include: dilute CNP antibody (500 μg / mL) stock to 2 μg / mL with coating buffer (20 μL VEGF stock into 5 mL coating buffer); add coating antigen at 50 μL / well to a 96-well microplate, cover and place plate at 2-8°C for approximately 12 hours or overnight; discard coating antigen and wash plate 3 times with 300 μL / well PBS-T wash buffer; add 300 μL / well blocking buffer and cover and incubate plate at 37±1°C for 120 minutes; repeat steps 1 and 2 as follows: Dilute P-Fc standard (8.3 mg / mL) to 50 ng / mL with dilution buffer: 8.3 mg / mL is diluted 83-fold to 100 μg / mL, 100 μg / mL is diluted 10-fold to 10 μg / mL, and 10 μg / mL is diluted 10-fold to 1 μg / mL, and 25 μL of 1 μg / mL solution is added to 475 μL of dilution buffer as the first standard point of 50 ng / mL; In Table 28, prepare the remaining CNP-Fc standards using two 1:2 serial dilution schemes; Or dilute process intermediate sample, e.g., cell culture supernatant of HEK293, to 1:2000, then 1:4000, 1:8000 and 1:16000 with dilution buffer. For ARPE19 cell culture supernatants, 1:250, 1:500, 1:1000 and 1:2000 dilutions can be performed in separate plates. Table 29 is a schematic of a HEK293 spent media quantification plate showing known CNP-Fc concentrations (columns 1A-1G and columns 2A-2G) to construct a standard curve, a buffer only blank (Blank) used as a negative control, and unknown samples for CNP-Fc concentration determination (columns 3-10). White wells represent empty wells in the 96-well plate. Unknown samples were tested in duplicate and the ratio in brackets is the dilution factor.

[0384] [Table 30]

[0385] [Table 31-1]

[0386] [Table 31-2]

[0387] Further ELISA procedures may include: Transfer diluted standards and unknown samples according to Table 29 to the plate, 50 μL per well for each dilution, in duplicate; Cover the plate and incubate the plate for 60 minutes at 37±1° C.; Discard the reaction mixture in the plate and wash six times with 300 μL / well of wash buffer; Dilute goat anti-human IgG Fc (biotin) preadsorbate 1:20,000 in dilution buffer and add 50 μL / well; Cover the plate and incubate for 60 minutes at 37±1° C.; Discard the reaction mix and wash the plate six times with 300 μL / well of wash buffer; Dilute streptavidin-HRP 1:10,000 in dilution buffer and add 50 μL / well. Cover and incubate plate for 60 min at 37±1°C; discard reaction in plate and wash 6 times with 300 μL / well wash buffer; add TMB substrate, 50 μL / well; cover plate and incubate for 15 min at 37±1°C; stop reaction by adding stop solution, 50 μL / well; read on microplate reader with 450 nm wavelength filter with 600 nm as reference wavelength; or copy data to Excel spreadsheet and construct standard curve for CNP-Fc. Unknown samples can be analyzed by first taking average of duplicates. Concentration of unknown samples can be determined by plugging obtained OD value into equation made from standard curve. Finally, concentration was adjusted for dilution factor. Assay is considered valid if the following criteria are met: Negative control has A of TMB substrate essentially similar to blank control reading. 450 or the standard curve is linear with an R value of ≧0.975.

[0388] 4. Data Analysis IOP measurements were performed three times a week at weeks 1, 2, and 4. DARC spots on in vivo images were counted automatically by an algorithm developed and validated in the Cordeiro lab. DARC counts were defined as the number of annexin-positive spots seen on the 120-minute retinal images at each time point after subtracting the baseline spots. A single DARC score was generated for each retina by subtracting the DARC spots observed at the baseline time point from those observed at the final time point. A linear transformation of +22 was applied to all DARC counts for visualization purposes only and did not affect the statistical properties of the data. Inflammation was assessed by manually counting vitreous inflammatory cells using OCT images. RBPMS in retinal whole-mount preparations + RGCs were automatically counted and analyzed by an algorithm recently developed and validated in the Cordeiro lab. To assess whether treatments showed regional differences, retinal whole-prep specimens were divided in half into superior and inferior halves. Microglial morphology in retinal whole-prep specimens was automatically analyzed by a machine learning approach recently developed and validated in the Cordeiro lab.

[0389] statistical analysis Statistical analysis and graphing of data were completed with IBM SPSS and GraphPad Prism9. Data are presented as mean ± SEM, and p<0.05 was considered statistically significant. Data were analyzed using ANOVA and t-tests, where appropriate. When this was not appropriate, the nonparametric Mann-Whitney U test was used instead. Multiple pairwise comparisons were always corrected using Tukey adjustments. To overcome the low n per treatment dose and to improve power, the medium and high concentration conditions were combined to evaluate the effect of treatment as a single group. Thus, FP, AAV-P, and AAV-FP consisted of both medium and high concentrations only.

[0390] exclusion Four rats (R3663, R3667, R3708, and R3719) died during the in vivo experiments. Two of these (R3663 and R3667) were treated with vector only, one (R3663) died before week 4, and the other (R3667) died after imaging at week 4. Therefore, there are no available data for DARC / OCT, histology, and blood samples from R3663. However, R3667 died after imaging at week 4 (just before sorting), so all data for DARC / OCT and histology were available, except for blood samples. One rat (R3708) treated with low dose AAV-FP died before week 4. Therefore, there are no available data for DARC / OCT, blood samples, and protein. One rat (R6719) treated with high dose pure FP peptide protein died before week 1. Therefore, no data are available for DARC / OCT, histology and blood samples. Some rats were excluded from data analysis for various reasons listed in Table 30.

[0391] [Table 32]

[0392] result IOP analysis in both Fc4-CNP36 (FP) and AAV treatments. To evaluate whether intravitreal administration of treatments in the left eye affected the IOP profile, IOP measurements were performed three times a week at weeks 1, 2, and 4. IOP data was analyzed by subtracting the OD (right eye) from the OS (left eye) for each animal at each time point. The results showed that FP had a significant effect on IOP reduction after intravitreal injection of 2, 20, and 80 μg / eye of affinity purified FP. Analysis of the FP group alone showed a significant effect of FP on IOP after pONT (p<0.01). Figure 16 shows the effect of Fc4-CNP36 (FP) on intraocular pressure (IOP) changes in a rat partial optic nerve transection (pONT) model. Figure 16A shows the IOP of animals monitored during the course of the study. Figure 16B shows the IOP changes of animals after intravitreal administration (IVT) of Fc4-CNP36. FIG. 16C shows the effect of Fc4-CNP36 concentration on IOP change after intravitreal administration. Vehicle: 10 mM phosphate, pH 7.3, 180 mM NaCl, 0.001% Pluronic F68; FP(L)=2 μg / eye IVT; FP(M)=20 μg / eye IVT; and FP(H)=80 μg / eye IVT. A significant reduction in IOP (p<0.01) for all FP concentrations was observed on day 1 compared to baseline (day 0) or day 7 after pONT surgery. IOP reduction was not significantly affected by 2 μg / eye to 80 μg / eye IVT of FP-4-CNP36 (FP), showing that IPO reduction reaches a maximum of 2 μg / eye. Table 31 shows the calculated delivered dose per eye.

[0393] [Table 33]

[0394] Effect of FP on DARC number and retinal ganglion cell (RGC) protection. DARC (detection of apoptotic retinal cells) is a retinal imaging technique developed within the last 20 years from basic experimental science to phase 2 clinical trials. Higher DARC numbers indicate more severe retinal cell apoptosis. Figure 17 shows the effect of Fc4-CNP36 on RGC protection in a rat pONT model. Figure 17A shows the effect of Fc4-CNP36 concentration on the reduction of detection of apoptotic retinal cells (DARC). Figure 17B shows the effect of Fc4-CNP36 concentration on RGC number. Vehicle: 10 mM phosphate, pH 7.3, 180 mM NaCl, 0.001% Pluronic F68; FP(L)=2 μg / eye IVT; FP(M)=20 μg / eye IVT; and FP(H)=80 μg / eye IVT. The FP condition showed significantly fewer DARC counts with the FP (high) concentration, 80 μg / eye. The FP condition showed significantly fewer DARC counts with the FP (high) concentration than the AAV-negative (p=0.0078). The DARC counts were analyzed by subtracting the baseline for each animal, and the results showed that the DARC reduction correlated with the intravitreally injected dose, such that 80 mg / eye IVT showed complete inhibition of DARC formation (FIG. 17A). The RGC counts increased with increasing dose (FIG. 17B).

[0395]

[0342] The protein levels of FP4-CNP36 (FP) in the eyes and serum samples of the animals were analyzed by ELISA assay, and the results are shown in Figure 18, which shows the FP4-CNP36 levels detected by ELISA in the treated eyes and control right eyes of the animals. The levels of FP4-CNP36 in the treated eyes (OS) correlated with the dose administered.

[0396] Effect of FP on DARC and RGC numbers. DARC number analysis showed that AMI273, a test article of AAV2.N54-Fc4-CNP36 encoding Fc4-CNP36 (AAV-FP), and AMI302, AAV2.N54-CNP36 encoding only the CNP36 peptide (AAV-P), increased DARC counts by 10% 3 weeks after intravitreal administration of AMI273 and AMI302 compared to sham vector.9 and 10 10 vg / eye (Figure 19). Figures 19A-B show the effect of AAV2.N54-Fc4-CNP36 and AAV2.N54-CPNP36 on retinal DARC reduction in the rat pONT model. DARC values ​​were subtracted from baseline. Final DARC numbers were plotted. Figure 19A: AMI273 is an AAV2.N54-Fc4-CNP36 vector that expressed Fc4-CNP36 protein. Figure 19B: AMI302 is an AAV2.N54-CNP36 vector that expressed CNP36 peptide. In AMI273, 10 10 Animals receiving vg / eye IVT injections had a marked decrease in DARC counts, whereas 10 10 Animals that received vg / eye IVT did not show a significant reduction in DARC, indicating that the high levels of Fc4-CNP36 bioavailable levels were higher than CNP36, the peptide alone. As seen in Figures 18 and 19, there was a clear effect of Fc4-CNP36 (FP) protein on reducing the levels of RGC apoptosis in this model in vivo. All AAV treatments showed an increase in DARC counts compared to FP, indicating that the AAV group had elevated baseline levels. This correlated with the timing of DARC imaging after intravitreal administration of AAV, indicating that persistent inflammation in the AAV group was associated with higher DARC counts. A comparison of the individual concentrations of the treatment groups is shown in Figure 19. It is important to note that there is a dose-dependent effect of FP on reducing DARC counts, with the highest concentration, 80 μg / eye of FP, showing a significant reduction compared to the sham control, which is the same vector AMI189, containing the same DNA sequence but with the open reading frame (ORF) disrupted and resuspended in the same buffer.

[0397]

[0344] Effect on RGC survival. In this surgical model of pONT, it was shown that the initial injury in the superior retina causes primary degeneration, but secondary effects are seen in the inferior retina due to secondary degeneration. Although primary damage is inevitable, secondary damage can be prevented if therapeutic intervention is successful. To evaluate the effect of AAV-P and AAV-FP on RGC survival, regional differences were examined. RBPMS in retinal whole-mount preparations + Density of RGCs (cells / mm 2 ) was analyzed by hemisection of the upper and lower retina (Figure 20 and Figure 21). Figure 20A-B shows the effect of AAV2.N54-Fc4-CNP36 (AAV-FP) and AAV2.N54-CPNP36 (AAV-P) on RGC protection in the rat pONT model. Figure 20A: AMI273 was a vector of AAV2.N54-Fc4-CNP36 and expressed the Fc4-CNP36 protein. Figure 20B: AMI302 was a vector of AAV2.N54-CNP36 and expressed the CNP36 peptide. The difference in cell density between the upper and lower retina was then calculated. By looking at the top RGC count subtracted from the bottom RGC count (difference data) as an index of protection against secondary degeneration, we found that the AAV-FP condition provided significantly more protection than the AAV-P (p=0.002) and sham vector (p=0.005). Other comparisons were not significant. p values ​​were adjusted for 5 comparisons. By subtracting top from bottom, RGC density was significantly higher in the AAV-FP condition than the AAV-P (p=0.002) and sham vector (AAV-negative, p=0.005) conditions (Figure 20). Figure 21 shows the RGC count in treated eyes of animals administered Fc4-CNP36 protein (FP) and AAV vector in the rat pONT model, and the RGC density (cells / mm 2 ) are shown in the upper and lower regions according to different treatment conditions in the following order: buffer, FP, sham vector (AAV-negative), AAV-positive, AAV-FP, and AAV-P.

[0398]

[0345] Expression of Fc4-CNP36 (Figure 22) and CNP36 (Figure 23) in ocular tissues and serum was detected by ELISA in AAV-treated eyes (OS) and untreated (right) eyes. Fc4-CNP36 concentration in ocular homogenates correlated well with increasing AAV dose levels in the left eye (OS), whereas no transgene expression was detected in the right eye (OD). Figure 22 shows Fc4-CNP36 concentration in ocular and serum samples after administration of AAV vector (AAV-FP) IVT. The Fc4-CNP36 GOI was expressed in AMI273-treated eyes but not in untreated eyes. The transgene product, Fc4-CNP36, or CNP36 was not detected in untreated eyes, right (OD) eyes. The transgene was not detected in any of the serum samples of animals administered with either AAV-FP or AAV-P independently. FIG. 23 shows CNP36 concentrations in ocular and serum samples following administration of AAV vector (AAV-P) IVT.

[0399] Summary and Conclusion DARC analysis revealed that the FP condition showed the lowest DARC count, followed by the AAV-FP group. RGC counts increased in eyes after treatment with FP, AAV-FP and AAV-P, respectively. IOP reduction was also observed in animals after IVT injection of FP (Fc4-CNP36), reaching the maximum IOP-lowering effect at 2 μg / eye, with no further reduction detectable with increasing FP.

[0400]

[0347] However, DARC number appears to be closely correlated with RGC survival despite secondary neurodegenerative effects. The protective effects of AAV-FP and FP on RGC survival in the inferior retina were consistent with the DARC data. A comparison of DARC number and RGC area density is shown in Figure 24, where the decrease in DARC number correlated with the increase in RGC density (lower minus upper) in the FP and AAV-FP conditions in the analysis of secondary degeneration. Such data support that FP and AAV-FP promote neuroprotection and produce secondary neurodegenerative effects.

[0401] Example 5. EC of CNP-Fc constructs 50 EC of CNP-Fc described herein 50 was measured by ELISA. 2.5E+05 NIH3T3 cells / well were plated. After 2 days, -10M or -5M of different CNP-Fc (Fc4-CNP22; Fcl-CNP36; Fc4-CNP36; or aflibercept-Fc4-CNP36) or comparable natriuretic peptides (ANP or CNP-22 as control) were added to specific wells in the presence of 1.8mM IBMX (an inhibitor of cyclic nucleotide phosphodiesterase). After 30 minutes, the supernatants were collected for cGMP measurement by ELISA. Figure 25A shows the standard curve of cGMP. Figure 25B shows the EC of various CNP fusions (e.g., CNP-Fc) and comparable natriuretic peptides described herein based on cGMP secreted by cells. 50 The data fitting and calculation of (EC 50 Values: CNP-22 ≡ Fc4-CNP36 > aflibercept-Fc4-CNP36 > Fc1-CNP36 >> Fc4-CNP22).

[0402] Example 6. Evaluation of neuroprotective effects and treatment efficacy of AAV-FP in an experimental glaucoma model Glaucoma is the leading cause of irreversible blindness worldwide and is characterized by the degeneration and loss of retinal ganglion cells (RGCs) and their axons due to apoptosis. Although elevated intraocular pressure (IOP) is currently the only modifiable risk factor, a proportion of glaucoma patients continue to experience loss of vision despite effective IOP control. Thus, non-IOP risk factors are increasingly believed to play a role in glaucoma pathology, and targeting these factors represents a potential treatment for glaucoma. Neuroprotection and reduction of neuroinflammation have attracted considerable interest in recent years as therapeutic approaches to prevent functional deficits in glaucoma.

[0403] DARC (detection of apoptotic retinal cells) is a biomarker that binds to exposed phosphatidylserine, allowing for the identification of disease, stress, and apoptotic cells. DARC can be used as a platform to evaluate the neuroprotective effects and treatment efficacy of drugs in both preclinical and clinical trials. DARC can be used to evaluate gene therapy delivered by the methods described herein or engineered polynucleotides in a model of partial optic nerve transection.

[0404] This study aims to investigate the efficacy of two doses of AAV-FP compared to sham AAV and NGF positive controls using well-established and translatable endpoints, including DARC to an ocular hypertension model. Using a well-established rat model of glaucoma (ocular hypertension or OHT), we can investigate: whether AAV-FP can prevent or reduce RGC (retinal ganglion cell) apoptosis in vivo by DARC imaging, and whether DARC imaging can be used to evaluate dose-response relationships; whether AAV-FP can promote RGC survival and reduce inflammation by evaluation of histological labeling and microglial morphometry throughout disease induction in response to AAV intervention.

[0405] Experimental design Treatment Groups (Table 31): Group 1: OHT only (n=6) Group 2: OHT + vehicle (n = 6) Group 3: OHT+IVT NGF (n=6) Group 4: OHT+IVT Sham AAV-FP (n=6) Group 5: OHT+IVT administration 1 AAV-FP (n=6) Group 6: OHT+IVT administration 2 AAV-FP (n=6)

[0406] [Table 34]

[0407]

[0353] In vivo schedule (4 months): ·Animals: 36 Dark Agouti (DA) male rats (n=6, 6 groups) weighing 150–200 g.

[0408] Treatment: Animals may be randomly grouped into two groups (see above). Groups 2, 4, 5, and 6 could receive intravitreal injections 4 weeks prior to IOP elevation. Group 3 will receive IVT NGF at the time of surgery · OHT surgery: As was well established in the group, injection of hypertonic saline into the episcleral vein could increase intraocular pressure (IOP) in the left eye only.

[0409] IOP measurements: IOP may be measured using a tonometer prior to surgery (baseline) and at day 1, week 1, and week 3 after surgery. Only animals with elevated IOP (>5mmHg compared to baseline) on day 1 in the induced eye will be enrolled in the treatment group.

[0410] In vivo DARC imaging: RGC apoptosis in both eyes of each animal could be assessed by DARC at baseline and 3 weeks after OHT induction by intranasal injection of fluorescently labeled Annexin V 2 hours prior to imaging with a confocal scanning laser ophthalmoscope (cSLO) [1, 2, 3]. Retinal images were then collected and the number of apoptotic RGCs was counted.

[0411] In vivo OCT images can also be recorded at the same time points as DARC to assess for vitritis.

[0354] Histological evaluation (2 months) Animals were culled 3 weeks after OHT induction, and both eyes were enucleated and whole retinal preparations were dissected.

[0412] Retinal whole-tissue preparations could be stained with RBPMS and IBA-1 to label viable RGCs and microglial cells and imaged under a confocal microscope.

[0355] Data analysis (1 month): RGC apoptosis in DARC images of each eye can be analyzed.

[0413] · RGC survival in each eye can be assessed in whole retinal preparations. Primary and secondary degeneration of each eye can be analyzed. Analysis of microglial morphology in each eye can be assessed.

[0414]

[0356] The foregoing disclosure has been described in some detail for purposes of clarity and understanding, but it will be apparent to those skilled in the art from a reading of this disclosure that various changes in form and detail may be made without departing from the true scope of the present disclosure. For example, all of the techniques and devices described above may be used in various combinations. All publi...

Claims

1. An engineered polynucleotide comprising a viral vector, wherein the viral vector comprises an expression cassette, and the expression cassette encodes an engineered polypeptide comprising a natriuretic peptide covalently linked to an antibody or a fragment thereof.

2. The engineered polynucleotide according to claim 1, wherein the natriuretic peptide comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 1 to 5.

3. The engineered polynucleotide according to claim 1, wherein the natriuretic peptide comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

4.

4. The engineered polynucleotide according to claim 1, wherein the antibody or a fragment thereof comprises a crystallizable fragment (Fc) region.

5. The engineered polynucleotide according to claim 1, wherein the antibody or a fragment thereof comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 6 to 8.

6. The engineered polynucleotide according to claim 1, wherein a natriuretic peptide is covalently linked to an antibody or a fragment thereof by a peptide linker.

7. The engineered polynucleotide according to claim 1, wherein the engineered polypeptide comprises a protease cleavage site, and the protease cleavage site may comprise a furinprotease site.

8. The engineered polynucleotide according to claim 1, wherein the engineered polypeptide comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs. 131 to 140.

9. The engineered polynucleotide according to claim 1, wherein the natriuretic peptide comprises a C-type natriuretic peptide (CNP) or a fragment thereof.

10. The engineered polynucleotide according to claim 1, wherein the AAV vector comprises AAV serotypes including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof.

11. The engineered polynucleotide according to claim 10, wherein the AAV serotype comprises AAV2.

12. The engineered polynucleotide according to claim 10, wherein the AAV vector encodes a modified AAV capsid.

13. The engineered polynucleotide according to claim 1, wherein the AAV vector comprises a second expression cassette.

14. The engineered polynucleotide according to claim 13, wherein the second expression cassette encodes a therapeutic agent.

15. The engineered polynucleotide according to claim 14, wherein the therapeutic agent comprises a hormone, a natriuretic peptide receptor (NPR) agonist, or a VEGF inhibitor.

16. A pharmaceutical composition comprising an engineered polynucleotide according to any one of claims 1 to 15.

17. The pharmaceutical composition according to claim 16, which is formulated for administration to a subject requiring administration of the pharmaceutical composition via intrathecal cavity, intraocular, intravitreous, retinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, subretinal, superchoroidal, intratumor, lung, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray, intraluminal-GI route, or a combination thereof.

18. A pharmaceutical composition according to claim 16 or 17, for treating an eye disease or condition.

19. Use of an engineered polynucleotide according to any one of claims 1 to 15 in the manufacture of a pharmaceutical product for a method of treating a disease or condition in a subject.

20. The use according to claim 19, wherein the disease or condition includes a disease of the eye.

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