Compositions and methods for expressing therapeutic agents

Abstract

Described herein are compositions and methods for expressing therapeutic agents. Compositions comprise an engineered polypeptide comprising a natriuretic peptide linked to an antibody or fragment thereof. Also disclosed are AAV vectors encoding the engineered polypeptides, and therapeutic uses thereof for the treatment of diseases such as glaucoma.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 319,233, filed on March 11, 2022; U.S. Provisional Application Serial No. 63 / 440,858, filed on January 24, 2023; and U.S. Provisional Application Serial No. 63 / 441,643, filed on January 27, 2023, the entire texts of which are hereby incorporated by reference herein.

[0003] Incorporation by reference

[0004] 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. To the extent that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. Background Art

[0005] 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 version called C-type natriuretic peptide (CNP), where CNP can be any of the following 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, the membrane-associated guanylyl cyclase (GC) is activated and catalyzes the enzymatic conversion of GTP to cyclic guanylyl monophosphate (cGMP), a functional effector or second messenger. Due to its ability to activate cGMP, CNP has been used as a therapeutic agent. Summary of the invention

[0006] However, the half-life of cleaved CNPs such as CNP53, CNP36 or CNP22 is only a few minutes, which limits their use in treating diseases or conditions. Therefore, there is still a need to engineer CNPs to increase their stability. There is also still a need to engineer CNPs for treating diseases or conditions.

[0007] In some aspects, an engineered polynucleotide is described herein, comprising a viral vector comprising an expression cassette, wherein the expression cassette encodes 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 having at least 80% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 95% identity 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 crystallizable fragment (Fc) region. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 6-8. In some embodiments, the antibody or its fragment comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO:6-8. In some embodiments, the antibody or its fragment comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO:6-8. In some embodiments, the antibody or its fragment comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO:6-8. In some embodiments, the natriuretic peptide is covalently linked to the N-terminus of the antibody or its fragment. In some embodiments, the natriuretic peptide is covalently linked to the C-terminus of the antibody or its fragment. In some embodiments, the natriuretic peptide is covalently linked to the antibody or its fragment via a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence including (GGGGS)n, wherein n is an integer between 0-20. In some embodiments, n is an integer four. 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 having at least 80% identity with any one of SEQ ID NO:131-140. In some embodiments, the engineered polypeptide comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO:131-140. In some embodiments, the engineered polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 131-140. In some embodiments, the natriuretic peptide comprises a C-type natriuretic peptide (CNP) or a fragment thereof. In some embodiments, the CNP or a fragment thereof comprises 22 consecutive bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 22 consecutive bases at the C-terminus of SEQ ID NO: 1.In some embodiments, CNP or a fragment thereof comprises 36 consecutive bases of SEQ ID NO: 1. In some embodiments, CNP or a fragment thereof comprises 36 consecutive bases at the C-terminus of SEQ ID NO: 1. In some embodiments, CNP or a fragment thereof comprises 53 consecutive bases of SEQ ID NO: 1. In some embodiments, CNP or a fragment thereof comprises 53 consecutive bases at the C-terminus 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, and the AAV serotype comprises 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 a natriuretic peptide receptor (NPR). In some embodiments, the therapeutic agent comprises an agonist of a cyclic GMP (cGMP) signaling pathway. In some embodiments, the therapeutic agent comprises a VEGF inhibitor.

[0008] In some aspects, an engineered polypeptide is described herein, comprising an antibody or fragment thereof operably coupled to a natriuretic peptide, wherein the antibody or fragment thereof comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 6-8. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 95% identity 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 an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 6-8. In some embodiments, the antibody or its fragment comprises the amino acid sequence of any one of SEQ ID NO:6-8. In some embodiments, the natriuretic peptide is covalently linked to the N-terminus of the antibody or its fragment. In some embodiments, the natriuretic peptide is covalently linked to the C-terminus of the antibody or its fragment. In some embodiments, the natriuretic peptide is covalently linked to the antibody or its fragment through a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence including (GGGGS)n, wherein n is an integer between 0-20. In some embodiments, n is an integer four. 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 having at least 80% identity with any one of SEQ ID NO:131-140. In some embodiments, the engineered polypeptide comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO:131-140. In some embodiments, the engineered polypeptide comprises an amino acid sequence of any one of SEQ ID NO:131-140. In some embodiments, the engineered polypeptide comprises an amino acid sequence of any one of SEQ ID NO:131-140. In some embodiments, the natriuretic peptide comprises a C-type natriuretic peptide (CNP) or a fragment thereof. In some embodiments, the CNP or a fragment thereof comprises 22 consecutive bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 22 consecutive bases at the C-terminus of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 36 consecutive bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 36 consecutive bases at the C-terminus of SEQ ID NO: 1.In some embodiments, the CNP or a fragment thereof comprises 53 consecutive bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 53 consecutive bases at the C-terminus of SEQ ID NO: 1.

[0009] In some aspects, a cell is described herein comprising an engineered polynucleotide as described herein. In some embodiments, at least a fragment of an engineered polynucleotide is integrated into the genome of the cell. In some aspects, a cell is also described herein comprising an engineered polypeptide as described herein.

[0010] In some aspects, described herein 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.

[0011] In some aspects, a pharmaceutical composition is described herein, 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 intrathecal, intraocular, intravitreal, retinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intracerebral parenchyma, subcutaneous, subretinal, suprachoroidal, intratumoral, pulmonary, intratracheal, intraperitoneal, intravesical, intravaginal, intrarectal, oral, sublingual, transdermal, by inhalation, by inhalation atomization form, by intraluminal-GI route or a combination thereof to a subject in need thereof. In some embodiments, the pharmaceutical composition is formulated for intravitreal, subretinal or suprachoroidal administration. In some embodiments, the pharmaceutical composition is used to treat 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 thereof.

[0012] In some aspects, described herein is a method for treating a disease or condition in a subject in need, the method 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. In some aspects, described herein is a method for treating a disease or condition in a subject in need, the method 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 a single administration cures the disease or condition. In some aspects, described herein is a method for treating a disease or condition in a subject in need, the method 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 administration does not include daily administration. In some aspects, described herein is a method of treating a disease or condition in a subject in need thereof, the method 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 administration comprises weekly administration, biweekly administration, monthly administration, bimonthly administration, semi-annual administration, annual administration, or a combination thereof. In some embodiments, the disease or condition comprises an ocular disease.In some embodiments, the ocular diseases include ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, color blindness, 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 Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, Oguchi disease (Oguchi disease), Malattia leventinese (familial dominant drusen), blue cone monochromacy, or a combination thereof. In some embodiments, the engineered polynucleotides described herein, the engineered polypeptides described herein, the cells described herein (e.g., cells transduced with the engineered polynucleotides described herein), the viral particles described herein, the pharmaceutical compositions described herein, or a combination thereof, increase 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 engineered polynucleotides described herein, the engineered polypeptides described herein, the cells described herein (e.g., cells transduced with the engineered polynucleotides described herein), the viral particles described herein, the pharmaceutical compositions described herein, or a combination thereof, increase the half-life of an agonist for 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 two-fold, at least five-fold, at least ten-fold, at least twenty-fold, at least fifty-fold, at least one hundred-fold, or more in a subject compared to the half-life of an endogenous agonist for natriuretic peptide receptor B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof.

[0013] In some aspects, described herein is a method for treating a disease or condition in a subject, comprising administering an engineered polynucleotide to the subject, wherein the engineered polynucleotide comprises a viral vector comprising an expression cassette for expressing an engineered polypeptide, the engineered polypeptide comprising an antibody or fragment thereof operably coupled to a natriuretic peptide in the subject's cells, and wherein the cell expresses the engineered polypeptide, thereby treating the disease or condition. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NO: 1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NO: 1-5. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 95% identity 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 its fragment comprises a crystallizable fragment (Fc) region. In some embodiments, the antibody or its fragment comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO:6-8. In some embodiments, the antibody or its fragment comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO:6-8. In some embodiments, the antibody or its fragment comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO:6-8. In some embodiments, the antibody or its fragment comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO:6-8. In some embodiments, the natriuretic peptide is covalently linked to the N-terminus of the antibody or its fragment. In some embodiments, the natriuretic peptide is covalently linked to the C-terminus of the antibody or its fragment. In some embodiments, the natriuretic peptide is covalently linked to the antibody or its fragment via a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence including (GGGGS)n, wherein n is an integer between 0-20. In some embodiments, n is an integer four. 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 having at least 80% identity with any one of SEQ ID NO:131-140. In some embodiments, the engineered polypeptide comprises an amino acid sequence having at least 95% identity 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 a C-type natriuretic peptide (CNP) or a fragment thereof. In some embodiments, CNP or a fragment thereof comprises 22 consecutive bases of SEQ ID NO: 1.In some embodiments, the CNP or a fragment thereof comprises 22 consecutive bases at the C-terminus of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 36 consecutive bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 36 consecutive bases at the C-terminus of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 53 consecutive bases of SEQ ID NO: 1. In some embodiments, the CNP or a fragment thereof comprises 53 consecutive bases at the C-terminus 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, and the AAV serotype comprises 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 a natriuretic peptide receptor (NPR). In some embodiments, the therapeutic agent comprises an agonist of a cyclic GMP (cGMP) signaling pathway. In some embodiments, the therapeutic agent comprises a VEGF inhibitor. The engineered polynucleotide is administered intrathecally, intraocularly, intravitreally, retinaly, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymal, subcutaneously, subretinally, suprachoroidally, intratumorally, intratracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhalation of aerosolized form, by intraluminal-GI route or a combination thereof to a subject. In some embodiments, the disease or condition comprises an ocular disease.In some embodiments, the ocular diseases comprise ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color blindness, 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, Budd-Bieder syndrome, Best disease, choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, O'Sullivan's disease, Leventinese disease (familial dominant drusen), blue cone monochromacy, or a combination thereof.

[0014] In some aspects, described herein is a method for treating glaucoma in a subject, the method comprising administering an AAV2 vector to the subject, 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 having at least 80% identity to SEQ ID NO: 4. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 95% identity 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 crystallizable fragment (Fc) region. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NO: 6-8. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NO: 6-8. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence of any one of SEQ ID NO: 6-8. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence of any one of SEQ ID NO: 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 linked to the C-terminus of the antibody or fragment thereof. In some embodiments, the natriuretic peptide is covalently linked to an antibody or a fragment thereof by a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence comprising (GGGGS) n, wherein n is an integer between 0-20. In some embodiments, n is an integer of four. In some embodiments, a protease cleavage site flanks the natriuretic peptide and the antibody or a fragment thereof. In some embodiments, the protease cleavage site comprises a furin protease site. In some embodiments, the natriuretic peptide comprises a C-type natriuretic peptide (CNP) or a fragment thereof. In some embodiments, CNP or a fragment thereof comprises 22 consecutive bases of SEQ ID NO: 1. In some embodiments, CNP or a fragment thereof comprises 22 consecutive bases at the C-terminus of SEQ ID NO: 1. In some embodiments, CNP or a fragment thereof comprises 36 consecutive bases of SEQ ID NO: 1. In some embodiments, CNP or a fragment thereof comprises 36 consecutive bases at the C-terminus of SEQ ID NO: 1. In some embodiments, CNP or a fragment thereof comprises 53 consecutive bases of SEQ ID NO: 1. In some embodiments, CNP or a fragment thereof comprises 53 consecutive 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 a natriuretic peptide receptor (NPR). In some embodiments, the therapeutic agent comprises an agonist of a cyclic GMP (cGMP) signaling pathway.In some embodiments, the therapeutic agent includes a VEGF inhibitor. In some embodiments, the AAV2 vector is encapsulated in an AAV virus particle before administration. In some embodiments, the AAV2 vector is administered intrathecally, intraocularly, intravitreally, retinaly, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymal, subcutaneously, subretinally, suprachoroidally, intratumorally, intratracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhalation of aerosolized form, by intraluminal-GI route or a combination thereof to a subject in need. In some embodiments, the AAV2 vector is administered intravitreally, subretinaly or suprachoroidally. In some embodiments, the administration of the AAV2 vector increases natriuretic peptide receptor B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling or a combination thereof in the subject. In some embodiments, a single administration of the AAV2 vector cures glaucoma. In some embodiments, the administration of the AAV2 vector does not include daily administration. In some embodiments, the administration of the AAV2 vector includes weekly administration, biweekly administration, monthly administration, bimonthly administration, semi-annual administration, annual administration, or a combination thereof. In some embodiments, the administration of the AAV2 vector increases the half-life of an agonist for natriuretic peptide receptor B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof in a subject. In some embodiments, the half-life is increased to at least two times, at least five times, at least ten times, at least twenty times, at least fifty times, at least one hundred times, or more times compared to the half-life of an endogenous agonist for natriuretic peptide receptor B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof in a subject. In some embodiments, glaucoma includes neovascular glaucoma (NG), glaucoma, traumatic glaucoma, or a combination thereof.

[0015] In some aspects, an engineered polynucleotide comprising an AAV vector is described herein, and the AAV vector comprises one or more expression cassettes, wherein one or more expression cassettes encode peptides. In some aspects, an engineered polynucleotide comprising an AAV vector is also described herein, and the AAV vector comprises one or more expression cassettes, wherein one or more expression cassettes encode engineered polypeptides, and the engineered polypeptides include: antibodies or fragments thereof operably coupled to peptides. In some embodiments, the AAV vector includes an AAV serotype, and the AAV serotype includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV serotype includes AAV2. In some embodiments, the peptide includes CNP. In some embodiments, CNP includes at least 22 amino acid residues. In some embodiments, CNP includes at least 36 amino acid residues. In some embodiments, CNP includes at least 53 amino acid residues. In some embodiments, the CNP comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1-5. In some embodiments, the peptide is covalently linked to the N-terminus of the antibody or fragment thereof. In some embodiments, the peptide is covalently linked to the C-terminus of the antibody or fragment thereof. In some embodiments, the peptide is operably coupled to the antibody or fragment thereof via a peptide linker. In some embodiments, the peptide linker comprises (GGGGS) n wherein n is an integer between 0 and 10. In some embodiments, the AAV vector encodes an engineered AAV capsid.

[0016] In some aspects, an engineered polypeptide is described herein, comprising an antibody or fragment thereof operably coupled to a peptide, wherein the antibody or fragment thereof comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 6-8. In some embodiments, wherein the peptide comprises a 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 having at least 80% identity to SEQ ID NOs: 1-5. In some embodiments, the peptide is covalently linked to the N-terminus of the antibody or fragment thereof. In some embodiments, the peptide is covalently linked to the C-terminus of the antibody or fragment thereof. In some embodiments, the peptide is operably coupled to the antibody or fragment thereof via a peptide linker. In some embodiments, the peptide linker comprises (GGGGS) n An amino acid sequence of wherein n is an integer between 0 and 10.

[0017] In some aspects, an engineered polynucleotide encoding an engineered polypeptide described herein is 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 includes an AAV vector. In some embodiments, the AAV vector includes an AAV serotype, and the AAV serotype includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV serotype includes 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, one or more expression cassettes encode continuous polypeptides, wherein the continuous polypeptides include the engineered polypeptides described herein. In some embodiments, the continuous polypeptides include a protease cleavable sequence. In some embodiments, the continuous polypeptides include a furin protease cleavable sequence. In some embodiments, the continuous polypeptides include a self-cleaving polypeptide sequence. In some embodiments, one or more expression cassettes express at least one additional therapeutic agent. In some embodiments, at least one additional therapeutic agent includes a hormone. In some embodiments, at least one additional therapeutic agent comprises an agonist of a natriuretic peptide receptor (NPR). In some embodiments, at least one additional therapeutic agent comprises an agonist of a cyclic GMP (cGMP) signaling pathway. In some embodiments, 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 divalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), bis-scFv, (scFv)2, a double-chain antibody, a mini antibody, a nano antibody, a three-chain antibody, a four-chain antibody, a disulfide-stabilized Fv protein ("dsFv"), a single domain antibody (sdAb), an Ig NAR, a camel 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 includes soluble VEGFR1, soluble VEGFR2, soluble VEGFR3, a soluble fragment thereof, or a combination thereof. In some embodiments, the non-antibody VEGF inhibitor includes a VEGF capture agent (VEGF-Trap) or a modified version thereof.

[0018] In some aspects, described herein is a cell comprising an engineered polynucleotide described herein.

[0019] In some aspects, described herein is a cell comprising an engineered polypeptide described herein.

[0020] In some aspects, a pharmaceutical composition is described herein, which comprises an engineered polynucleotide described herein, an engineered polypeptide described herein, or a cell described herein. In some embodiments, the pharmaceutical composition is formulated for intrathecal, intraocular, intravitreal, retinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intracerebral parenchyma, subcutaneous, subretinal, suprachoroidal, intratumoral, pulmonary, intratracheal, intraperitoneal, intravesical, intravaginal, intrarectal, oral, sublingual, transdermal, by inhalation, by inhalation atomization form, by intraluminal-GI route or a combination thereof to a subject in need thereof. In some embodiments, the pharmaceutical composition is formulated for intravitreal, subretinal or suprachoroidal administration. In some embodiments, the pharmaceutical composition is used to treat 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 thereof.

[0021] In some aspects, described herein is a method of treating a disease or condition in a subject in need thereof, the method 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 eye disease. In some embodiments, the ocular diseases comprise ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color blindness, 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, Budd-Bieder syndrome, Best disease, choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, O'Sullivan's disease, Leventinese disease (familial dominant drusen), blue cone monochromacy, or a combination thereof.

[0022] In some aspects, described herein is a method of treating a disease or condition in a subject in need thereof, the method 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 one administration cures the disease or condition. In some embodiments, the disease or condition comprises an eye disease. In some embodiments, the ocular diseases comprise ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color blindness, 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, Budd-Bieder syndrome, Best disease, choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, O'Sullivan's disease, Leventinese disease (familial dominant drusen), blue cone monochromacy, or a combination thereof.

[0023] In some aspects, described herein is a method of treating a disease or condition in a subject in need thereof, the method 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 administering does not include daily administration. In some embodiments, the disease or condition comprises an eye disease. In some embodiments, the ocular diseases comprise ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color blindness, 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, Budd-Bieder syndrome, Best disease, choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, O'Sullivan's disease, Leventinese disease (familial dominant drusen), blue cone monochromacy, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] This patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0025] Figure 1A -F illustrates an exemplary construct for encoding and expressing a natriuretic peptide (eg, CNP) described herein.

[0026] Figure 2A Exemplary AAV vectors encoding the natriuretic peptides described herein are illustrated.

[0027] Figure 2B Exemplary arrangements of CNPs fused to fusion partners described herein are illustrated. The left panel depicts unfused CNPs. The right panel depicts CNPs fused to fusion partners to form CNP fusion proteins (FPs).

[0028] Figure 3A -C illustrates an exemplary arrangement of a natriuretic peptide operably coupled (eg, covalently attached) to an antibody or fragment thereof (eg, a crystallizable fragment region or Fc of an antibody).

[0029] Figure 4A An exemplary anti-human CNP antibody titration standard curve is illustrated.

[0030] Figure 4B An exemplary comparison of antibody dilution assays is illustrated.

[0031] Figure 4C Exemplary standard curves for the abundance of CNP-Fc encoded by AMI061, AMI087, or AMI088 are illustrated.

[0032] Figure 5 Exemplary AAV vector expression profiles in HEK293, HCH or SkMC cells transduced with an AAV vector described herein (AMI061, AMI087 or AMI088) are illustrated.

[0033] Fig. 6A -B illustrates an exemplary SDS-PAGE and peptide sequencing demonstrating CNP or CNP-Fc expression in AAV vector-transduced cells.

[0034] Fig. 7A It was demonstrated that AAV CNP-Fc36 encoded and expressed by 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 level of cGMP was produced by CNP-Fc36 encoded by the AAV vector AMI088.

[0035] Figure 7BAffinity binding between the CNP fusions described herein and the NPR-B protein as measured by the BiaCore assay is demonstrated.

[0036] Figure 7C A single cycle kinetic (SCK) assay of the binding between CNP and NPR-B is illustrated.

[0037] Fig.7D BiaCore assay of AMI263 (Fc4-CNP36) against NPR-B is illustrated.

[0038] Fig. 7E BiaCore assay of CNP-Fc encoded by AMI088 against NPR-B is illustrated.

[0039] Figure 7F A BiaCore assay of ANPs against NPR-B is illustrated.

[0040] Figure 7G The BiaCore assay of BNP against NPR-B is demonstrated.

[0041] Figure 7H BiaCore assay of CNP-Fc encoded by AMI263 against NPR-B is illustrated.

[0042] Fig.7I BiaCore assay of CNP-Fc encoded by AMI087 against NPR-B is illustrated.

[0043] Fig. 8A -B illustrates that AAV CNP-Fc36 encoded and expressed by AAV vectors is functional in vitro in a time course measurement (as determined by cGMP production stimulated by expressed CNP36).

[0044] Fig.9A Body weight measurements of mice before (day 0) or seven days after administration of AAV vectors for in vivo functional studies are illustrated. On the day of intravitreal (IVT) injection, all animals weighed 20-22 g, and all animals generally maintained body weight over the course of the study.

[0045] Fig. 9B Representative images from central retinas stained with hematoxylin and eosin (H&E) are illustrated.

[0046] Fig. 9C H&E stained retinal ganglion cell (RGC) counts are illustrated. The mean RGC counts in the right eye (OD; experimental eye) were lower than those in the left eye (OS; control eye), and there was no difference in the mean RGC counts in OD and OS between Group 1 and Group 2.

[0047] Fig. 10A Flatmount analysis of RGC cell counts for Group 1 and Group 2 mice in an in vivo functional study is illustrated. Groups 1 and 2 were stained with 4',6-diamidino-2-phenylindole (DAPI) and a neuron-specific class III β-tubulin antibody (TUJ-1), and images were captured at 20x. RGCs were counted in four quadrants, and cells / mm were compared across groups. 2 Take the average.

[0048] Fig. 10B Explains from Fig. 10A When RGC counts were quantified across groups, the mean count in the control group (Group 1 OS; PBS, no NMDA) was 11,000 ± 700 cells / mm 2 The reduced RGC counts in the NMDA control group (Group 1 OD; PBS + NMDA) were 8,000 ± 1,000 cells / mm 2 The RGC count of group 2OD was slightly higher than that of group 1OD, which was 9,000±500 cells / mm 2 .

[0049] Fig. 10C Flat mount analysis of RGC cell counts for groups 3-8 of mice in an in vivo functional study is illustrated. Groups 3-8 were stained for DAPI and four images were captured for each retina at 20x approximately 600 μm from the optic nerve head. RGCs were counted in the central 248.1 x 325.7 μm area and cells / mm were plotted on each group. 2 Take the average.

[0050] Fig. 10D Explains from Fig. 10C When calculating the RGC count, the control group (Group 7 OS; PBS, no NMDA) had an average count of 12,000 ± 700 cells / mm 2 The reduced RGC counts in the NMDA control group (Group 7OD; PBS+NMDA) were 9,000 ± 300 cells / mm 2 The RGC counts of group 8 OD were similar to those of group 7 OS, which was 12,000 ± 300 cells / mm 2 The RGC counts at group 3 OD were similar to those at group 7 OD, which were 9,000 ± 1,000 cells / mm 2 The RGC count at OD of group 4 was 10,000 ± 1,400 cells / mm 2The RGC count at OD 5 of group A was 10,000 ± 600 cells / mm 2 The RGC count at group 6 OD was 10,000 ± 1,500 cells / mm 2 .

[0051] Fig.11A -B illustrates the statistical analysis of groups 3-8 (one-way ANOVA followed by multiple comparisons using Dunnett's statistical hypothesis comparison). Treatment of PBS-injected eyes with NMDA caused a statistically significant decrease in RGC counts (P<0.0001). Increasing the dose of MK-801 to 100 μM rescued group 8 RGC counts 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 groups 4-6 were compared to group 3 NMDA / sham vehicle controls, both AMI 182 (group 5) and AMI 088 (group 6) reached statistical significance (P<0.0001). Fig.11A ; P = 0.0332 for Group 5 vs. Group 3; P = 0.0021 for Group 6 vs. Group 3). When RGC counts of AAV-treated groups were compared to NMDA / PBS controls (Group 7 OD), only the AMI 088 construct maintained statistical significance (P = 0.002; Fig. 11B ).

[0052] Fig. 12A Immunohistochemistry images obtained from retinal flat mounts are illustrated. Groups 3-6 were stained for CNP36 in addition to DAPI to assess the location and level of expression of AAV vectors, and images were captured (4x and 20x).

[0053] Fig. 12B Phase 1 immunohistochemistry (Groups 1 and 2) is illustrated. Qualitatively, there appeared to be no difference in the number of TUJ-1+ and cone arrestin+ cells between the groups. All groups had clear cone arrestin staining with some TUJ-1+ cells observed.

[0054] Figure 13 illustrates the quantification of CNP-Fc or CNP. Quantification of CNP36-Fc in eye and serum samples: Animals from Group 3 (AMI189) and Group 6 (AMI088) were analyzed using CNP36-Fc ELISA. Group 3 (sham vehicle) animals served as negative controls ( Fig.13A). Group 6 animals 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 in GraphPad Prism software using one-way ANOVA followed by Dunnett's multiple comparisons (****=<0.0001). Statistically significant differences were observed between Group 3 and Group 6. In samples where the Fc fusion was not present, CNP36 levels were low due to its short half-life. Ocular samples from Groups 3, 4, and 5 were analyzed using the commercial ELISA kit described above ( Fig. 13B , left figure). Although compared with group 3 (false carrier; negative control), groups 4 and 5 show slightly higher levels of CNP36, but the observed differences are not significant. The low level observed on ELISA indicates that only a small portion of the total expressed CNP36 is quantified, because most of the protein can be proteolyzed before quantification. Because the test articles of group 4 and group 5 show efficacy, CNP36 is expressed, but is not protected from proteolysis. Neither CNP36-Fc nor CNP36 is expressed in serum samples. Fig. 13B The right panel illustrates exemplary in vivo expression of CNP fusions detected by ELISA.

[0055] Fig.14 RGC protection by AAV vector encoding CNP is illustrated. Mouse eyes were injected with 1 μl of AAV construct at 4E+8 vg / eye via IVT 28 days before NMDA injection. Images indicate that NMDA induced a reduction in RGC#, which was rescued by MK-801. Sham vector showed reduced RGCs in the NMDA-only treatment group, but AMI182 and AMI088 groups showed higher RGC counts than sham vector and NMDA treatment groups.

[0056] Fig.15 An exemplary SDS-PAGE showing patterns associated with VP1, VP2 or VP3 expression is illustrated. All AAV samples were: produced in Sf-9 cells; 2 Purify by ultracentrifugation, buffer exchange; and sterile filtration. Approximately 1e11 vg / lane was loaded on the gel.

[0057] FIG. 16 illustrates the effect of Fc4-CNP36 (FP) on changes in intraocular pressure (IOP) in the rat partial optic nerve transection (pONT) model. Fig.16A The IOP of the animals monitored during the course of the study is illustrated. Fig. 16B IOP changes in animals following intravitreal (IVT) administration of Fc4-CNP36 are shown. Fig. 16CThe effect of Fc4-CNP36 concentration on IOP changes after intravitreal administration is illustrated. 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.

[0058] FIG. 17 illustrates the effect of Fc4-CNP36 on RGC protection in the rat pONT model. Fig.17A Detection of Fc4-CNP36 concentrations that reduce apoptotic retinal cells (DARC) is illustrated. Fig. 17B Fc4-CNP36 concentrations for RGC counts are indicated. 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.

[0059] Fig.18 Depicted are protein concentrations at terminal samples of the rat pONT model. Only the OS (left eye) was injected, and the OD (right eye) was not injected.

[0060] Fig.19A -B illustrates the effect of AAV2.N54-Fc4-CNP36 and AAV2.N54-CPNP36 on retinal DARC reduction in the rat pONT model. DARC values ​​have been subtracted from baseline. Terminal DARC counts are plotted. Fig.19A : AMI273 is a vector of AAV2.N54-Fc4-CNP36, which expresses Fc4-CNP36 protein. Fig.19B : AMI302 is a vector of AAV2.N54-CNP36, which expresses the CNP36 peptide.

[0061] Fig. 20A -B illustrates the effects of AAV2.N54-Fc4-CNP36 (AAV-FP) and AAV2.N54-CPNP36 (AAV-P) on RGC protection in the rat pONT model. Fig. 20A : AMI273 is a vector of AAV2.N54-Fc4-CNP36, which expresses Fc4-CNP36 protein. Fig. 20B : AMI302 is a vector of AAV2.N54-CNP36, which expresses the CNP36 peptide.

[0062] Fig.21 Depicted are RGC counts in treated eyes of animals dosed with Fc4-CNP36 protein (FP) and AAV vector in the rat pONT model.

[0063] Fig. 22 The concentration of Fc4-CNP36 in ocular and serum samples after IVT administration of an AAV vector (AAV-FP) is shown. The GOI of Fc4-CNP36 was expressed in eyes that were dosed with AMI273 but not in eyes that were not dosed.

[0064] Fig.23 CNP36 concentrations in ocular and serum samples following IVT administration of an AAV vector (AAV-P) are shown.

[0065] Fig.24 Comparison of DARC counts and RGC regional density is illustrated.

[0066] Fig.25A -B illustrates the EC of the CNP-Fc described in this paper. 50 of determination. Fig.25A A standard curve for cGMP is illustrated. Fig.25B The EC values ​​of different CNP fusions (e.g., CNP-Fc) and comparable natriuretic peptides described herein are shown. 50 Data fitting and calculation.

[0067] 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 which sets forth illustrative embodiments. DETAILED DESCRIPTION

[0068] Overview

[0069] In some aspects, described herein is an engineered polynucleotide comprising an AAV vector comprising one or more expression cassettes for encoding a peptide. Also described herein is an engineered polynucleotide comprising an AAV vector comprising one or more expression cassettes for encoding an engineered polypeptide comprising an antibody or fragment thereof operably coupled to a peptide. For example, an engineered polypeptide comprises an antibody or fragment thereof operably coupled to a CNP described herein. Figure 2A Exemplary AAV vectors for encoding peptides (eg, CNP) or fusion proteins (eg, CNP-Fc fusion proteins) are described. Figure 2BExemplary arrangements of CNPs fused with fusion partners described herein are described. In some embodiments, the peptide encoded by the engineered polynucleotide is a natriuretic peptide. In some embodiments, the peptide is CNP. In some embodiments, CNP is full-length CNP. In some embodiments, CNP is cut. In some embodiments, CNP comprises at least 22 amino acid residues, at least 36 amino acid residues, or at least 53 amino acid residues. In some embodiments, CNP is CNP-22 (SEQ ID NO: 5). In some embodiments, CNP is CNP-36 (SEQ ID NO: 4). In some embodiments, CNP is CNP-53 (SEQ ID NO: 3). In some embodiments, CNP comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity with any one of SEQ ID NO: 1-5. In some embodiments, CNP comprises an amino acid sequence having 100% identity with any one of SEQ ID NO: 1-5.

[0070] In some embodiments, the peptide is operably coupled to an antibody or a fragment thereof (e.g., an Fc fragment). In some embodiments, the antibody or a fragment thereof comprises a signaling peptide (SP). For example, the engineered polypeptide may comprise an amino acid having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 9 or SEQ ID NO: 10. In some embodiments, the antibody or a fragment thereof comprises a variable region (e.g., an immunoglobulin heavy chain variable region or Vh). For example, the engineered polypeptide may comprise an amino acid having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 11 or SEQ ID NO: 12.

[0071] In some embodiments, the peptide is covalently linked to the N-terminus of an antibody or its fragment. In some embodiments, the peptide is covalently linked to the C-terminus of an antibody or its fragment. In some aspects, the operable coupling of a peptide to an antibody or its fragment increases the stability of the peptide. In some aspects, the operable coupling of a peptide to an antibody or its fragment increases the half-life of the peptide. In some aspects, the operable coupling of a peptide to an antibody or its fragment increases the half-life of the peptide in vivo. In some aspects, the operable coupling of a peptide to an antibody or its fragment increases the half-life of the peptide in circulation. In some aspects, the operable coupling of a peptide to an antibody or its fragment increases the half-life of the peptide in cells. In some embodiments, the antibody or its fragment comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity with any one of SEQ ID NO:6-8. In some embodiments, the antibody or its fragment comprises an amino acid sequence having 100% identity with any one of SEQ ID NO:6-8. In some embodiments, the peptide operably coupled 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 coupled 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 coupled 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 coupled to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to any one of SEQ ID NOs: 9-12. Figure 3A -C illustrates an exemplary description of the CNP-Fc fusion protein described herein.

[0072] In some embodiments, the engineered polynucleotides described herein include vectors. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV vector. In some embodiments, the AAV vector includes an AAV serotype, and the AAV serotype includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV vector includes an AAV serotype, and the AAV serotype includes AAV2. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the viral vector or the AAV vector comprises one or more expression cassettes. In some embodiments, one or more expression cassettes can encode CNP or CNP operably coupled to an antibody or fragment thereof (e.g., CNP fusion protein) and at least one additional therapeutic agent. For example, at least one additional therapeutic agent can be a hormone or agonist for stimulating a natriuretic peptide receptor (NPR) or activating a cyclic GMP (cGMP) signaling pathway. In some embodiments, at least one additional therapeutic agent includes a VEGF inhibitor. For example, the at least one additional therapeutic agent can be an antibody targeting VEGF or an shRNA targeting a VEGF transcript. In some embodiments, the at least one additional therapeutic agent comprises a cytokine inhibitor (eg, a tumor necrosis factor inhibitor).

[0073] In some embodiments, the engineered polynucleotides described herein are encapsulated or as part of a viral particle. In some embodiments, the viral particle is at least partially encoded by an engineered polynucleotide. In some embodiments, the viral particle is not encoded by an 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 for the treatment of a disease or condition. In some embodiments, the viral particle can be formulated into a pharmaceutical composition described herein.

[0074] In some aspects, described herein is a method for treating a disease or condition in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide described herein, an engineered polypeptide encoded by an engineered polynucleotide described herein, a cell transduced with an engineered polynucleotide described herein, or a pharmaceutical composition described herein. In some embodiments, the engineered polynucleotide encodes an engineered polypeptide described herein. In some embodiments, the method cures a disease or condition or significantly reduces the severity associated with a disease or condition. In some embodiments, the method confers protection against a disease or condition. In some embodiments, the method cures a disease or condition or significantly reduces the severity associated with a disease to a condition after a single administration. In some embodiments, the method cures a disease or condition or significantly reduces the severity associated with a disease to a condition without the need for daily administration. In some aspects, the disease or condition comprises an ocular disease, the ocular disease comprising ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color blindness, 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, Budd-Bieder syndrome, Best disease, choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, O'Sullivan's disease, Leventinese disease (familial dominant drusen), blue cone monochromacy, or a combination thereof.

[0075] Engineered polynucleotides

[0076] In some aspects, an engineered polynucleotide encoding a peptide or fusion protein is described herein. In some embodiments, the engineered polynucleotide encodes a natriuretic peptide, such as a C-type natriuretic peptide (CNP) or a fragment thereof. In some embodiments, CNP or a fragment thereof comprises 22 consecutive bases of SEQ ID NO:1. In some embodiments, CNP or a fragment thereof comprises 22 consecutive bases at the C-terminus of SEQ ID NO:1. In some embodiments, CNP or a fragment thereof comprises 36 consecutive bases of SEQ ID NO:1. In some embodiments, CNP or a fragment thereof comprises 36 consecutive bases at the C-terminus of SEQ ID NO:1. In some embodiments, CNP or a fragment thereof comprises 53 consecutive bases of SEQ ID NO:1. In some embodiments, CNP or a fragment thereof comprises 53 consecutive bases at the C-terminus of SEQ ID NO:1.

[0077] In some embodiments, the engineered polynucleotide encodes CNP or CNP fusion protein (e.g., CNP-Fc fusion protein described herein). In some embodiments, the engineered polynucleotide includes a viral vector such as an AAV vector, which includes one or more expression cassettes for encoding CNP or CNP fusion protein. In some embodiments, the engineered polynucleotide includes a vector. In some embodiments, the vector is a viral vector. In some embodiments, the engineered polynucleotide includes an AAV vector. In some embodiments, the engineered polynucleotide includes an AAV vector encoding an engineered AAV capsid. In some embodiments, the AAV vector includes one or more expression cassettes for encoding an engineered polypeptide, which includes: a peptide or a fusion protein, which includes an antibody or fragment thereof operably coupled to a peptide. In some embodiments, one or more expression cassettes encode an engineered polypeptide including CNP. In some embodiments, one or more expression cassettes encode an engineered polypeptide including an antibody or fragment thereof operably coupled to CNP. In some embodiments, CNP includes at least 22 amino acid residues. In some embodiments, CNP includes at least 36 amino acid residues. In some embodiments, CNP includes at least 53 amino acid residues. In some embodiments, the CNP comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the CNP comprises an amino acid sequence having 100% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the engineered polynucleotide encodes a natriuretic peptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 4. In some embodiments, the engineered polynucleotide encodes a natriuretic peptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 4. In some embodiments, the engineered polynucleotide encodes a natriuretic peptide comprising an amino acid sequence of SEQ ID NO: 4.

[0078] In some embodiments, the CNP encoded by the engineered polynucleotide is operably coupled to an antibody or its fragment. In some embodiments, an antibody or its fragment includes a crystallizable fragment (Fc) region. In some embodiments, the CNP encoded by the engineered polynucleotide is operably coupled to the N-terminus of an antibody or its fragment. In some embodiments, the CNP encoded by the engineered polynucleotide is operably coupled to the C-terminus of an antibody or its fragment. In some embodiments, the CNP encoded by the engineered polynucleotide is covalently connected to an antibody or its fragment. In some embodiments, the CNP encoded by the engineered polynucleotide is covalently connected to the N-terminus of an antibody or its fragment. In some embodiments, the CNP encoded by the engineered polynucleotide is covalently connected to the C-terminus of an antibody or its fragment. In some embodiments, the CNP encoded by the engineered polynucleotide is covalently connected to the C-terminus of an antibody or its fragment. In some embodiments, the CNP is covalently connected to an antibody or its fragment through a peptide linker.

[0079] 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 coupled 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 coupled 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 coupled 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 coupled to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to any one of SEQ ID NOs: 9-12.

[0080] 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, and the AAV serotype includes 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.

[0081] In some embodiments, the engineered polynucleotide encodes a continuous polypeptide, wherein the continuous polypeptide comprises an engineered polypeptide described herein. In some embodiments, the continuous polypeptide comprises a protease cleavable sequence. In some embodiments, the continuous polypeptide comprises a furin protease cleavable sequence. In some embodiments, the continuous polypeptide comprises a self-cleaving polypeptide sequence.

[0082] In some embodiments, the engineered polynucleotide comprises one or more expression cassettes, wherein the one or more expression cassettes encode CNP or CNP fusion protein and at least one additional therapeutic agent. In some embodiments, at least one additional therapeutic agent comprises a hormone. In some embodiments, at least one additional therapeutic agent comprises an agonist of a natriuretic peptide receptor (NPR). In some embodiments, at least one additional therapeutic agent comprises an agonist of a cyclic GMP (cGMP) signaling pathway. In some embodiments, at least one additional therapeutic agent comprises a cytokine inhibitor. In some embodiments, at least one additional therapeutic agent comprises a VEGF inhibitor, wherein 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 is an antibody. In some embodiments, the VEGF inhibitor is not an antibody.

[0083] In some cases, the engineered polynucleotide includes additional features. The additional features may include sequences such as tags, signal peptides, intron sequences, promoters, stuffer sequences, etc.

[0084] In some cases, the engineered polynucleotide encodes a signal peptide. Signal peptides are sometimes referred to as signal sequences, targeting signals, localization signals, localization sequences, transit peptides, leader sequences or leader peptides, and are short peptides present at the N-terminus of most newly synthesized proteins that are destined for the secretory pathway. These proteins include those present in certain organelles (endoplasmic reticulum, Golgi apparatus or endosomes), secreted from cells or inserted into most cell membranes. In some cases, the nucleic acids provided herein may include a signal peptide. The signal peptide may be of any length, but is typically 15-30 amino acids long. The signal peptide may be approximately: 10-15, 10-20, 10-30, 15-20, 15-25, 15-30, 20-30 or 25-30 amino acids long. Various signal peptides may be used, and include, but are not limited to: human antibody heavy chain (Vh), human antibody light chain (Vl) and aflibercept.

[0085] In some cases, the engineered polynucleotide comprises an intron sequence. An intron is any nucleotide sequence in a sequence that can be removed by RNA splicing during the maturation of the final RNA product. In other words, an intron is a non-coding region of the RNA transcript or the DNA encoding it, which is eliminated by splicing before translation. Although introns do not encode protein products, they are participants in gene expression regulation. Some introns themselves encode functional RNA by 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 a vital role in a wide range of gene expression regulation functions, such as degradation and mRNA output of nonsense mediation. In an embodiment, an 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-1α gene intron 1, intervening sequence intron, human growth hormone intron and / or human β-globin intron. Any intron sequence number is considered. In an embodiment, the intron sequence is SV40. In some cases, the nucleic acid may include 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.

[0086] In an embodiment, the engineered polynucleotide comprises additional features including a promoter. A promoter is a DNA sequence that binds to a protein and initiates transcription of a single RNA from its downstream DNA. The RNA may encode a protein or may have its own function (such as tRNA, mRNA or rRNA). The promoter is located near the gene transcription start site, upstream of the DNA (towards the 5' region of the sense strand). The promoter may be about 100-1000 base pairs long. Various promoters are contemplated and may be employed in the engineered polynucleotides of the present disclosure. In embodiments, the promoter is: cytomegalovirus (CMV) promoter, elongation factor 1 alpha (EF1 alpha) promoter, simian vacuolating virus (SV40) promoter, phosphoglycerate kinase (PGK1) promoter, ubiquitin C (Ubc) promoter, human β-actin promoter, CAG promoter, tetracycline response element (TRE) promoter, UAS promoter, actin 5c (Ac5) promoter, polyhedron promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKIIa) promoter, GAL1 promoter, GAL 10 promoter, TEF1 promoter, glyceraldehyde 3-phosphate dehydrogenase (GDS) promoter, ADH1 promoter, CaMV35S promoter, Ubi promoter, human polymerase III RNA (H1) promoter, U6 promoter, polyadenylation constructs thereof, and any combination thereof. In some cases, the promoter is a CMV promoter.

[0087] Any of the engineered polynucleotides provided can include a viral vector sequence. The viral vector can be, but is not limited to, a lentivirus, a retrovirus, or an adeno-associated virus. The viral vector can be an adeno-associated virus (AAV) vector. In some cases, the viral vector is an adeno-associated virus vector. Many serotypes of AAV vectors are contemplated, and include, but are 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 can be engineered to make it more suitable for biological function, tissue, or cell selection. In some embodiments, the AAV vector is AAV2 and variants AAV2.N53 and AAV2.N54. It is also contemplated that a chimeric AAV vector containing at least 2 AAV serotypes can be contained. 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 in a chimeric AAV vector. In some cases, only a portion of AAV is chimeric. For example, a suitable portion may include a capsid, VP1, VP2 or VP3 domain and / or Rep. In some cases, at least one of VP1, VP2 and VP3 has at least one amino acid substitution compared to an otherwise comparable wild-type AAV capsid protein. In some cases, mutations may occur in VP1 and VP2, in VP1 and VP3, in VP2 and VP3, or in VP1, VP2 and VP3. In some embodiments, at least one of VP1, VP2 and VP3 has one to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2 and VP3, for example, about one 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 cases, VP can be removed. For example, in some embodiments, mutant AAV does not include at least one of VP1, VP2 or VP3.

[0088] In some cases, the AAV vector can be modified. For example, the AAV vector can include modifications such as insertions, deletions, chemical changes, or synthetic modifications. In some cases, a single nucleotide is inserted into the AAV vector. In other cases, multiple nucleotides are inserted into the vector.

[0089] Codon optimization

[0090] In embodiments, the engineered polynucleotides described herein include modifications that confer enhanced expression of biological agents such as CNP or CNP fusion proteins described herein. For example, some biological agents are derived from natural gene sequences and contain unmodified sequences that are not optimized for introduction and expression in target cells. In embodiments, the engineered polynucleotides separated are codon optimized. Codon optimization can be specific for cell type-specific codon usage. Different organisms and cell types show a tendency to use certain codons rather than other codons for the same amino acid. Some species are known to almost completely avoid using certain codons. Similarly, some cell types tend to use certain codons rather than other codons for the same amino acid. In embodiments, the method for optimizing the codons of the engineered polynucleotides may include reallocating codon usage based on the usage frequency of each codon in the target cell. In some cases, the target cell may be some tissues or organs. In some cases, modifications are performed to increase guanine and / or cytosine content.

[0091] In embodiments, the engineered polynucleotide sequence can be modified to replace at least one codon with another codon encoding the same amino acid. In some cases, the codon is modified in the coding region of the sequence. In some cases, the codon is modified in 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 the codon adaptation index.

[0092] Various modifications are contemplated herein. In some cases, codons can be interchanged. For example, a sequence can be modified to replace AGA with AGG. In other cases, CCC is replaced with CCT. In other cases, AGC is replaced with TCC. In other cases, CCC is replaced with CCG. Any non-limiting replacement provided in Table 1 can be applied to modify nucleic acids. Any number of codons can be interchanged in nucleic acids. In some cases, 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 can be replaced. In embodiments, the engineered polynucleotide comprises 3 codon modifications. In embodiments, the engineered polynucleotide comprises 16 codon modifications. In embodiments, 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 embodiments, the engineered polynucleotide comprises two codon modifications, which are: AGA to AGG and at least one of: CCT to CCC, AGC to TCC or CCC to CCG. In embodiments, the engineered polynucleotide comprises three codon modifications, which are: AGA to AGG and at least two of: CCT to CCC, AGC to TCC or CCC to CCG. In embodiments, the engineered polynucleotide comprises four codon modifications, which are: AGA to AGG, CCT to CCC, AGC to TCC and CCC to CCG. Additional modifications may include any codon modification provided in Table 1 with the above codons and / or any possible additional modifications from Table 1. In an embodiment, the nucleic acid is modified such that AGA is replaced with AGG and CCT is replaced with CCC. In an embodiment, the nucleic acid is modified such that AGA is replaced with AGG and AGC is replaced with TCC. In an embodiment, the nucleic acid is modified such that AGA is replaced with AGG and CCC is replaced with CCG.

[0093] Table 1. Non-limiting exemplary codons that can be used interchangeably to modify nucleic acids. Thymine can be replaced with uracil in the following codons

[0094]

[0095] In some embodiments, the engineered polynucleotide sequence may include 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 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 an AAV2 serotype. In some embodiments, the viral vector sequence may include sequences of at least 2 AAV serotypes. In some embodiments, at least two serotypes may be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV11, and AAV12.

[0096] In some cases, modification can also include chemical modification. Modified nucleic acid can include modification of its backbone, sugar or core base, and even novel base or base pair. Modified nucleic acid can have improved chemical and / or biological stability. Decoration with different chemical substituents (e.g., hydrophobic groups) can also produce improved properties and functions such as new structural motifs and enhanced target binding.

[0097] 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-ribulose nucleic acid; TNA, α-L-threose nucleic acid; tPhoNA, 3'-2' phosphonomethylthreosyl nucleic acid; dXNA, 2'-deoxyxylose nucleic acid; PS, phosphorothioate; phNA, alkylphosphonate nucleic acid; PNA and peptide nucleic acid.

[0098] Dual Expression

[0099] In some aspects, an engineered polynucleotide is described herein, which comprises 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, one or more expression cassettes encode continuous polypeptides. In some embodiments, the continuous polypeptide comprises a protease peptide sequence. In some embodiments, the protease peptide sequence can be cut by a protease endogenously expressed in a cell. Non-limiting examples of proteases can include serine endoproteases, aspartic acid endoproteases, cysteine ​​thiol endoproteases, metalloendoproteases, or glutamic acid and threonine endoproteases. In some embodiments, the protease peptide sequence can be cut by a serine endoprotease. In some embodiments, the protease peptide sequence can be cut by furin. In some embodiments, the continuous polypeptide comprises a protease cleavable sequence. In some embodiments, the protease cleavable sequence can be cut by any protease described herein. In some embodiments, the protease cleavable sequence can be cut 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 can include T2A, P2A, E2A, F2A or a combination thereof. In some embodiments, the self-cleaving polypeptide sequence includes 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 can include a furin protease-F2A fusion polypeptide sequence. In some embodiments, the engineered polynucleotide includes a viral vector such as an AAV vector.

[0100] 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 site (IRES). In some embodiments, the expression cassette is subject to expression control of the promoter. In some embodiments, the expression cassette is subject to expression control of the promoter. In some embodiments, the expression cassette can further apply expression control via at least one IRES.

[0101] 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, CNP or CNP fusion protein (e.g., CNP-Fc fusion protein) and at least one additional therapeutic agent are each expressed from an expression cassette.

[0102] In some embodiments, at least one additional therapeutic agent is an inhibitor of a targeted cytokine (e.g., tumor necrosis factor). In some embodiments, at least one additional therapeutic agent can be an antibody targeting a cytokine or an inhibitory nucleic acid targeting a transcript of a cytokine. For example, at least one additional therapeutic agent can be an inhibitory RNA such as a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a double-stranded RNA (dsRNA), a transfer RNA (tRNA), a ribosomal RNA (rRNA), or a heterogeneous nuclear RNA (hnRNA), which targets a transcript of a cytokine for degradation, thereby reducing the expression of the cytokine in the cell.

[0103] In some embodiments, at least one additional therapeutic agent is a VEGF inhibitor. For example, Fig.25B Functional analysis of a dual-expressed CNP fusion (Fc4-CNP36) and a VEGF inhibitor (aflibercept) is described. In some embodiments, the VEGF inhibitor includes an inhibitory RNA for targeting and degrading VEGF transcripts. In some embodiments, the VEGF inhibitor includes an antibody or a fragment thereof. In some embodiments, the VEGF antibody binds to VEGF to reduce neovascularization signaling including the VEGF signal transduction transduction 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, (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 camel 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 having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identity to any one of SEQ ID NOs: 21-27 (Table 2), or a combination thereof, or a fragment thereof.

[0104] Table 2. Non-limiting examples of polypeptide sequences of VEGF antibodies

[0105]

[0106]

[0107] 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 signal transduction 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, a soluble VEGF receptor may include soluble VEGFR1, soluble VEGFR2, soluble VEGFR3, a soluble fragment thereof, or a combination thereof. In some embodiments, a non-antibody VEGF inhibitor includes at least one of FLT1, KDR / FLK1, FLT4, a fragment thereof, or a combination thereof. In some embodiments, a 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, a non-antibody inhibitor VEGF includes a VEGF capture agent. 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).

[0108] Table 3. Non-limiting examples of non-antibody VEGF inhibitors

[0109]

[0110]

[0111]

[0112]

[0113] Modified capsid

[0114] Provided herein are compositions containing modified adeno-associated virus (AAV) capsids and methods of use thereof. The modified AAV capsids may contain exogenous sequences compared to otherwise comparable unmodified AAV capsids. Exogenous sequences may refer to exogenous polypeptide sequences. AAV capsids may be modified to impart improved function to them and any compositions and / or methods utilizing them, thereby obtaining better therapeutic agents, particularly for ocular use.

[0115] 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 encode a protein with a support 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 transcriptional control and replication and for packaging the viral genome into newly generated, pre-assembled capsids. The second half of the AAV genome contains the cap gene, which encodes the viral proteins (VP) VP1, VP2, and VP3 and the assembly activation protein (AAP). The transcription of all VPs as capsid monomers is controlled by a single promoter (p40 in the case of AAV2), resulting in a single mRNA. Splicing (VP1) and an unusual translation start codon (VP2) make the presence of VP1 and VP2 as low as about 1 / 10 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 in addition, VP2 and VP1 share approximately 65 amino acids (the "common VP1 / VP2 region"). Only VP1 contains a unique sequence at its N-terminus (approximately 138 amino acids, unique to VP1). AAP was identified in 2010 as a 23 kDa protein encoded in an alternative cap ORF. It serves to stabilize newly produced VP proteins and transport them from the cytoplasm to the nucleus. Although AAV serotypes 1-3, 6-9, and rh10 fail to produce capsids in the absence of AAP, capsid production from AAV4 and AAV5 is reported to be low but detectable.

[0116] In one aspect, AAV may comprise modifications. The modification may be a rep, cap and / or X encoded polypeptide sequence of AAV. In some cases, the modification may be a cap polypeptide. The cap polypeptide may be modified in any VP domain, such as VP1, VP2 and / or VP3. In some cases, VP1 is modified. In some cases, VP2 is modified. In some cases, VP3 is modified. In some aspects, two or all of the VP domains may be modified. In some cases, VP1 and VP2 are modified. In some cases, 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 modifications in Rep and Cap, Cap and X, Rep and X, and / or Rep, Cap and X. Any combination of domains may be modified, such as a combination of any of the aforementioned VP modifications with Rep and / or X modifications. In some cases, Rep and VP1 and / or VP2 are modified. In some aspects, the subject Rep is modified. The rep modifications can include modifications as provided herein, and can be in at least one of Rep 78, Rep 68, Rep 52, or Rep 40. In some cases, the Rep is a different AAV serotype than the subject capsid.

[0117] In some cases, the AAV capsid is modified. The capsid of an AAV serotype is assembled from 60 VP monomers, of which approximately 50 copies of VP3, 5 copies of VP2, and 5 copies of VP1. Topologically prominent capsid surface structures are each five-fold hole or "channel-like structure", each two-fold depression, and three protrusions around each three-fold symmetry axis. These holes allow exchange between the inside and outside of the capsid. The depression, more precisely, the bottom layer of each two-fold axis, is the thinnest part of the viral capsid. The protrusions around the three-fold axis contain five of the nine so-called variable regions (VRs). Specifically, VR-IV, VR-V, and VR-VIII form a ring (loop 1-4) at the top of the protrusion, while VR-VI and VR-VII are present at its base. VRs differ between serotypes and are responsible for serotype-specific variations in antibody and receptor binding. Due to their exposed position and their function in receptor binding, VRs that form protrusion rings are ideal locations for capsid modifications aimed at redirecting or expanding AAV tropism (cell surface targeting). While redirected tropism (vector retargeting) combines ablation of native receptor binding (e.g., by site-directed mutagenesis) with insertion of ligands that mediate transduction through novel, non-native AAV receptors, AAV vectors with expanded tropism gain the ability to transduce cells through additional receptors while maintaining their native receptor binding capacity.

[0118] In some aspects, modification of the AAV capsid may refer to the insertion of an exogenous polypeptide sequence. In other aspects, modification may refer to a deletion in a polypeptide sequence. Modification may also refer to the modification of at least one amino acid residue (classical or non-classical) in a polypeptide sequence.

[0119] 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 classical amino acid or a non-classical amino acid. Any number of amino acid residues can be inserted. In some cases, the insertion site can be in the GH loop or loop IV of the AAV capsid protein, for example, in a solvent accessible portion of the GH loop or loop IV of the AAV capsid protein.

[0120] In some cases, the modification includes inserting an exogenous polypeptide sequence comprising a sequence of Formula 1: X0-X1-X2-X1-X3-X1-X1-X4 (SEQ ID NO: 41). In some cases, X0 is valine (V), isoleucine (I), leucine (L), phenylalanine (F), tryptophan (W), tyrosine (Y) or methionine (M). In some cases, 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 cases, X2 is V, I, L or M, wherein X3 is E, S or Q. In some cases, X4 is K, R, E or A. In some cases, Formula 1 further comprises X5. X5 can be proline (P) or R.

[0121] In some cases, 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 cases, Formula 1 comprises VKLG-X3-X1-X1-X4 (SEQ ID NO: 45). In some cases, the exogenous polypeptide is VKLG-X3-X1-T-X4 (SEQ ID NO: 46) and / or VKLG-X3-X1-X1-K (SEQ ID NO: 47). In some cases, the exogenous polypeptide comprises LALG-X3-X1-X1-X4 (SEQ ID NO: 48). In some cases, 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 cases, 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 cases, the exogenous polypeptide comprises LALG-X3-X1-SK (SEQ ID NO: 56). In some cases, the exogenous polypeptide comprises LKLG-X3-X1-X1-X4 (SEQ ID NO: 57). In some cases, the exogenous polypeptide comprises LKLG-X3-X1-T-X4 (SEQ ID NO: 58). In some cases, the exogenous polypeptide comprises LKLG-X3-X1-TK (SEQ ID NO: 59).

[0122] In some cases, the exogenous polypeptide comprises a sequence of Formula 1. In some cases, 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 cases, the exogenous polypeptide is one of Table 4 with 0-2 modifications to residues.

[0123] In some cases, at least two exogenous polypeptides (such as those described in Formula 1) are inserted into the capsid sequence of the AAV provided herein. The at least two exogenous polypeptides can be inserted into the same position or different positions. In one aspect, any one of the exogenous polypeptide sequences provided in Table 4 can be inserted into an unmodified AAV capsid sequence, such as those wild-type sequences provided in Table 5, to generate a modified AAV capsid.

[0124] Table 4. Exemplary exogenous polypeptide sequences that can be inserted into the AAV capsid (exemplary insertion sites for AAV2 are shown, but similar locations for other AAV serotypes are also contemplated)

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139] Table 5. Exemplary wild-type AAV capsid polypeptide sequences

[0140]

[0141]

[0142]

[0143] Similarly, the deletion may comprise the deletion of at least one amino acid residue in the sequence encoding the AAV capsid. Any number of amino acids may be deleted. In some cases, 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 may be inserted or deleted in the polypeptide sequence encoding the AAV capsid. In some cases, at least or at most 1-5, 5-10, 10-15, 15-20 or a combination thereof of exogenous amino acid residues can be inserted and / or deleted in the polypeptide sequence encoding the AAV capsid. In some cases, about or at most about 5 amino acids to about 11 amino acids are inserted in the insertion site in the GH loop or loop IV of the capsid protein relative to the corresponding unmodified AAV capsid protein. For example, the insertion site can 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 insertion sites 587-588 are based on AAV2 capsid protein. About 5 amino acids to about 11 amino acids can be inserted in the corresponding sites of AAV serotypes other than AAV2 (e.g., AAV5, AAV6, AAV8, AAV9, etc.).

[0144] In some embodiments, the insertion site is a single insertion site between two adjacent amino acids between amino acids 570-614 of VP1 of any AAV serotype, e.g., the insertion site is between two adjacent amino acids located in 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 insertion site 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 insertion site 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.

[0145] In some aspects, 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 588 and 589 of AAV10.

[0146] As another example, the insertion site can be between amino acids 450 and 460 of the AAV capsid protein, as shown in Table 5. For example, the insertion site can be at (e.g., immediately adjacent to its N-terminus) 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.

[0147] In some embodiments, a subject capsid protein comprises a GH loop comprising 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 shown in Table 5. One skilled in the art would know, based on a comparison of the amino acid sequences of capsid proteins of various AAV serotypes, that the insertion site "corresponding to amino acids 587-588 of AAV2" is in the capsid protein of any given AAV serotype.

[0148] In some cases, the exogenous polypeptide may have 0 to 4 spacer amino acids (Y 1 -Y 4 ). Suitable spacer amino acids include, but are not limited to, leucine, alanine, glycine and / or serine.

[0149] Modification of the AAV capsid can include modification of at least one amino acid residue in the polypeptide sequence. In some cases, the modification can be performed at any AAV capsid position as described herein, and can include any number of modifications. In some cases, the modification can include mutations. Mutations can include: point mutations, missense mutations, nonsense mutations, deletions, duplications, frameshifts, and / or repeat sequence expansions.

[0150] In one aspect, the amino acid can 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, 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.

[0151] In some cases, the mutation is a point mutation. A point mutation comprises a change from a charged amino acid residue to a polar or non-polar amino acid residue. In some cases, the charged amino acid is positively charged. In some cases, the charged amino acid is negatively charged.

[0152] Point mutations can be conservative mutations. Non-limiting examples of conservative mutations include: non-polar aliphatic amino acids to non-polar aliphatic amino acids; polar amino acids to polar amino acids; positively charged amino acids to positively charged amino acids; negatively charged amino acids to negatively charged amino acids; and aromatic amino acids to aromatic amino acids. For example, 20 naturally occurring amino acids can share similar properties. Aliphatic amino acids can be: glycine, alanine, valine, leucine, or isoleucine. Amino acids containing hydroxyl groups or sulfur / selenium can be: serine, cysteine, selenocysteine, threonine, or methionine. Cyclic amino acids can be proline. Aromatic amino acids can be phenylalanine, tyrosine, or tryptophan. Basic amino acids can be histidine, lysine, and arginine. Acidic amino acids can be aspartic acid, glutamic acid, asparagine, or glutamic acid. Conservative mutations can be serine to glycine, serine to alanine, serine to serine, serine to threonine, serine to proline. Conservative mutations may be arginine to asparagine, arginine to lysine, arginine to glutamine, arginine to arginine, arginine to histidine. Conservative mutations may be leucine to phenylalanine, leucine to isoleucine, leucine to valine, leucine to leucine, leucine to methionine. Conservative mutations may be proline to glycine, proline to alanine, proline to serine, proline to threonine, proline to proline. Conservative mutations may be threonine to glycine, threonine to alanine, threonine to serine, threonine to threonine, threonine to proline. Conservative mutations may be alanine to glycine, alanine to threonine, alanine to proline, alanine to alanine, alanine to serine. Conservative mutations may be valine to methionine, valine to phenylalanine, valine to isoleucine, valine to leucine, valine to valine. Conservative mutations may be glycine to alanine, glycine to threonine, glycine to proline, glycine to serine, glycine to glycine. Conservative mutations may be isoleucine to phenylalanine, isoleucine to isoleucine, isoleucine to valine, isoleucine to leucine, isoleucine to methionine. Conservative mutations may be phenylalanine to tryptophan, phenylalanine to phenylalanine, phenylalanine to tyrosine. Conservative mutations may be tyrosine to tryptophan, tyrosine to phenylalanine, tyrosine to tyrosine. Conservative mutations may be cysteine ​​to serine, cysteine ​​to threonine, cysteine ​​to cysteine. Conservative mutations may be histidine to asparagine, histidine to lysine, histidine to glutamine, histidine to arginine, histidine to histidine. Conservative mutations may be glutamine to glutamic acid, glutamine to asparagine, glutamine to aspartic acid, glutamine to glutamine. Conservative mutations may 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.

[0153] Non-limiting examples of additional amino acid mutations can be: 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 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 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 any previously described mutations, and vice versa.

[0154] Any of the aforementioned modifications (insertions, deletions and / or mutations) may be made at any residue in the AAV sequence. The sequence may be a capsid sequence. In other cases, the sequence may not be a capsid sequence, but a Rep and / or X sequence. As previously described, the sequence may be in VP1, VP2 and / or VP3. In some cases, sequence modifications are made to loops of the capsid sequence (such as loop 3 and / or loop 4). In some cases, residues of the sequences in Table 5 are modified.

[0155] In some cases, the residues of the capsid polypeptide sequences in Table 5 are modified, such as inserted, deleted and / or mutated. In some cases, the modification is from 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800 or a combination thereof. In some cases, the modification is in residues at positions 200-300, 300-400, 400-500, 500-600 or a combination thereof. In some cases, the modification is in residues at positions 300-500 or a combination thereof. In one aspect, the insertion site is in the GH loop or loop IV of the AAV capsid protein, for example, in the solvent accessible 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.

[0156] 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 cases, the modification is at position 452, 453, 466, 467, 468, 471, 585, 586, 587, and / or 588 of AAV2. In some cases, the modification is at position 452 or 453 of AAV2. In some cases, the modification is at position 587 or 588 of AAV2. In some cases, the modification is an insertion at position 452, 453, 466, 467, 468, 471, 585, 586, 587 and / or 588 of any one of SEQ ID NOs: 121-126. In some cases, the modification is an insertion at position 452, 453, 466, 467, 468, 471, 585, 586, 587 and / or 588 of SEQ ID NOs: 121. In some cases, the modification is a mutation, and the mutation is R585A or R588A of any one of SEQ ID NOs: 121-126. In some cases, the modification is a mutation, and the mutation is R585A or R588A of SEQ ID NOs: 121.

[0157] In some embodiments, the subject modified AAV capsid does not contain any other amino acid modification (mutation, substitution, insertion or deletion) except for the insertion of about 5 amino acids to about 11 amino acids in the loop (loop 3 and / or 4) relative to the corresponding unmodified AAV capsid protein. In other embodiments, in addition to the insertion of about 5 amino acids to about 11 amino acids in loop 3 and / or loop 4 relative to the unmodified AAV capsid protein, the subject variant AAV capsid also contains 1 to about 25 amino acid insertions, deletions or substitutions compared to the unmodified AAV capsid protein. In one embodiment, in addition to the insertion of about 7 amino acids to about 10 amino acids in the GH loop or loop IV relative to the corresponding parent AAV capsid protein, the subject AAV virion capsid does not contain any other amino acid substitutions, insertions or deletions. In other embodiments, in addition to the insertion of about 7 amino acids to about 10 amino acids in the GH loop or loop IV relative to the corresponding parent AAV capsid protein, the subject AAV virion capsid also contains 1 to about 25 amino acid insertions, deletions or substitutions compared to the parent AAV capsid protein. For example, in some embodiments, in addition to an insertion of about 7 amino acids to about 10 amino acids in the GH loop or loop IV relative to the corresponding parent AAV capsid protein, the subject AAV virion capsid further comprises 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 insertions, deletions, or substitutions compared to the parent AAV capsid protein.

[0158] In some cases, chimeric AAV capsids are provided herein. The chimeric capsid comprises polypeptide sequences of at least 2 AAV serotypes. The chimeric capsid may comprise a mixture of sequences selected from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and / or AAV12. In some cases, the chimeric serotype is different between VP1, VP2 and / or VP3. In some cases, the chimeric capsid comprises sequences of at least 2 serotypes selected from the following: AAV4 and AAV6, AAV5 and AAV6, AAV11 and AAV6, AAV12 and AAV6 and any combination thereof. In some cases, the first AAV serotype may be AAV4, and the second serotype may be AAV6. In some cases, the first AAV serotype and the second AAV serotype of the chimeric AAV vector may be AAV11 and AAV6. In some cases, the first AAV serotype and the second AAV serotype of the chimeric AAV vector can be AAV12 and AAV6. In some cases, the chimeric capsid comprises the following sequences: AAV2 and AAV5 or AAV2 and AAV6. In some cases, the chimeric capsid comprises the following sequences: AAV2 and AAV5, AAV2 and AAV6, AAV2 and AAV8, AAV2 and AAV9, AAV2 and AAV1, and AAV2 and AAV12.

[0159] The modifications to the AAV provided herein can confer enhanced activity to the modified AAV compared to the original unmodified or wild-type AAV. The modifications provided herein can improve cell transduction, tropism and / or reduce immunogenicity associated with the capsid.

[0160] In some cases, 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 into a cell.

[0161] In some cases, the modifications provided herein enhance tropism. Enhanced tropism refers to the ability to transduce cells through additional receptors compared to the original unmodified AAV. In some aspects, the enhanced tropism can improve the infectivity of ocular cells, thereby improving gene therapy by utilizing modified AAV. In some cases, the modifications provided herein can improve the tropism of ocular cells selected from the following: bipolar cells, retinal ganglion cells, horizontal cells, amacrine cells, epithelial cells, retinal pigment cells, photoreceptors or any combination thereof. In some cases, the modification improves the tropism of retinal cells.

[0162] AAV vectors are also provided herein. AAV vectors contain: inverted terminal repeats (ITRs), Rep, Cap, AAP, and X sequences. Typically, the AAV viral genome is flanked by ITRs, which serve as packaging signals and replication origins. The rep gene encodes a family of multifunctional proteins (Rep proteins) responsible for controlling viral transcription, replication, packaging, and integration in AAVS1. For AAV2, four Rep proteins are 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 number indicates molecular weight. The expression of AAP and viral capsid proteins VP1 (90kDa), VP2 (72kDa), and VP3 (60kDa) (all encoded in the cap gene) is controlled by the p40 promoter. The X gene is located in the region shared with the cap gene at the 3' end of the genome and has its own promoter (p81). While the X protein appears to enhance viral replication, the AAP is essential for capsid assembly. Three different VPs contribute to the icosahedral AAV2 capsid in a ratio of 1 (VP1):1 (VP2):10 (VP3).

[0163] The modified capsid proteins disclosed herein can be isolated, for example, purified. In some embodiments, the modified capsids disclosed herein are contained in AAV vectors or AAV virions (e.g., recombinant AAV virions, rAAV or AAV viral particles). In other embodiments, such modified AAV vectors and / or AAV variant virions are used in vivo or in vitro methods for treating eye diseases of primate retina (e.g., human retina).

[0164] Also provided herein are vectors comprising modified AAV capsids. Any of the previously described modifications may be encompassed in the vectors provided herein. In some cases, the AAV vector comprises a modified capsid comprising exogenous sequences in at least two loops of the VP domain compared to an otherwise comparable AAV capsid sequence lacking exogenous sequences. In some aspects, the vectors provided herein may also comprise transgenic sequences.

[0165] Engineered Peptides

[0166] Engineered polypeptides are described herein, including peptides operably coupled to antibodies or fragments thereof. In some embodiments, the engineered polypeptides are encoded by engineered polynucleotides described herein. In some embodiments, the engineered polypeptides include natriuretic peptides. In some embodiments, the engineered polypeptides include CNPs. 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, 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, CNP comprises 22 amino acid residues. In some embodiments, CNP comprises 36 amino acid residues. In some embodiments, CNP comprises 53 amino acid residues. In some embodiments, CNP comprises 61 amino acid residues. In some embodiments, 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 having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 1. In some embodiments, the CNP comprises an amino acid sequence having 100% identity to SEQ ID NO: 1. In some embodiments, the CNP comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 2. In some embodiments, the CNP comprises an amino acid sequence having 100% identity to SEQ ID NO: 2. In some embodiments, the CNP comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 3. In some embodiments, the CNP comprises an amino acid sequence having 100% identity 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.

[0167] In some embodiments, the engineered polypeptide includes an antibody or fragment thereof operably coupled to a peptide or CNP. In some embodiments, the antibody or fragment thereof includes the Fc region of an antibody. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% identity to any one of SEQ ID NO: 6-8. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% identity to SEQ ID NO: 6. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence having 100% identity to SEQ ID NO: 6. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% identity 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.

[0168] In some embodiments, the engineered polypeptide comprises a peptide covalently linked to the N-terminus of an antibody or fragment thereof. In some embodiments, the engineered polypeptide comprises a peptide covalently linked to the C-terminus of an antibody or fragment thereof. In some embodiments, the engineered polypeptide comprises a peptide covalently linked or operably coupled to an antibody or fragment thereof via 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 of at least one glycine followed by a serine. For example, a subunit may be represented as (GS) n ,in n is an integer between 0 and 20. In this case, n Equal to two would be equivalent to having (GS) 2or GSGS. In some embodiments, the subunits may include one glycine followed by serine (e.g., GS), two glycines followed by serine (e.g., GGS), three glycines followed by serine (e.g., GGGS), four glycines followed by serine (e.g., GGGGS), five glycines followed by serine (e.g., GGGGGS), or six glycines followed by serine (e.g., GGGGGGS). In some embodiments, the peptide linker comprises at least one subunit of glycine followed by serine, at least two subunits of glycine followed by serine, at least three subunits of glycine followed by serine, at least four subunits of glycine followed by serine, or at least six subunits of glycine followed by serine. In some embodiments, n is an integer of one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 16, 17, 18, 19, or 20. In some embodiments, n is an integer greater than 20. In some embodiments, n is the integer four. In some embodiments, n It is the integer five.

[0169] In some embodiments, the engineered polypeptide comprises a peptide covalently linked to an antibody or fragment thereof via a peptide linker, wherein the peptide linker comprises (GGGGS) n The amino acid sequence of n is an integer between 0 and 20. In some embodiments, the engineered polypeptide comprises a peptide covalently linked to an antibody or fragment thereof via a peptide linker, wherein the peptide linker comprises (GGGGS) n The amino acid sequence of n is an integer between 0 and 5. In some embodiments, the engineered polypeptide comprises a peptide covalently linked to an antibody or fragment thereof via a peptide linker, wherein the peptide linker comprises (GGGGS) n The amino acid sequence of n is an integer of four. In some embodiments, the engineered polypeptide comprises a peptide covalently linked to an antibody or fragment thereof via a peptide linker, wherein the peptide linker comprises (GGGGS) n The amino acid sequence of n It is the integer five.

[0170] In some embodiments, the peptide operably coupled 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 coupled 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 coupled 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 coupled to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 131. In some embodiments, the peptide operably coupled 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: 132. In some embodiments, the peptide operably coupled to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 132. In some embodiments, the peptide operably coupled 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: 133. In some embodiments, the peptide operably coupled to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 133. In some embodiments, the peptide operably coupled 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: 134. In some embodiments, the peptide operably coupled to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 134. In some embodiments, the peptide operably coupled 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: 135. In some embodiments, the peptide operably coupled to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 135.In some embodiments, the peptide operably coupled 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: 136. In some embodiments, the peptide operably coupled to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 136. In some embodiments, the peptide operably coupled 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: 137. In some embodiments, the peptide operably coupled 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 coupled 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 coupled 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 coupled 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 coupled 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 coupled 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 coupled to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 140.

[0171] In some embodiments, the peptide operably coupled to the antibody or its fragment comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NOs: 9-12. In some embodiments, the peptide operably coupled to the antibody or its fragment comprises an amino acid sequence having 100% identity to any one of SEQ ID NOs: 9-12. In some embodiments, the peptide operably coupled to the antibody or its fragment comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 9. In some embodiments, the peptide operably coupled to the antibody or its fragment comprises an amino acid sequence having 100% identity to SEQ ID NO: 9. In some embodiments, the peptide operably coupled 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, the peptide operably coupled to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 10. In some embodiments, the peptide operably coupled 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: 11. In some embodiments, the peptide operably coupled to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 11. In some embodiments, the peptide operably coupled 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: 12. In some embodiments, the peptide operably coupled to the antibody or fragment thereof comprises an amino acid sequence that is 100% identical to SEQ ID NO: 12.

[0172] In some embodiments, compared with uncoupled CNP, the engineered polypeptide comprising the CNP that is operably coupled to an antibody or its fragment increases the half-life of the coupled CNP. In some embodiments, compared with the half-life of uncoupled CNP, the half-life of the CNP that is operably coupled to an antibody or its fragment increases by at least 0.1 times, at least 0.2 times, at least 0.3 times, at least 0.4 times, at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, to at least 2.0 times, to at least 5.0 times, to at least 10.0 times, to at least 20.0 times, to at least 50.0 times or to at least 100.0 times. In some embodiments, the half-life of the CNP operably coupled to the antibody or fragment thereof is increased 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 the uncoupled CNP. In some embodiments, the in vivo half-life of the CNP operably conjugated to the antibody or fragment thereof is increased 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 the unconjugated CNP.

[0173] In some embodiments, compared with uncoupled CNP, the engineered polypeptide comprising the CNP that is operably coupled to an antibody or its fragment increases the protection for degradation of the coupled CNP. In some embodiments, compared with the degradation of uncoupled CNP, the CNP that is operably coupled to an antibody or its fragment will increase the protection for degradation by at least 0.1 times, at least 0.2 times, at least 0.3 times, at least 0.4 times, at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, to at least 2.0 times, to at least 5.0 times, to at least 10.0 times, to at least 20.0 times, to at least 50.0 times or to at least 100.0 times.

[0174] In some embodiments, the engineered polypeptide can be administered to a subject to treat a disease or condition. In some embodiments, the engineered polypeptide can be formulated into a pharmaceutical composition to be administered to a subject to treat a disease or condition. In some embodiments, the engineered polypeptide comprising a CNP and an antibody or a fragment thereof can be administered to a subject to treat a disease or condition. In some embodiments, the engineered polypeptide comprising a CNP and an antibody or a fragment thereof can be formulated into a pharmaceutical composition to be administered to a subject to treat a disease or condition.

[0175] In some embodiments, the engineered polypeptide can increase the activity or signaling cascade associated with the natriuretic peptide receptor (NPR). In some embodiments, the engineered polypeptide can increase the activity or signaling cascade associated with the cyclic GMP (cGMP) signaling pathway. In some embodiments, the engineered polypeptide comprising CNP and an antibody or a fragment thereof can increase the activity or signaling cascade associated with the natriuretic peptide receptor (NPR). In some embodiments, the engineered polypeptide comprising CNP and an antibody or a fragment thereof can increase the activity or signaling cascade associated with the cyclic GMP (cGMP) signaling pathway.

[0176] In some embodiments, the engineered polypeptide can be administered to a subject to treat a disease or condition by increasing the activity or signal cascade associated with a natriuretic peptide receptor (NPR). In some embodiments, the engineered polypeptide can be administered to a subject to treat a disease or condition by increasing the activity or signal cascade associated with a cGMP signaling pathway. In some embodiments, an engineered polypeptide comprising a CNP and an antibody or a fragment thereof can be administered to a subject to treat a disease or condition by increasing the activity or signal cascade associated with a natriuretic peptide receptor (NPR). In some embodiments, an engineered polypeptide comprising a CNP and an antibody or a fragment thereof can be administered to a subject to treat a disease or condition by increasing the activity or signal cascade associated with a cGMP signaling pathway.

[0177] Pharmaceutical composition

[0178] Pharmaceutical compositions are described herein, comprising engineered polynucleotides, AAV vectors comprising engineered polynucleotides, engineered polypeptides, cells transduced by AAV vectors comprising engineered polynucleotides, viral particles comprising engineered polynucleotides, or combinations thereof. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable: carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition comprises two or more active agents as disclosed herein. In some embodiments, pharmaceutical compositions comprising engineered polynucleotides, AAV vectors comprising engineered polynucleotides, or AAV vectors comprising engineered polynucleotides treat diseases or conditions described herein. In some embodiments, diseases or conditions include eye diseases. In some embodiments, the disease or condition comprises ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color blindness, age-related macular degeneration (nAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, traumatic glaucoma, Budd-Bieder syndrome, Best disease, choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, O'Sullivan's disease, Leventinese disease (familial dominant drusen), blue cone monochromacy, or a combination thereof.

[0179] For in vivo delivery, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the engineered polypeptide, the cell transduced by the AAV vector comprising the engineered polynucleotide, or a combination thereof can be formulated into a pharmaceutical composition and can generally be administered intravitreally or parenterally (e.g., via intramuscular, subcutaneous, intratumoral, transdermal, intrathecal, etc. routes of administration). In some embodiments, the pharmaceutical composition is formulated for administration to a subject in need thereof intrathecally, intraocularly, intravitreally, retinaly, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonary, intratracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhalation of aerosolized form, by intraluminal-GI route, or a combination thereof.

[0180] In some aspects, pharmaceutical composition can be used to treat objects in need such as humans or mammals. In some cases, objects can be diagnosed with diseases, for example, eye diseases. In some aspects, subject pharmaceutical composition is co-administered with secondary therapy. Secondary therapy can include any therapy for eye purposes. In some cases, secondary therapy includes nutritional therapy, vitamins, laser therapy such as laser photocoagulation, photodynamic therapy, Visudyne, anti-VEGF therapy, glasses, eye drops, numbing agents, vision correction therapy, behavior / perception therapy, etc. In some aspects, any biological agent previously described can be considered as secondary therapy.

[0181] 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, for example, the rate of loss, and therefore disease progression, is reduced to 1 / 2, 1 / 3, 1 / 4, or 1 / 5 or less, for example, the rate of loss, and therefore disease progression, is reduced to 1 / 10 or less.

[0182] In some embodiments, an effective amount of the pharmaceutical composition reduces neovascularization 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 neovascularization signaling in a cell not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition reduces neovascularization in a subject in need thereof 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 neovascularization in the subject if the subject was not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition reduces vascular leakage in a subject in need thereof 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 if the subject was not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition reduces inflammation in a subject in need thereof 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 if the subject was not treated with the pharmaceutical composition.

[0183] In some embodiments, an effective amount of a subject rAAV virion results in a gain in visual function, retinal function, retinal anatomy or health, and / or eye movement, and / or neurological function, such as a 2-fold, 3-fold, 4-fold, or 5-fold or more improvement in retinal function, retinal anatomy or health, and / or eye movement, such as a 10-fold or more improvement in retinal function, retinal anatomy or health, and / or eye movement. As will be readily appreciated by those of ordinary skill, the dose required to achieve the desired therapeutic effect is typically in the range of 1 x 10 8 About 1x 10 15 The range of recombinant virions is usually referred to as 1 x 10 8 About 1x 10 15 A "vector genome".

[0184] In some aspects, the compositions provided herein, such as pharmaceutical compositions, are administered to a subject in need thereof. In some cases, administration comprises delivering a dose of about 0.5 x 10 vectors of AAV. 9 vg, 1.0x 10 9 vg, 1.0x 10 10 , 1.0x10 11 vg, 3.0x 10 11 vg, 6x 10 11 vg, 8.0x 10 11 vg, 1.0x 10 12 vg, 1.0x 10 13 vg, 1.0x 10 14 vg, 1.0x10 15 vg, 1.5x 10 15 For example, for in vivo injection, such as direct injection into the eye, a therapeutically effective dose may be about 10 6 to about 10 15 order of magnitude of the subject AAV virions, e.g., about 10 8 Up to 10 12 For in vitro transduction, the effective amount of engineered AAV virions to be delivered to cells will be about 10 8 to about 10 13 Other effective doses can be easily determined by one of ordinary skill in the art through routine experiments establishing dose response curves.

[0185] Administration can be repeated any time. In some aspects, administration is performed as follows: twice a day, every other day, twice a week, every two months, every three months, once a month, every other month, every six months, every year, or once every two years.

[0186] The dosage treatment can be a single dose regimen or a multiple dose regimen. In addition, the subject can be administered as many doses as appropriate. Those skilled in the art can easily determine the appropriate number of doses. In some aspects, the pharmaceutical composition is administered via intravitreal injection, subretinal injection, microinjection, or injection on the eyeball.

[0187] When practicing the treatment or use methods provided herein, a therapeutically effective amount of the pharmaceutical composition described herein is administered to a mammal suffering from a disease to be treated, a disorder or a condition (e.g., cancer). In some embodiments, the mammal is a human. The therapeutically effective amount can vary greatly depending on the severity of the disease, the age and relative health of the object, the effectiveness of the therapeutic agent used, and other factors. The therapeutic agent described herein and the composition in some cases can be used alone or in combination with one or more therapeutic agents as a component of a mixture.

[0188] The pharmaceutical compositions described herein can be administered to a subject by a suitable route of administration, including but not limited to intravenous, intraarterial, oral, parenteral, oral, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation or intraperitoneal 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, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multi-particulate formulations, and mixed immediate release formulations and controlled release formulations.

[0189] The pharmaceutical compositions may be manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping or compression processes.

[0190] In certain embodiments, the pharmaceutical compositions provided herein include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances, such as merfen and thiomersal; stable chlorine dioxide; and quaternary ammonium compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0191] 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, freeze-dried formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulse release formulations, multi-particulate formulations, and mixed immediate release and controlled release formulations. On the one hand, therapeutic agents as discussed herein, such as therapeutic agents, are formulated into pharmaceutical compositions suitable for intramuscular, subcutaneous, or intravenous injection. On the one hand, 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, hydrogenated castor oil (cremophor) and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using a surfactant. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preservatives, wetting agents, emulsifiers and dispersants. 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 also desirable to include isotonic agents such as sugars, sodium chloride and the like. The absorption of injectable drug forms can be prolonged by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0192] In another aspect, the dosage form includes a microencapsulated formulation. In some embodiments, one or more other compatible materials are present in the microencapsulated material. Non-limiting examples of materials include pH regulators, erosion accelerators, defoamers, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0193] Liquid dosage forms for oral administration are optionally aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. In addition to the therapeutic agent, the liquid dosage form optionally includes additives such as: (a) a disintegrant; (b) a dispersant; (c) a wetting agent; (d) at least one preservative, (e) a viscosity enhancer, (f) at least one sweetener, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersion also includes a crystal formation inhibitor.

[0194] In some embodiments, pharmaceutical compositions described herein are self-emulsifying drug delivery systems (SEDDS). Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets. In general, emulsions are produced by violent mechanical dispersion. SEDDS, in contrast to emulsions or microemulsions, spontaneously forms emulsions when added to excess water without any external mechanical dispersion or agitation. The advantage of SEDDS is that only gentle mixing is required to disperse the droplets throughout the solution. In addition, water or aqueous phase is optionally added before being applied, which ensures the stability of unstable or hydrophobic active ingredients. Therefore, SEDDS provides an effective delivery system for the oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provides an improvement in the bioavailability of hydrophobic active ingredients.

[0195] In addition, the pharmaceutical composition optionally includes one or more pH adjusters or buffers, 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 tris; and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in amounts required to maintain the pH of the composition within an acceptable range.

[0196] In addition, the pharmaceutical composition optionally includes one or more salts in an amount required to bring the osmotic pressure of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and 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.

[0197] Reagent test kit

[0198] In some embodiments, disclosed herein are kits for use, comprising engineered polynucleotides described herein, AAVs comprising engineered polynucleotides, engineered polypeptides, cells transduced by AAV vectors comprising engineered polynucleotides, viral particles comprising engineered polynucleotides, pharmaceutical compositions, or combinations thereof. In some embodiments, the kits disclosed herein can be used to treat a disease or condition in a subject. In some embodiments, the kits comprise a combination of materials or components other than an engineered polynucleotide, an AAV comprising an engineered polynucleotide, an engineered polypeptide, a cell transduced by an AAV vector comprising an engineered polynucleotide, or a pharmaceutical composition.

[0199] In some embodiments, the kit described herein includes components for selecting a homogeneous population of AAV containing engineered polynucleotides described herein. In some embodiments, the kit includes components for determining the number of units of biomolecules (e.g., AAV) synthesized and / or released or expressed on the surface by host cells. In some embodiments, the kit includes components for performing assays such as enzyme-linked immunosorbent assays (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 an object. In some embodiments, the kit is specially configured for the purpose of treating a mammalian subject. In some embodiments, the kit is specially configured for the purpose of treating a human subject.

[0200] Instructions for use may be included in the kit. In some embodiments, the kit includes instructions for applying engineered polynucleotides, AAV vectors comprising engineered polynucleotides, engineered polypeptides, AAVs comprising engineered polynucleotides, cells transduced with AAV vectors, pharmaceutical compositions, or combinations thereof to objects in need. In some embodiments, the kit includes instructions for further engineering cells to express biomolecules (e.g., engineered polynucleotides, AAV vectors comprising engineered polynucleotides, engineered polypeptides, AAVs comprising engineered polynucleotides, or cells transduced with AAV vectors). In some embodiments, the kit includes instructions for thawing or otherwise recovering the biological activity of engineered polynucleotides, AAV vectors comprising engineered polynucleotides, AAVs comprising engineered polynucleotides, and the engineered polynucleotides, AAVs comprising engineered polynucleotides, can be frozen or lyophilized during storage or transportation. In some embodiments, the kit includes instructions for measuring the viability of the recovered engineered polynucleotide, AAV vector comprising the engineered polynucleotide, AAV comprising the engineered polynucleotide to ensure efficacy for its intended purpose (e.g., therapeutic efficacy, if used to treat a subject).

[0201] Optionally, the kit also contains other available components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandage materials or other available appliances. The materials or components assembled in the kit can be stored and provided to the practitioner in any convenient and suitable manner to maintain their operability and practicality. For example, the components can be in dissolved, dehydrated or lyophilized form; they can be provided at room temperature, refrigerated or frozen temperatures. The components are usually contained in suitable packaging materials.

[0202] Delivery Method

[0203] The engineered polynucleotides can be easily introduced into host cells, such as mammals, bacteria, yeast or insect cells by any method in the art. For example, the engineered polynucleotides can be transferred to host cells by physical, chemical or biological means. In some embodiments, the engineered polynucleotides can be delivered to host cells by encapsulating the engineered polynucleotides in viral particles such as AAV particles. In some embodiments, the engineered polynucleotides can be delivered to cells by physical methods (such as calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, gene gun, electroporation, etc.).

[0204] Physical methods for introducing the encoded engineered polynucleotide into cells can include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, gene gun, electroporation, etc. One method for introducing engineered polynucleotides into host cells is calcium phosphate transfection.

[0205] Chemical means for introducing non-natural engineered polynucleotides into cells may 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 (SNA), liposomes, or lipid nanoparticles. Example colloidal systems used as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles). Other state-of-the-art nucleic acid targeted delivery methods are available, such as carriers delivering engineered polynucleotides or encoding engineered polynucleotides with targeted nanoparticles.

[0206] In the case of utilizing a non-viral delivery system, an example delivery vehicle is a liposome. It is contemplated that lipid formulations are used to introduce engineered polynucleotides or vectors encoding engineered polynucleotides into cells (in vitro, in vitro or in vivo). On the other hand, carriers can be associated with lipids. Carriers associated with lipids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, attached to liposomes via connecting molecules associated with liposomes and engineered polynucleotides, embedded in liposomes, compounded with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or otherwise associated with lipids. The compositions related to lipids, lipid / DNA or lipid / expression vectors are not limited to any specific structure in the solution. For example, in some embodiments, they exist in a double-layer structure, micelle or "collapse" structure. Alternatively, they are just dispersed in the solution and may form aggregates of uneven size or shape. Lipid is a fatty substance, and in some embodiments, it is a naturally occurring or synthetic lipid. For example, lipids include fat droplets naturally present in the cytoplasm as well as compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0207] Lipids suitable for use are obtained from commercial sources. Stock solutions of lipids in chloroform or chloroform / methanol are usually 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 covers a variety of monolayer and multilayer lipid vehicles produced by closed lipid bilayers or aggregates. Liposomes are generally characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure and entrap water and dissolved solutes between the lipid bilayers. However, compositions having structures different from normal vesicle structures in solution are also covered. For example, in some embodiments, the lipids present a micellar structure or exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0208] In some cases, non-viral delivery methods include lipofection, nucleofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, exosomes, polycations or lipid: cargo conjugates (or aggregates), naked polypeptides (e.g., recombinant polypeptides), naked DNA, artificial virosomes, and agents that enhance 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 may include aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylene oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxyesters, polyamide esters, amine-containing polyoxyesters, and poly(anhydrides). Such synthetic polymers can be homopolymers or copolymers (e.g., random, block, segmented, grafted) of multiple different monomers, such as two or more of lactic acid, lactide, glycolic acid, glycolide, ε-caprolactone, trimethylene carbonate, p-dioxanone, etc. In an example, the scaffold can be composed of a polymer comprising glycolic acid and lactic acid, such as a polymer having a ratio of glycolic acid to lactic acid of 90 / 10 or 5 / 95. Non-limiting examples of naturally occurring biocompatible, biodegradable polymers can include glycoproteins, proteoglycans, polysaccharides, glycosaminoglycans (GAGs) and fragments derived from these components, elastin, laminin, decrorin, fibrinogen / fibrin, fibronectin, osteopontin, integrin, hyaluronic acid, collagen, chondroitin sulfate, heparin, heparan sulfate, ORC, carboxymethylcellulose, and chitin.

[0209] In some cases, the engineered polynucleotides described herein can be packaged and delivered to cells via extracellular vesicles. Extracellular vesicles can be any membrane-bound particles. In some embodiments, extracellular vesicles can be any membrane-bound particles secreted by at least one cell. In some cases, extracellular vesicles can be any membrane-bound particles synthesized in vitro. In some cases, extracellular vesicles can be any membrane-bound particles synthesized in the absence of cells. In some cases, extracellular vesicles can be exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptotic bodies, proto-oncobodies, overflow bubbles (exopher), enveloped viruses, exosomes (exomere) or other very large extracellular vesicles.

[0210] In some embodiments, engineered polynucleotides can be delivered to cells via biological methods (such as using DNA and RNA vectors). Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammals (e.g., human cells). In some embodiments, other viral vectors are derived from slow viruses, poxviruses, herpes simplex virus type I, adenoviruses and adeno-associated viruses, etc. 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 cases, retroviral vectors include gamma retroviral vectors, such as vectors derived from Moloney murine leukemia virus (MoMLV, MMLV, MuLV or MLV) or mouse stem cell virus (MSCV) genomes. In some cases, retroviral vectors also include slow viral vectors, such as slow viral vectors derived from human immunodeficiency virus (HIV) genomes. In some cases, AAV includes serotypes including AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a combination thereof. Based on these initial serotypes, the AAV capsid of each serotype can be engineered to make it more suitable for biological function, tissue or cell selection. In some embodiments, 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 2 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 in the chimeric AAV. In some cases, only a portion of the AAV is chimeric. For example, a suitable portion may include a 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 an otherwise comparable wild-type AAV capsid protein. In some cases, the mutation may occur in VP1 and VP2, in VP1 and VP3, in VP2 and VP3, or in VP1, VP2, and VP3. In some embodiments, at least one of VP1, VP2, and VP3 has one to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3, such as about one 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 cases, VP can be removed. For example, in some embodiments, the mutant AAV does not include at least one of VP1, VP2, or VP3.

[0211] Methods of modifying cells

[0212] In one aspect, the present invention also provides a method for modifying cells to generate engineered cells. Cells can refer to primary cells, recombinant cells or cell lines. In some cases, cells are packaging cells. Packaging cells can be any of the following cells: HEK 293 cells, HeLa cells and African green monkey kidney cells (Vero cells) (just to name a few). Engineered cells can be primary cells. In some cases, engineered cells can be eye cells. Suitable eye cells include, but are not limited to: photoreceptors, ganglion cells, RPE cells, amacrine cells, horizontal cells, Muller cells, etc.

[0213] In some cases, cell is a packaging cell for generating viral particles. In order 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 by hybrid adenovirus / AAV vectors infect in cell lines such as human embryonic kidney cell line HEK 293 (human kidney cell line containing functional adenovirus E1 genes providing trans-acting E1 protein). Suitable transfection methods include calcium phosphate coprecipitation, direct microinjection, electroporation, liposome-mediated gene transfer and the delivery of nucleic acids using high-speed microprojectiles, which are all known in the art.

[0214] In order to engineer cells, a plurality of cells can be contacted with the engineered polynucleotides of separation. Contact can include any time length, and can include about 5min to about 5 days. Contact can last 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 cases, contact can last 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.

[0215] In some cases, the supernatant of the packaging cell line is treated with PEG precipitation for concentrating the virus. In other cases, a centrifugation step can be used to concentrate the virus. For example, during centrifugation, a column can be used to concentrate the virus. In some embodiments, precipitation occurs at no more than about 4°C (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, recombinant AAV is separated from the supernatant of PEG precipitation by low-speed centrifugation and then with a CsCl gradient. Low-speed centrifugation can be about 4000rpm, about 4500rpm, about 5000rpm, or about 6000rpm for about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes. In some cases, recombinant AAV is separated from the supernatant of PEG precipitation by centrifugation at about 5000rpm for about 30 minutes, and then with a CsCl gradient. In some cases, CsCl purification can be replaced by IDX gradient ultracentrifugation. The 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, or any time between these two time points after transfection. The supernatant can also be purified, concentrated, or a combination thereof. For example, the concentration or viral titer can be determined by qPCR or silver staining.

[0216] In one aspect, a plurality of AAV particles (containing the engineered polynucleotides described herein) isolated from engineered cells are also provided. The viral titer can be about 10 2 vp / mL, about 10 3 vp / mL, about 10 4 vp / mL, about 10 5 vp / mL, about 10 6 vp / mL, about 10 7 vp / mL, about 10 8 vp / mL or up to about 10 9 vp / mL. The virus titer can be about 10 2 GC / mL, about 10 3 GC / mL, about 10 4 GC / mL, about 10 5 GC / mL, about 10 6 GC / mL, about 10 7 GC / mL, about 10 8 GC / mL or up to about 10 9 GC / mL. In some cases, the virus titer can be about 10 2 TU / mL, about 10 3 TU / mL, about 10 4 TU / mL, about 105 TU / mL, about 10 6 TU / mL, about 10 7 TU / mL, about 10 8 TU / mL or up to about 10 9 TU / mL. The optimal viral titer may vary depending on the cell type to be transfected. The virus may range from 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, in order to infect 1 million cells using an MOI of 10,000, 10,000 x 1,000,000 = 10 10 GC.

[0217] In some cases, multiple AAV particles can be formulated into a unit dosage form. Various formulations are contemplated for adult or pediatric delivery and include, but are not limited to: 0.5 x 10 9 vg, 1.0x10 9 vg, 1.0x 10 10 , 1.0x 10 11 vg, 3.0x 10 11 vg, 6x 10 11 vg, 8.0x 10 11 vg, 1.0x 10 12 vg, 1.0x 10 13 vg, 1.0x 10 14 vg, 1.0x 10 15 vg or up to 1.5x10 15 The composition of viral particles may be frozen or otherwise stored in a suitable container.

[0218] The compositions and methods provided herein can be sufficient to enhance the delivery and / or expression of a subject biologic agent 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 comparable unmodified nucleic acid. In some cases, an otherwise comparable unmodified nucleic acid is a nucleic acid encoding a VEGF capture agent. In some cases, the modifications may be sufficient to enhance the delivery and / or expression of a subject biologic agent by 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 180 fold, about 181 fold, about 186 fold, about 187 fold, about 188 fold, about 189 fold, about 190 fold, about 191 fold, about 192 fold, about 193 fold, about 194 fold, about 195 fold, about 196 fold, about 197 fold, about 198 fold, about 199 fold, about 200 fold, about 201 fold, about 202 fold, about 203 fold, about 204 fold, about 205 fold, about 206 fold, about 207 fold, about 208 fold, about 209 about 181-fold, about 186-fold, 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 than an otherwise comparable unmodified nucleic acid. In embodiments, increased expression comprises at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, or at least 500-fold increase, as determined by in vitro assays. Suitable in vitro assays include ELISA, western blots, Luminex, microscopy, imaging, and / or flow cytometry.

[0219] The subject AAV virions can exhibit 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 more than 50-fold increased infectivity of retinal cells compared to the infectivity of AAV virions comprising otherwise comparable WT AAV capsid proteins to retinal cells (photoreceptors, ganglion cells, RPE cells, amacrine cells, horizontal cells, Muller cells, etc.).

[0220] Treatment

[0221] Provided herein is a method for treating a disease or condition described herein. In some aspects, the method confers protection for a disease or condition. The method of treatment may include introducing an engineered polynucleotide, an AAV vector comprising an engineered polynucleotide, an AAV comprising an engineered polynucleotide, a cell transduced with an AAV vector, a viral particle comprising an engineered polynucleotide, a pharmaceutical composition, or a combination thereof, to an object in need. A method for treating a disease or condition is also provided, which includes administering a pharmaceutical composition to an object in need. The pharmaceutical composition may include a sequence encoding a biological agent, which includes an engineered polynucleotide, an AAV vector comprising an engineered polynucleotide, an AAV vector comprising an engineered polynucleotide, a viral particle comprising an engineered polynucleotide, or a combination thereof. In some embodiments, administration is by any suitable mode of administration, including systemic administration (e.g., intravenous, intravitreal, subretinal or etc.). In some embodiments, the object is a person.

[0222] In some embodiments, the method includes treating a disease or condition in a subject in need by administering to the subject a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or a pharmaceutical composition described herein. In some embodiments, the method treats a disease or condition, wherein a single administration of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or a pharmaceutical composition described herein cures the disease or condition. In some embodiments, the method treats a disease or condition, wherein administration of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or a pharmaceutical composition described herein does not include daily administration. In some embodiments, the disease or condition includes an eye disease. Non-limiting examples of ocular diseases can include ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color blindness, 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, Budd-Bieder 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-cone dystrophy, cone-rod dystrophy, Okoro's disease, Leventinese disease (familial dominant drusen), blue cone 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.

[0223] In some embodiments, administering a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or a pharmaceutical composition described herein to a subject can protect the subject from a disease or condition. For example, administering a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or a pharmaceutical composition can protect the subject from a disease or condition that originates from damage. As shown in Example 4, the engineered polypeptide promotes the protection of retinal ganglion cells in the eye after transection injury. In some embodiments, administering a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or a pharmaceutical composition protects or promotes the survival of cells in the subject. In some embodiments, administering a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or a pharmaceutical composition protects or promotes the survival of ocular cells in the subject. In some embodiments, administering a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or a pharmaceutical composition protects or promotes the survival of retinal ganglion cells in the subject. In some embodiments, administration of a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or a pharmaceutical composition reduces intraocular pressure in a subject.

[0224] 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 over a period of time (e.g., every 2 days, twice a week, once a week, weekly, 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 twice or more over 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 includes a single administration. In some embodiments, the administration described herein does not include daily administration.

[0225] In some embodiments, the method includes applying (including, for example, oral or topical application) engineered polynucleotides, AAV vectors comprising engineered polynucleotides, AAVs comprising engineered polynucleotides, cells or pharmaceutical compositions transduced with AAV vectors in a therapeutically effective amount by various forms and approaches. In some embodiments, the composition can be applied by intravitreal, subretinal, suprachoroidal, parenteral, intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, subarachnoid, intraocular, intrasternal, eye, endothelial, local, intranasal, pulmonary, rectal, intraarterial, intrathecal, inhalation, intralesional, intradermal, epidural, intracapsular, subcapsular, intracardiac, transtracheal, subcutaneous, subarachnoid or spinal administration (e.g., injection or infusion). In some embodiments, the composition can be applied by absorption of epithelial or mucosal lining (e.g., oral mucosa, rectal and intestinal mucosa administration). In some embodiments, the composition is delivered via a variety of administration routes.

[0226] In some embodiments, the method includes administering 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 by intravenous infusion. In some embodiments, 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 are administered by slow continuous infusion over a long period of time (such as more than 24 hours). In some embodiments, 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 are administered as intravenous injections or short infusions. In some embodiments, 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 are administered via vitreous routes. In some embodiments, the engineered polynucleotide, AAV vector comprising the engineered polynucleotide, AAV comprising the engineered polynucleotide, cells transduced with an AAV vector, viral particles comprising the engineered polynucleotide, pharmaceutical compositions, or a combination thereof can be administered in a localized manner, for example, via injection of the agent directly into an organ, optionally in the form of a depot or slow release formulation or implant.

[0227] In some embodiments, 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 can be administered in conjunction with other therapies, such as antiviral therapy, chemotherapy, antibiotics, cell therapy, cytokine therapy, or anti-inflammatory agents. In some embodiments, 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 can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering a composition containing a therapeutic agent can vary. In some cases, the composition can be used as a prophylactic agent and can be continuously administered to an object (e.g., an object for immunity or an object for treatment) that is susceptible to coronavirus or has a tendency to a condition or disease associated with coronavirus. Prophylactic administration can reduce the likelihood of infection, disease, or condition, or can reduce the severity of infection, disease, or condition.

[0228] 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 can be administered to an object before the onset of symptoms. In some embodiments, 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 can be administered to an object (e.g., an object for immunization or an object for treatment) after a test result (e.g., as soon as possible after the test result), the test result is, for example, a test result providing a diagnosis, a test showing the presence of coronavirus in an object (e.g., an object for immunization or an object for treatment), or a test showing progression of the condition (e.g., a decrease in blood oxygen levels). The therapeutic agent can be administered after the onset of a disease or condition is detected or suspected (e.g., as soon as feasible). The therapeutic agent can be administered after potential exposure to a coronavirus (e.g., as soon as practicable), such as after a subject (e.g., a subject for immunization or a subject for treatment) has come into contact with an infected subject, or learns that they have come into contact with an infected subject who may be contagious.

[0229] The actual dosage level of the agent (e.g., engineered polynucleotide or pharmaceutical composition) of the present disclosure can be varied so as to obtain an amount of agent to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without causing toxicity to the subject (e.g., a subject for immunization or a subject for treatment). The selected dosage level can depend on a variety of pharmacokinetic factors, including the activity of the particular composition employed in the present disclosure, the route of administration, the time of administration, the rate of excretion, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0230] The dosage regimen can be adjusted to provide the best desired response (e.g., a therapeutic and / or preventive response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for objects (e.g., objects for immunization or objects for treatment); each unit contains a predetermined amount of active agent calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier. The specifications of the dosage unit form of the present disclosure can be determined by and directly depend on: (a) the unique characteristics of the active agent and the specific therapeutic effect to be achieved, and (b) the inherent limitations of such active agents in the art for the sensitivity of the treatment of individuals. The dosage can be determined by reference to the plasma concentration or local concentration of a cyclic polyribonucleotide or an antibody or an antigen-binding fragment thereof. The dosage can be determined by reference to the plasma concentration or local concentration of a linear polyribonucleotide or an antibody or an antigen-binding fragment thereof.

[0231] The engineered polynucleotides described herein, AAV vectors comprising engineered polynucleotides, AAVs comprising engineered polynucleotides, cells transduced with AAV vectors, viral particles comprising engineered polynucleotides, pharmaceutical compositions, or combinations thereof can be in unit dosage forms suitable for single administration of precise doses. In unit dosage forms, the preparation can be divided into unit doses containing an appropriate amount of the composition. In unit dosage forms, the preparation can be divided into unit doses containing an appropriate amount of one or more linear polyribonucleotides, antibodies or their antigen-binding fragments, and / or therapeutic agents. The unit dose can be in the form of a package containing a discrete amount of the preparation. Non-limiting examples are packaged injections, vials, and ampoules. The aqueous suspension compositions disclosed herein can be packaged in single-dose non-reclosable containers. A multi-dose reclosable container can be used, for example, with or without a preservative combination. The preparations for injection disclosed herein can be presented in unit dosage forms, for example, in ampoules with a preservative or in a multi-dose container.

[0232] In some cases, the increased level of a biologic agent in a subject is at least 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, or 500-fold increased as determined by a diagnostic assay.

[0233] Suitable diagnostic assays may include ophthalmic diagnostic assays. Ophthalmic diagnostic assays may include ophthalmic tests such as refractive testing, eye scans, ocular coherence tomography, Farnworth Munsell 100 chromatic test, computerized optic disc imaging and nerve fiber layer analysis (GDX, HRT, OCT), corneal topography, electroretinogram (ERG), electrooculogram (EOG), visual evoked potential (VEP), visual evoked response (VER), fluorescein angiography, ocular coherence tomography (OCT), retinal photography, fundus photography, corneal endothelial microscopy, Goldmann, Humphrey, FDT, Octopus, biometrics / IOL calculation, A-scan, B-scan, and combinations thereof.

[0234] In some cases, retinal tests can be used. Non-limiting methods for evaluating retinal function and changes therein include evaluating visual acuity (e.g., best corrected visual acuity [BCVA], walking, navigation, object detection and discrimination), evaluating visual fields (e.g., static and dynamic perimetry), performing clinical examinations (e.g., slit lamp examination of the anterior and posterior segments), evaluating electrophysiological responses to light and dark of all wavelengths (e.g., all forms of electroretinograms (ERGs) [full field, multifocal, and pattern], all forms of visual evoked potentials (VEPs), electrooculograms (EOGs), 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 laser confocal scanning ophthalmoscopy (AO-SLO), fluorescence and / or autofluorescence; measuring ocular movements and eye movements (e.g., nystagmus, gaze preference and stability), measuring reported outcomes (patient-reported changes in visually and non-visually guided behaviors and activities, patient-reported outcomes [PROs], questionnaire-based assessments of quality of life, daily activities, and measures of neurological function (e.g., functional magnetic resonance imaging (MRI)).

[0235] In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cells transduced with the AAV vector, the viral particles comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof, is 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 comparable cells not contacted with the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, or the pharmaceutical composition.

[0236] In some embodiments, the methods of treatment described herein can treat ocular diseases.Related ocular diseases and the patient's condition can include but are not limited to: blindness, color blindness, age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma, Budd-Bieder 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-cone dystrophy, cone-rod dystrophy, Okoshi disease, Leventinese disease (familial dominant drusen) and blue cone monochromacy.In embodiments, ocular disease or the patient's condition is AMD.AMD can be wet AMD or dry AMD.

[0237] In some cases, the use of pharmaceutical compositions is sufficient to at least alleviate the symptoms of a disease or condition, treat a disease or condition, and / or eliminate a disease or condition. In some cases, the improvement of a disease or condition can be determined by any diagnostic assay provided. In other cases, the improvement can be obtained via an interview with the treated object. For example, the object may be able to communicate with the attending physician that their vision is improved compared to their vision before the application of the subject drug. In other cases, an in vivo animal model can be used to determine the reduction of a disease or condition after treatment. Suitable animal models include mouse models, primate models, rat models, dog models, etc.

[0238] The use of absolute or sequential terms, such as "shall," "shall not," "should," "shall not," "must not," "must not," "first," "initially," "next," "subsequently," "before," "after," "last," and "ultimately," are not meant to limit the scope of the embodiments disclosed herein, but are intended to be exemplary.

[0239] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising".

[0240] As used herein, the phrases "at least one," "one or more," and "and / or" are open-ended expressions that are both conjunctions and antonyms in operation. For example, each of the expressions "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" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0241] As used herein, "or" may mean "and", "or", or "and / or", and may be used both exclusively and inclusively. For example, the term "A or B" may mean "A or B", "A but not B", "B but not A", and "A and B". In some cases, the context may determine the specific meaning.

[0242] Any systems, methods, software, and platforms described herein are modular. Therefore, terms such as "first" and "second" do not necessarily imply priority, order of importance, or order of action.

[0243] When referring to a number or a numerical range, the term "about" means that the number or numerical range mentioned is an approximate value within experimental variation (or within statistical experimental error), and the number or numerical range may vary, for example, between 1% and 15% of the stated number or numerical range. In the example, the term "about" refers to ±10% of the stated number or value.

[0244] The terms "increased", "increasing" or "increase" are used herein to generally mean an increase in 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 an increase of 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 up to and including a 100% increase or any increase between 10%-100%. Other examples of "increase" include an increase of at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, at least 1000 times, or more times compared to a reference level.

[0245] The term "reduced (decreased)", "reduced (decreasing)" or "reduced (decrease)" is used herein to generally mean a reduction in a statistically significant amount. In some embodiments, the term "reduced (decreased)" or "reduced (decrease)" means a reduction of at least 10% compared to a reference level, such as a reduction of 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% reduction (e.g., a level that is not present or an undetectable level compared to a reference level) or any reduction between 10%-100%. In the context of a marker or symptom, these terms mean that this level is statistically significantly reduced. Reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably drops to a level that is accepted as being within the normal range for individuals without a given disease.

[0246] The terms "AAV," "AAV construct," or "recombinant AAV," or "AAV" refer to any known serotype of adeno-associated virus, 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 a helper virus (such as adenovirus, herpes simplex virus, vaccinia virus, and CMV) to achieve replication. Wild-type AAV is common in the general population and is not associated with any known pathology. Hybrid AAV is an AAV that contains the capsid proteins of one AAV serotype and the genomic material of another AAV serotype. Chimeric AAV comprises genes and / or protein sequences derived from two or more AAV serotypes, and may include mutations to the gene sequences of those two or more AAV serotypes. Exemplary chimeric AAVs may comprise chimeric AAV capsids, for example, capsid proteins having one or more amino acid regions derived from two or more AAV serotypes. AAV variants are AAVs comprising one or more amino acid mutations (for example, comprising one or more amino acid mutations in its capsid protein compared to its parent AAV) in its genome or protein. As used herein, AAVs include avian AAVs, bovine AAVs, canine AAVs, equine AAVs, primate AAVs, non-primate AAVs, and ovine AAVs, wherein primate AAVs refer to AAVs infecting non-primates, and wherein non-primate AAVs refer to AAVs infecting non-primates, such as avian AAVs infecting avian animals. In some cases, wild-type AAV contains rep and cap genes, wherein the rep gene is required for viral replication and the cap gene is required for the synthesis of capsid protein. As used herein, the terms "recombinant AAV" and "rAAV" are interchangeable.

[0247] The term "recombinant AAV vector", or "AAV vector", or "AAV vector" refers to a vector derived from any AAV serotype mentioned above. In some cases, the AAV vector may contain one or more AAV wild-type genes that are completely or partially deleted, such as rep and / or cap genes, but contain functional elements required for packaging and using AAV viruses to achieve gene therapy. For example, it is known that the functional reverse terminal repeat sequence or ITR sequence flanked by the open reading frame or the cloned exogenous sequence is important for the replication and packaging of the AAV virion, but the ITR sequence can be modified from the wild-type nucleotide sequence, including the insertion, deletion or substitution of nucleotides, so that AAV is suitable for 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 bypass the need for viral second-strand DNA synthesis and can result in a higher expression rate of transgenic proteins. In some aspects, AAV vectors can be generated to allow the selection of optimal serotypes, promoters, and transgenes. In some cases, the vector can be a targeted vector or a modified vector that selectively binds or infects immune cells.

[0248] The term "AAV virion" or "AAV virion" refers to a viral particle comprising a capsid comprising at least one AAV capsid protein encapsulating an AAV vector as described herein, wherein in some embodiments the vector may further comprise a heterologous polynucleotide sequence or a transgene. The virion may be an engineered virion.

[0249] The terms "subject", "host", "individual" and "patient" are used interchangeably herein to refer to animals, typically mammals. Any suitable mammal can be administered a composition as described herein (such as an engineered guide RNA) or treated by a method as described herein. The subject can be a vertebrate or an invertebrate. The subject can be a laboratory animal. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal can be of any age or at any stage of development (e.g., an adult, teenager, child, infant, or mammal in utero). The mammal can be male or female. In some embodiments, the subject is a human. The subject can be a patient. The subject can suffer from a disease. The subject can show symptoms of a disease. The subject can not show symptoms of a disease, but still suffer from the disease. A subject may be receiving medical care from a caregiver (eg, a subject is hospitalized and treated by a physician).

[0250] The terms "protein", "peptide" and "polypeptide" are used interchangeably and refer to compounds of two or more subunit amino acids, amino acid analogs or peptide mimetics in their broadest sense. The term also encompasses amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other operation, such as conjugation with a labeling component. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptide mimetics. The subunits may be connected by peptide bonds. In another embodiment, the subunits may be connected by other bonds (e.g., 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 can constitute a protein sequence or a peptide sequence. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and D and L optical isomers, amino acid analogs and peptide mimetics. As used herein, the term "fusion protein" refers to a protein comprising domains from more than one naturally occurring or recombinantly produced protein, wherein typically each domain has a different function. In this regard, the term "linker" refers to a protein fragment used to connect these domains together, optionally to retain the conformation of the fusion protein domain and / or to prevent adverse interactions between the fusion protein domains that can impair their respective functions.

[0251] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, meaning that when aligned, the percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of 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) software package.

[0252] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific embodiments provided in this specification. Although the present invention has been described with reference to the above specific description, the description and explanation of the embodiments herein are not intended to be interpreted in a restrictive sense. Without departing from the present invention, those skilled in the art will now think of various variations, changes and substitutions. In addition, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations or relative proportions described herein, which depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein can be used to practice the present invention. Therefore, it is considered that the present invention should also cover any such alternatives, modifications, variations or equivalents. The following claims are intended to define the scope of the present invention, and thus cover methods and structures and their equivalents within the scope of these claims.

[0253] Implementation

[0254] 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.

[0255] Embodiment 2. An engineered polynucleotide comprising an AAV vector, wherein the AAV vector comprises one or more expression cassettes, wherein the one or more expression cassettes encode an engineered polypeptide, wherein the engineered polypeptide comprises: an antibody or fragment thereof operably coupled to a peptide.

[0256] Embodiment 3. An engineered polynucleotide as described in embodiment 1 or 2, wherein the AAV vector comprises an AAV serotype, and the AAV serotype comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or any combination thereof.

[0257] Embodiment 4. The engineered polynucleotide of embodiment 3, wherein the AAV serotype comprises AAV2.

[0258] Embodiment 5. The engineered polynucleotide of any one of embodiments 1-4, wherein the peptide comprises a CNP.

[0259] Embodiment 6. The engineered polynucleotide of embodiment 5, wherein the CNP comprises at least 22 amino acid residues.

[0260] Embodiment 7. The engineered polynucleotide of embodiment 5, wherein the CNP comprises at least 36 amino acid residues.

[0261] Embodiment 8. The engineered polynucleotide of embodiment 5, wherein the CNP comprises at least 53 amino acid residues.

[0262] 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.

[0263] Embodiment 10. The engineered polynucleotide of embodiment 2, wherein the peptide is covalently linked to the N-terminus of the antibody or fragment thereof.

[0264] Embodiment 11. The engineered polynucleotide of embodiment 2, wherein the peptide is covalently linked to the C-terminus of the antibody or fragment thereof.

[0265] Embodiment 12. The engineered polynucleotide of embodiment 2, wherein the peptide is operably coupled to the antibody or fragment thereof via a peptide linker.

[0266] Embodiment 13. The engineered polynucleotide of embodiment 12, wherein the peptide linker comprises an amino acid sequence comprising (GGGGS) n , where n It is an integer between 0 and 10.

[0267] Embodiment 14. The engineered polynucleotide of any one of embodiments 1-4, wherein the AAV vector encodes an engineered AAV capsid.

[0268] Embodiment 15. An engineered polypeptide comprising an antibody or fragment thereof operably coupled 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.

[0269] Embodiment 16. An engineered polypeptide as described in Embodiment 15, wherein the peptide comprises CNP.

[0270] Embodiment 17. The engineered polypeptide of embodiment 16, wherein the CNP comprises at least 22 amino acid residues.

[0271] Embodiment 18. The engineered polypeptide of embodiment 16, wherein the CNP comprises at least 36 amino acid residues.

[0272] Embodiment 19. The engineered polypeptide of embodiment 16, wherein the CNP comprises at least 53 amino acid residues.

[0273] 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.

[0274] Embodiment 21. An engineered polypeptide as described in any of Embodiments 15-20, wherein the peptide is covalently linked to the N-terminus of the antibody or fragment thereof.

[0275] Embodiment 22. An engineered polypeptide as described in any of embodiments 15-20, wherein the peptide is covalently linked to the C-terminus of the antibody or fragment thereof.

[0276] Embodiment 23. An engineered polypeptide as described in any of embodiments 15-22, wherein the peptide is operably coupled to the antibody or fragment thereof via a peptide linker.

[0277] Embodiment 24. An engineered polypeptide as described in embodiment 23, wherein the peptide linker comprises an amino acid sequence comprising (GGGGS) n , where n It is an integer between 0 and 10.

[0278] Embodiment 25. An engineered polynucleotide encoding an engineered polypeptide as described in any one of embodiments 15-24.

[0279] Embodiment 26. The engineered polynucleotide of embodiment 25, wherein the engineered polynucleotide is a vector.

[0280] Embodiment 27. The engineered polynucleotide of embodiment 26, wherein the vector is a viral vector.

[0281] Embodiment 28. The engineered polynucleotide of embodiment 27, wherein the viral vector comprises an AAV vector.

[0282] Embodiment 29. An engineered polynucleotide as described in Embodiment 28, wherein the AAV vector comprises an AAV serotype, and the AAV serotype comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or any combination thereof.

[0283] Embodiment 30. The engineered polynucleotide of embodiment 29, wherein the AAV serotype comprises AAV2.

[0284] Embodiment 31. An engineered polynucleotide as described in any of embodiments 28-30, wherein the AAV vector encodes an engineered AAV capsid.

[0285] Embodiment 32. An engineered polynucleotide as described in any of embodiments 27-31, wherein the viral vector comprises one or more expression cassettes.

[0286] Embodiment 33. An engineered polynucleotide as described in embodiment 2 or embodiment 32, wherein the one or more expression cassettes encode a continuous polypeptide, wherein the continuous polypeptide comprises an engineered polypeptide as described in any one of embodiments 2-33.

[0287] Embodiment 34. An engineered polynucleotide as described in embodiment 33, wherein the continuous polypeptide comprises a protease cleavable sequence.

[0288] Embodiment 35. The engineered polynucleotide of embodiment 33, wherein the contiguous polypeptide comprises a furin cleavable sequence.

[0289] Embodiment 36. An engineered polynucleotide as described in embodiment 33, wherein the continuous polypeptide comprises a self-cleaving polypeptide sequence.

[0290] 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.

[0291] Embodiment 38. The engineered polynucleotide of embodiment 37, wherein the at least one additional therapeutic agent comprises a hormone.

[0292] 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).

[0293] 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.

[0294] Embodiment 41. The engineered polynucleotide of Embodiment 37, wherein the at least one additional therapeutic agent comprises a VEGF inhibitor.

[0295] 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.

[0296] Embodiment 43. The engineered polynucleotide of embodiment 41 or embodiment 42, wherein the VEGF inhibitor comprises an antibody.

[0297] Embodiment 44. An engineered polynucleotide as described in Embodiment 43, wherein the VEGF inhibitor comprises a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single-chain variable fragment (scFv), bis-scFv, (scFv)2, a double-chain antibody, a minibody, a nanobody, a three-chain antibody, a four-chain antibody, a disulfide-stabilized Fv protein ("dsFv"), a single domain antibody (sdAb), an Ig NAR, a camel antibody, or a combination thereof, a binding fragment thereof, or a chemically modified derivative thereof.

[0298] Embodiment 45. The engineered polynucleotide of embodiment 41 or embodiment 42, wherein the VEGF inhibitor comprises a non-antibody VEGF inhibitor.

[0299] Embodiment 46. An engineered polynucleotide as described in 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.

[0300] Embodiment 47. An engineered polynucleotide as described in Embodiment 45, wherein the non-antibody VEGF inhibitor comprises soluble VEGFR1, soluble VEGFR2, soluble VEGFR3, a soluble fragment thereof, or a combination thereof.

[0301] Embodiment 48. An engineered polynucleotide as described in Embodiment 45, wherein the non-antibody VEGF inhibitor comprises a VEGF capture agent or a modified version thereof.

[0302] Embodiment 49. A cell comprising the engineered polynucleotide of any one of embodiments 1-14 or 25-48.

[0303] Embodiment 50. A cell comprising the engineered polypeptide of any one of embodiments 15-24.

[0304] Embodiment 51. A pharmaceutical composition comprising an engineered polynucleotide as described in any one of embodiments 1-14 or 25-48, an engineered polypeptide as described in any one of embodiments 15-24, or a cell as described in embodiment 49 or embodiment 50.

[0305] Embodiment 52. The pharmaceutical composition of embodiment 51, wherein the pharmaceutical composition is formulated for administration to a subject in need thereof intrathecally, intraocularly, intravitreally, retinaly, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymalally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonary, intratracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhalation of an aerosolized form, by an intraluminal-GI route, or a combination thereof.

[0306] Embodiment 53. The pharmaceutical composition of embodiment 52, wherein the pharmaceutical composition is formulated for intravitreal, subretinal, or suprachoroidal administration.

[0307] Embodiment 54. The pharmaceutical composition of embodiment 52, wherein the pharmaceutical composition is used to treat an eye disease or condition.

[0308] Embodiment 55. The pharmaceutical composition of embodiment 52, wherein 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 thereof.

[0309] Embodiment 56. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide as described in any one of embodiments 1-14 or 25-48, an engineered polypeptide as described in any one of embodiments 15-24, a cell as described in embodiment 47 or embodiment 48, or a pharmaceutical composition as described in embodiments 49-52.

[0310] Embodiment 57. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide as described in any one of embodiments 1-13 or 24-46, an engineered polypeptide as described in any one of embodiments 14-23, a cell as described in embodiment 49 or embodiment 50, or a pharmaceutical composition as described in embodiments 49-52, wherein one administration cures the disease or condition.

[0311] Embodiment 58. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide as described in any of embodiments 1-13 or 24-46, an engineered polypeptide as described in any of embodiments 14-23, a cell as described in embodiment 47 or embodiment 48, or a pharmaceutical composition as described in embodiments 51-55, wherein the administration does not comprise daily administration.

[0312] Embodiment 59. The method of any one of Embodiments 56-58, wherein the disease or condition comprises an eye disease.

[0313] Embodiment 60. A method as described in embodiment 59, wherein the eye disease comprises ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color blindness, 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-Bieder syndrome, Best disease, choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, O'Sullivan's disease, Leventinese disease (familial dominant drusen), blue cone monochromacy or a combination thereof.

[0314] Example

[0315] The following illustrative examples represent embodiments of the stimulation, systems, and methods described herein and are not meant to be limiting in any way.

[0316] Example 1. Design and Experiments for Expression of CNP and CNP Fusion Proteins

[0317] A series of CNP and CNP fusion proteins (CNP with human antibody heavy chain secretion signal peptide at the 5' end, IgG 1 Fc fragment or IgG 4 The Fc fragment was fused at the N-terminus or C-terminus of CNP, as illustrated in FIG3 ). The designed CNP22 or CNP36 and CNP fusion proteins were reverse translated into DNA sequences with Homo sapiens codon output. The CNP and CNP fusion protein DNA sequences were further manually modified to adjust the GC content for synthesis into three overlapping DNA fragments to generate the AAV constructs described herein (e.g., any of the AAV constructs in FIGS. 1-2 ).

[0318] For example, to create AMI061-pFB-scCMV-Vh-leader-CNP36-Fc, AMI059 was first cut with StuI and SphI to remove the aflibercept coding sequence and the poly A signal. The codon-optimized Vh-leader-CNP36-Fc fragment was then PCR amplified using primers A056, A057, A025, and AMI014 as templates. Finally, the PCR fragments were assembled into the StuI and SphI sites of AMI059 to create AMI061 using the NEBuilder High-Fidelity DNA Assembly Kit. To create AMI087-pFB-scCMV-Vh-leader-CNP36-4xGGGGS-Fc-WPREmini, AMI061 was first cut with SnaBI and SphI to remove part of the CMV promoter and the CNP36-4xGGGS-Fc fragment. Then use primer A051 and A166 and AMI061 as template amplification partial CMV promoter and CNP36-4xGGGGS fragment.Use primer A167 and A025 and AMI061 as template amplification Fc fragment.These two fragments are joined together by PCR with primer A051 and A025, and are assembled into SnaBI and SphI sites of AMI061 with NEBuilder high-fidelity DNA assembly kit to create AMI087.In order to create AMI088-pFB-scCMV-Vh-leader-Fc-4xGGGGS-CNP36-WPREmini, first cut AMI060 AMI059 with AflII and XhoI to remove CNP-VGGRK-Fc fragment. Then use primer A180 and A181 to amplify Fc fragment, and use primer A182 and A183 and AMI060 as template amplification 4xGGGGS-CNP36 fragment.These two PCR fragments are joined together with primer A180 and A183, and finally are assembled into AflII and XhoI sites of AMI060 with NEBuilder high-fidelity DNA assembly kit to create AMI088.In order to create AMI182-pFB-scCMV-Vh-leader-CNP36-2xSTOP-4xGGGGS-Fc-WPREmini, first use primer A618 and A619 and AMI087 as template PCR amplification 4xGGGGS-Fc fragment.Then use NEBuilder high-fidelity DNA assembly kit, PCR fragment is assembled into XcmI and EcoNI sites of AMI087 to create AMI182. To create 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 sequence.CNP and a partial Fc fragment with three incorporated stop codons were then amplified using primers A620, A621, A622 and A619 and AMI087 as templates. Finally, CNP and a partial Fc fragment with three stop codons were assembled into the StuI and EcoNI sites of AMI087 using the NEBuilder High-Fidelity DNA Assembly Kit to create AMI183. The identity of the AAV constructs was confirmed by PCR amplification and sequencing analysis using the primers listed in Table 6.

[0319] Table 6. DNA primers used for PCR amplification and DNA sequencing analysis

[0320]

[0321]

[0322] Generation of recombinant baculovirus for AAV vector production

[0323] Recombinant baculovirus (rBV) was generated using the Bac-to-Bac baculovirus expression system according to the manufacturer's instructions. Briefly, pFB shuttle plasmids containing the target gene 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 bacmid DNA molecules with cathepsin gene deletions and incubated on ice for 30 min, followed by heat shock at 42°C for 30 seconds. After incubation on ice for 2 minutes, the bacteria were cultured at 37°C for 4 hours to recover, 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 incubation at 37°C for 48 hours, 2 white colonies containing recombinant bacmid DNA were picked, and the small-scale 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 diluted separately in 100 μL of ESFAF medium and then mixed together for about 30 min to form a transfection mixture. Sf9 cells were plated in 6-well plates at 1.5e+6 cells / well in 2 mL of ESFAF medium for about 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 incubation overnight at 28°C, an additional 1 mL of ESFAF medium was added to each well. After a total incubation time of 4 days, the medium containing the rBV was collected and amplified at a 1:200 ratio to generate sufficient rBV to be ready for use in the AAV production process.

[0324] AAV production and purification

[0325] rBV carrying AAV2 Rep and mutant capsid genes and target expression cassettes were used to co-infect Sf9-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 lines at a density of approximately 5e+6 cells / mL in a shaker incubator at 28°C with an oscillation speed of 180 revolutions per minute (rpm) for 3 days with 50% fresh ESFAF medium. At the end of infection, cell pellets were collected by centrifugation at 3,000 rpm for 10 min. Cells were cultured in a 50 mM Tris-HCl, pH 8.0, 2 mM MgCl 2 , 1% sodium lauryl sarcosine, 1% TritonX-100 and 125 units / mL Benzonase in Sf9 lysis buffer and vortex vigorously, then lyse at 37°C with 350rpm for 1 hour. At the end of the oscillation, the salt concentration is increased to 500mM by vortexing, and the lysate is clarified by centrifugation at 8,000rpm for 20 minutes at 4°C. The clarified lysate is transferred to an ultra-clean centrifuge tube of a SW28 swing bucket rotor, containing 5mL of 1.50g / cc and 10mL of 1.30g / cc cesium chloride solution. After centrifugation at 28,000rpm for about 18 hours at 15°C, the AAV band is collected with a syringe and transferred to an ultra-clean centrifuge tube of a 70ti centrifuge rotor. The centrifuge tube is filled with 1.38g / cc cesium chloride solution and heat-sealed. The AAV samples were subjected to a second round of ultracentrifugation at 65,000 rpm at 15°C for about 18 hours, and the AAV band was collected with a syringe. The purified AAV sample buffer was exchanged into PBS buffer containing 0.001% Pluronic F-68 and sterilized by filtration with a 0.22 μm syringe filter. The sterilized AAV samples were stored at 4°C for one month and then transferred to -80°C for long-term storage. AAV titer was determined by real-time PCR method.

[0326] Transient expression of constructs in mammalian cell culture systems

[0327] at 37°C in CO 2 Human HEK293 cells were cultured in DMEM medium with 10% FBS in an incubator. For maintenance passages, cells were split 1:10 twice a week. For transfection, cells were plated at 2 x 10 610 cells / dish were seeded on a 10 cm cell culture dish overnight. 14 μg of plasmid DNA and 22 μL of Lipofectamine 3000 were each diluted in 0.5 mL of Opti-medium and mixed together. After incubation at room temperature for 5 minutes, the mixture was added dropwise to the cells and incubated at 37°C in CO 2 Incubate in an incubator for 48 hours and harvest the culture medium for further experiments.

[0328] One day before transient transfection, HEK293 cells were plated at 2 x 10 6 The density of 100 μg / dish was inoculated on a 10 cm tissue culture dish. Each transfection of CNP22 or CNP36 or CNP fusion variant plasmid was carried out using 14 μg / dish DNA with Lipofectamine 3000 reagent following the manufacturer's protocol. Cell culture supernatant was collected and protein expression was analyzed by western blot 48 hours after transfection. All transfections were performed in triplicate in at least three independent experiments.

[0329] CNP fusion (CNP-Fc or CNP-Fc) variant protein is determined by SDS-PAGE and western blot analysis. HEK293 cell culture medium (supernatant) is collected 48 hours or 72 hours after vector transduction. The total volume is that 30 μ L of cell supernatant is mixed with 10ul4x loading buffer and loaded on NuPAGE 10% Tris-glycine gel for electrophoresis. The protein is subsequently transferred to a PVDF membrane. The membrane is treated with casein blocker in PBS for at least one hour at room temperature and stained with goat anti-human IgG. 1 Fc antibody biotin conjugate probing was followed by incubation with streptavidin conjugated to horseradish peroxidase.

[0330] Purification of CNP and CNP fusion proteins

[0331] All functional protein sequences are converted into DNA sequence and cloned into plasmid for expression, and cloned into baculovirus vector for recombinant AAV packaging. Amplify expressible plasmid and prepare DNA preparation, and for transient transfection with HEK293 cells. Cell culture harvest is used for purification of CNP36-Fc protein by protein A affinity column chromatography. These fusion proteins are purified to homogeneity, and purity is characterized by SDS-PAGE.

[0332] By protein A affinity column chromatography, expressed CNP, CNP-Fc or CNP-Fc protein are purified from HEK293 cell culture harvest. The serum-free medium harvested is filtered with 0.2 μm filter to remove particulate and loaded onto protein A column (1-mL size) with a flow rate of 1.5~2.0 mL / min. Post is washed with washing 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). Neutralized protein buffer is exchanged to 1xPBS, and sterilized by filtration with 0.2 μm syringe filter pre-moistened with PBS, and stored at -80 °C. A. When the pH reaches 3-4.0, the column chromatography shows a sharp peak of CNP fusion protein outside the column ( Fig. 6A Similar observations were made for FP-CNP expression based on chromatogram and SDS-PAGE analysis ( Figure 6B The protein concentration of each preparation was determined by BCA protein assay ( Figure 5 Table 7 shows the correct size of CNP-Fc or CNP-Fc expressed by transduced HEK293 cells. The N-terminal sequence of CNP-Fc protein (AMI088) was determined by Edman degradation ( Fig. 6A The purified protein preparations were used for in vitro biological functional assays. Protein AMI263 was also used for optic nerve injury (ONC) to evaluate its role in protecting retinal ganglion cells (RGC) from damage (Table 8).

[0333] Table 7. Summary of CNP-Fc protein purified from HEK293 cell culture supernatant

[0334]

[0335] Fc1=IgG1 Fc;Fc4=IgG4 Fc

[0336] Table 8. Usage of CNP-Fc protein

[0337]

[0338] The biological function of these purified proteins in stimulating cGMP production was determined using NPR-B receptor positive cell line, NIH / 3T3, and NPR-B negative cell line HEK293 cells. cGMP was determined by ELISA using commercial CNP as standard curve. Fig. 7A and Fig. 8A- B (Time Course) illustrates the simulation of cGMP production by CNP-Fc36. Fig. 7A cGMP release resulting from the binding of the CNP fusions described herein to NPR-B is demonstrated. Figure 7B The kinetic affinity binding between the CNP fusion described herein and the NPR-B protein as measured by the BiaCore assay is illustrated. At 25 ° C, the kinetic affinity binding analysis was performed in HBS running buffer (20mM NaH2PO4-Na2HPO4·H2O, 150mM NaCl, pH 7.4) supplemented with 0.005% (v / v) surfactant P20, using a Biacore 3000 optical biosensor equipped with a research-grade CM5 sensor chip. Surface preparation. NBR-B is fixed at different levels in three flow cells via standard amine coupling, leaving unmodified Fc 1 to serve as a reference. All surface plasmon resonance (SPR) analyses were performed on a BIAcore 3000 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 on the reference flow cell and blank sample. Human NPR-B (R&D system) was covalently attached to the CM5 chip via amine coupling. A surface density of 6600 RU was used for measurements with natriuretic peptides, and sequential injections of CNPs (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 binding sites was observed, and the surface was regenerated by 2 injections of 0.5 M NaCl (60 s each). Kinetic parameters were calculated assuming simple 1:1 (Langmuir) binding. Figure 7C Single cycle kinetic (SCK) assays of binding between CNP and NPR-B are demonstrated (KD = 36.3 Pm; ka = 1.31e7; and kd = 4.74E-4). Fig.7DBiacore assays of AMI263 (Fc4-CNP36) against NPR-B are described (KD = 0.36 pM; ka = 1.38E6; and kd = 4.92E-6). All SPR analyses were performed on a BIAcore 3000 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 on the reference flow cell and blank samples. Human NPR-B (R&D system) was covalently attached to the CM5 chip via amine coupling. A surface density of 3100 RU was used for measurement with natriuretic peptides, and sequential injections of AMI263 (FC4-CNP) (0.125–2 nM) were performed at a flow rate of 30 μl / min (300 s each), followed by a dissociation time of 900 s. Binding site saturation was observed. Kinetic parameters were calculated assuming simple 1:1 (Langmuir) binding. Fig. 7E Biacore assays of AMI088 for NPR-B are described (KD = 34.1 pM; ka = 1.37E7; and kb = 4.47E-4). All SPR analyses were performed on a BIAcore 3000 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 on the reference flow cell and blank samples. Human NPR-B (R&D system) was covalently attached to the CM5 chip via amine coupling. The surface density of 3100 RU was used for measurement with natriuretic peptides, and sequential injections of AMI088 (FC1-CNP) (0.31–5 nM) were performed at a flow rate of 30 μl / min (300 s each), followed by a dissociation time of 900 s. Binding site saturation was observed. Kinetic parameters were calculated assuming simple 1:1 (Langmuir) binding. Figure 7F Biacore assay of ANP against NPR-B is described (KD=52.3nM; ka=2.36E3; and kd=1.24E-4). All SPR analyses were performed on a BIAcore 3000 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 on the reference flow cell and blank samples. Human NPR-B (R&D system) was covalently attached to the CM5 chip via amine coupling. The surface density of 3100RU was used for measurement with natriuretic peptides, and sequential injections of AMI263 (FC4-CNP) (0.5–300nM) were performed at a flow rate of 30μl / min (300s each), followed by a dissociation time of 900s. Binding site saturation was observed. Kinetic parameters were calculated assuming simple 1:1 (Langmuir) binding. Figure 7GBiacore assays of BNP against NPR-B are described (KD = 664pM; ka = 9.4E5; and kb = 6.24E-4). All SPR analyses were performed on a BIAcore 3000 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 on the reference flow cell and blank samples. Human NPR-B (R&D system) was covalently attached to the CM5 chip via amine coupling. The surface density of 3100RU was used for measurement with natriuretic peptides, and sequential injections of AMI263 (FC4-CNP) (0.5–300nM) were performed at a flow rate of 30μl / min (300s each), followed by a dissociation time of 900s. Binding site saturation was observed. Kinetic parameters were calculated assuming simple 1:1 (Langmuir) binding. Figure 7H Biacore assays of AMI263 for NPR-B are described (KD = 13.1 nM; ka = 1.14E4; and kd = 1.49E-3). All SPR analyses were performed on a BIAcore 3000 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 on the reference flow cell and blank sample. Human NPR-B (R&D system) was covalently attached to the CM5 chip via amine coupling. The surface density of 3100RU was used for measurement with natriuretic peptides. Sequential injections of AMI263 (FC4-CNP) (9.37-150 nM) were performed at a flow rate of 30 μl / min (300 s each), followed by a dissociation time of 900 s. Binding site saturation was observed. Kinetic parameters were calculated assuming simple 1:1 (Langmuir) binding. Fig.7IBiacore assays of AMI087 for NPR-B are described (KD = 41.9 nM; ka = 3.65E3; and kd = 1.53E-3). All SPR analyses were performed on a BIAcore 3000 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 on the reference flow cell and blank sample. Human NPR-B (R&D system) was covalently attached to the CM5 chip via amine coupling. The surface density of 3100RU was used for measurement with natriuretic peptides. Sequential injections of AMI087 (FC4-CNP) (9.37-150 nM) were performed at a flow rate of 30 μl / min (300 s each), followed by a dissociation time of 900 s. Binding site saturation was observed. Kinetic parameters were calculated assuming simple 1:1 (Langmuir) binding. Table 32 illustrates a summary of the affinity between NPR-B and the CNPs or CNP fusions described herein. Table 33 illustrates the corrected concentrations of proteins used for the Biacore assays.

[0339] Table 32. Summary of the affinity between NPR-B and CNP or CNP fusions

[0340]

[0341] Table 33. Corrected concentrations of proteins used for Biacore assays

[0342]

[0343] Stimulation of cyclic guanosyl monophosphate (cGM) production by CNP

[0344] The biological function of the purified CNP fusion protein was evaluated in a cell-based assay using a natriuretic peptide C-type receptor B (NPR-B) positive cell line, NIH / 3T3 and HEK293 cells negative cell lines. The test was also performed in the presence of natriuretic peptide A (ANP) and CNP22 controls ( Fig. 7A -B and Fig. 8A -B).

[0345] Production and delivery of adeno-associated viral (AAV) vectors

[0346] Several AAV2.N54-CNP and AAV2.N54-CNP-FC constructs were generated for delivery of different forms of CNP22, CNP36 or CNP fusions (CNP fused to the N-terminus or C-terminus of the Fc fragment). AAV2.N54-CNP and AAV2.N54-CNP-FC constructs are capable of transducing airway epithelial cells via sinus, nasal and / or lung delivery methods. Other serotypes of AAV may also be used, depending on the target tissue or cells to be delivered. For example, AAV6 has a tendency to preferentially transduce lung cells.

[0347] Sf9-derived insect cell line V432A cells were cultured in ESF AF medium supplemented with 100 units / ml penicillin and 100 μg / ml streptomycin at 28°C in stock flasks. Once the cell density reached 7 x 10 6 Cells were split 1:4 for maintenance at 1:10 cells / ml. Recombinant baculovirus (rBV) was generated according to the manufacturer's protocol. Briefly, the constructs were used to transduce DH10Bac, and the recombinant bacmid DNA was isolated. The bacmid DNA was transduced into V432A cells to generate rBV. 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 CNP and CNP fusion proteins used in the experiments of Examples 1-3. Table 11 lists the nucleic acid sequences of AAV constructs encoding CNP and CNP fusion proteins used in the experiments of Examples 1-3.

[0348] The purity of AAV vectors was determined by SimplyBlue staining assay. Briefly, 26 μl of AAV sample was mixed with 10 μL of 4x loading buffer plus 4 μL of 10x reducing agent and incubated at 95°C for 2 min. Approximately 1E+11 vg of each AAV sample was loaded onto each lane of a 10% SDS-PAGE gel and run at 100 volts until the dye reached the bottom of the gel. The gel was stained according to the manufacturer's protocol. The SDS-PAGE gel profile was obtained with the expected levels of VP1, VP2, and VP3 components ( Fig.15 ).

[0349] Suspension HEK293 cell cultures were used to further evaluate the production of protein for each of the AAV2.N54-CNP or AAV2.N54-CNP-FC constructs listed in Table 2. V432A cells were cultured to 7 x 10 6Cells / ml and diluted 1:1 with fresh ESF AF medium. About 200 viruses per cell of rBV containing the specified rep-cap gene and about 100 viruses per cell of rBV containing the DNA sequence encoding CNP-Fc or CNP-Fc protein were added separately to infect V432A cells at 28°C in a shaking incubator for 3 days. The infected V432A cells were harvested by centrifugation at 3,000 rpm for 10 minutes. The cell pellet was lysed in SF9 lysis buffer (50mM Tris-HCl, pH 7.8, 50mM NaCl, 2mM MgCl2, 1% sodium lauryl sarcosyl, 1% Triton X-100 and 140 units / ml ) was dissolved. Genomic DNA was digested by incubation at 37°C for one hour. At the end of the incubation, sodium chloride was added to adjust the salt concentration of the lysate to about 1M to further dissociate the AAV vector from the cell matrix. Cell debris was removed by centrifugation at 8,000rpm for 30 minutes. The clarified lysate was loaded onto a CsCl step gradient and ultracentrifuged at 28,000rpm for 20 hours in a swing-out rotor. The virus band was extracted through a syringe with an 18-gauge needle and loaded onto a second CsCl gradient, and linear ultracentrifuged at 65,000rpm for 20 hours. Then, the virus band was extracted and passed through two PD-10 desalting columns to remove CsCl and detergent, and simultaneously exchanged to buffer B (1xPBS, 0.1M sodium citrate and 0.001% Pluronic F-68). Quantitative real-time PCR (qPCR) was performed with ITR primers and probes to determine the AAV vector genome copy number (Table 12).

[0350] Table 9. Summary of AAV constructs encoding various forms of CNP and CNP fusion proteins

[0351]

[0352]

[0353] sc: self-complementary, ss: single-stranded; a IgG1 Fc, b IgG4 Fc

[0354] Table 10. Amino acid sequences of CNP and CNP fusion proteins

[0355]

[0356]

[0357]

[0358]

[0359] Table 11. Nucleic acid sequences of AAV constructs

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421] Table 12. AAV vector yields determined by ITR-qPCR

[0422]

[0423]

[0424] Example 2. Development and standardization of CNP-Fc ELISA

[0425] C-type natriuretic peptide (CNP) has two isoforms, CNP-22 and CNP-53. Example 2 illustrates that CNP-36 fused to Fc increases the half-life of CNP and also facilitates 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 It is a picture depiction of three carriers. Figure 1B -F illustrates other vectors that can encode CNP or CNP fusion protein. Develop enzyme-linked immunosorbent assay (ELISA) for quantitative CNP-Fc fusion protein in cell culture supernatant. ELISA is used to measure to quantify CNP-Fc expressed in HEK293 cells, human chondrocyte (HCH) cells and human skeletal muscle cells (hSkMC) when transduced with one of three AAV constructs described herein. Table 13 illustrates the materials and instruments for performing ELISA described herein.

[0426] Table 13. Materials and instruments used to perform ELISA to quantify CNP and CNP-Fc proteins

[0427]

[0428]

[0429] Buffers and substrates. Coating buffer: 3.7 g sodium bicarbonate, 0.64 g sodium carbonate in 1 L Milli Q water, pH 9.6. Store at room temperature (RT); Blocking buffer: Commercial casein blocking buffer in PBS + 0.1% Tween 20 added. Store at 4°C; Standard diluent: Same as blocking buffer; Wash buffer: 1X PBS with 0.1% Tween 20 (expired 30 days from date of manufacture); Coating antibody (Ab): Human CNP antibody (monoclonal), coating concentration 2 μg / mL; Detection Ab: Goat pAb anti-human Fc. Dilute to 1:20,000 final dilution in wells. Store at 4°C; HRP: 1 mg / mL Streptavidin-HRP stock solution, diluted to 1:10,000. Store at 4°C; Substrate: TMB. Store at 4°C; and Stop solution: 2N HCl. Store at ambient temperature.

[0430] program.

[0431] 1. Coat a 96-well plate with 2 μg / mL anti-CNP antibody at a final concentration per well in 50 μL of coating buffer and cover the plate with a sealing lid. Place the plate at 4°C overnight.

[0432] 2. The next day, remove the plate, wash three times with wash buffer, and tap the plate on a paper towel to remove excess solution.

[0433] 3. Use a multichannel pipette to add 300 μL of blocking buffer to each well. Place the plate with the sealed lid in a 37°C incubator for 2 hours.

[0434] 4. After incubation, discard the blocking buffer and tap on a paper towel to remove excess buffer.

[0435] 5. Prepare standard and sample dilutions in blocking buffer and add 50 μL to each well according to the experimental protocol.

[0436] 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 must all be 50 μL in addition to the coating and wash buffers (300 μL).

[0437] 6. Cover the plate with a sealing lid and place it back in the incubator for 1 hour.

[0438] 7. After 1 hour, discard the solution and wash the plate 6 times with 300 μL of wash buffer. Remove excess solution by tapping on paper towels as described above.

[0439] 8. Add detection antibody at a 1:20,000 dilution in blocking buffer and incubate at 37°C for 1 hour.

[0440] 9. Discard the solution and repeat the washing procedure as described in step 7.

[0441] 10. Add Streptavidin-HRP at a 1:10,000 dilution in blocking buffer for 45 min at 37°C.

[0442] 11. Discard the solution and wash the plate as described in step 7.

[0443] 12. Add 50 μL of TMB and avoid placing the plate under direct light for 15-20 min (it can be < 15 min) or until you start to see saturation in the highest concentration wells.

[0444] 13. Add 50 μL of stop solution and read the plate at 450 nm with 600 as reference within 15 min.

[0445] ELISA development.

[0446] 1. Coating / capture antibody titration: Different concentrations of anti-CNP antibody were coated onto wells (5, 2, 1 and 0.5 μg / mL) and plated onto 96-well plates. AMI061, AMI087 and AMI088 (CNP constructs) were also titrated from 0-200 ng / mL by serial dilution. The detection Ab was diluted to 1:4000 and the streptavidin HRP was diluted to 1:10,000.

[0447] 2. Detection antibody titration: The results from the above experiments show that 2 μg / mL coated Ab concentration is optimal for the assay. However, all constructs showed a steady level at 100 ng / mL and 200 ng / mL concentrations. Therefore, along with the detection antibody, the standard concentration was reduced from 200 ng / mL to 50 ng / mL. Now, the standard was serially diluted from 50 ng / mL to 0.8 ng / mL.

[0448] result.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 the designated wells. The scheme of addition has been depicted in Table 14. Wells with numbers show the concentration of CNP construct added to the well in ng / mL. Figure 4A Titration of the coated Ab for each construct is illustrated. The obtained standard curve was fitted on GraphPad Prism software using a hyperbola fit. At coating concentrations of 0.5 and 1 μg / mL concentrations, the absorbance was less than 1 O.D. The 5 μg / mL coating concentration showed saturation, and 2 μg / mL showed an OD greater than 1. Therefore, 2 μg / mL may be an appropriate concentration for the standard curve. The detection Ab was titrated to achieve a better fit of the standard curve. Titration of the detection antibody: At a 1:4000 dilution of the detection Ab, the curve fit was not optimal. This may lead to inaccurate sample estimates in future experiments. Therefore, the detection Ab was diluted to 1:5000, 1:10,000, 1:20,000, and 1:40,000. The coating Ab concentration was 2 μg / mL, the titration of CNP constructs was reduced from 200 ng / mL to 50 ng / mL and serially diluted to give concentrations of 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, 1.56 ng / mL, 0.76 ng / mL along with a blank, which was the assay diluent. As used in previous experiments, streptavidin-HRP was diluted 1:10,000. Figure 4B The standard curves of all three CNP constructs fitted with a hyperbola are shown. Compared with other dilutions, 1:20,000 detection Ab titration gives a good fit. Anti-CNP-Fc ELISA for quantitative expression of CNP in HEK293 cell supernatant: 1E+06 HEK293 cells are plated on 6-well plates. Cells are transduced with 100,000 MOI of AVV2 (N54-AMI061, N54-AMI087 and N54-AMI088) constructs carrying CNP fused to Fc. Constructs are designed and GC content is optimized, which contributes to better expression. qPCR is used to determine the viral vector titer after purification. Titer and other parameters are shown in Table 15. On day 5 after transduction, cell supernatants were collected and CNP-Fc ELISA was performed at different sample dilutions (no dilution, 1:5, 1:10, 1:50, 1:100, 1:200, 1:500, 1:1000, 1:2000 and 1:4000 dilutions). The experiment was performed in duplicate. The obtained standard curve ( Figure 4C). The intra-assay precision of CNP-Fc ELISA has been calculated and is shown in Table 16. The percentage coefficient of variance (CV) values ​​for all standards are less than 10%. Indicating good precision of the assay. No diluted samples showed saturated signals. For AMI061, dilutions of 1:500, 1:1000 and 1:2000 showed readings within the detectable range. For AMI087 and AMI088, the OD values ​​were within the detectable range of 1:4000 dilution. In addition, when compared with other CNP constructs, AMI088 showed the highest expression. These results are summarized in Table 17. Cells not transduced with CNP constructs showed very little expression, which was 6.2ng / mL, 2.5ng / mL and 6.4ng / mL for AMI061, AMI087 and AMI088, respectively.

[0449] Table 14. Schematic representation of the addition of coated Ab and CNP constructs in a 96-well plate

[0450]

[0451] Table 15. CNP construct titers calculated using qPCR

[0452]

[0453] Table 16. Intra-assay precision of CNP-Fc ELISA for AMI061, AMI087 and AMI088

[0454]

[0455] Table 17. Expression of AMI061, AMI087 and AMI088 in HEK293 cells

[0456] CNP-Fc construct Concentration (μg / mL) Standard Deviation AMI061 6.5 0.3 AMI087 33 4.5 AMI088 75 9.2

[0457] The standard curve of CNP-Fc ELISA is hyperbolic and needs to be fitted using hyperbolic fitting. The ELISA method described herein can be used for quantitative cell culture supernatant, preclinical and clinical samples in CNP-Fc. A brief overview of the conditions for standardizing this ELISA includes: using 2 μg / mL anti-CNP antibody coating concentration; CNP-Fc purified protein is serially diluted from 50ng / mL to 0.78ng / mL; and the detection Ab is diluted to 1:20,000 and the streptavidin-HRP is diluted to 1:10,000.

[0458] Example 3. Efficacy of evaluating retinal degeneration in the NMDA excitotoxicity mouse model

[0459] This study was used to evaluate the effect of adeno-associated virus (AAV) vectors on retinal ganglion cell protection following injury caused by N-methyl-D-aspartate (NMDA) administration in mice. Injection volumes and treatment options can be found in Table 18. Groups 7 and 8 were added to test higher concentrations of MK-801. Tissues for bioanalysis were whole-frozen rather than homogenized. A method for adding IBMX to whole blood was established and demonstrated. This study was conducted in wild-type mice (Mus Musculus, C57BL / 6).

[0460] Table 18. Experimental design used for NMDA mouse studies

[0461]

[0462]

[0463] Abbreviations: IVT – intravitreal; OU – both eyes; OD – right eye; OS – left eye; PBS – phosphate buffered saline. Identification of AAV2.N54 vectors and genes of interest:

[0464] AMI189: The same vector contains all DNA sequences, but the open reading frame (ORF) is interrupted in AMI263 encoding IgG4 CNP-Fc36. No protein production is expected in vitro and in vivo;

[0465] AMI169: AAV2.N54 vector encoding anti-VEGF scFv CNP36 fusion protein

[0466] AMI088: AAV2.N54 vector encoding Fc-VNP36.

[0467] AAV constructs were resuspended in formulation buffer 1, PBS, 0.01%

[0468] NMDA preparations

[0469] The molecular weight of NMDA is 147.3 g / mol. On the day of administration, 20 mg of NMDA was weighed and added to 2.7 mL of PBS to obtain a 50 mM solution [stock solution A]. The stock solution A solution was diluted 1:9 in PBS (100 μL stock solution A + 900 μL PBS) to obtain a final 5 mM NMDA solution. The final solution was filtered into a sterile vial with a 0.22 μm filter syringe. The solution was prepared on the day of injection, protected from light and stored refrigerated until administration.

[0470] MK-801 preparation

[0471] The molecular weight of MK-801 is 337.37 g / mol. The received MK-801 container has 5 mg of MK-801. On the day of administration, 1.0 mL of PBS was added to MK-801 to obtain a solution with a concentration of 14.82 mM [stock solution A]. The stock solution was serially diluted as follows: 10 μL stock solution A + 731 μL PBS: 0.2 mM (200 μM) MK-801 [stock solution B]. The final solution was filtered into a sterile vial with a 0.22 μm filter syringe. The solution was prepared on the day of injection, protected from light and stored refrigerated until administration.

[0472] Group 1 drug administration

[0473] Combine 300 μL of 5 mM NMDA stock with 300 μL of sterile PBS and inject 1 μL on day 0 to give a final concentration of 2.5 mM NMDA.

[0474] Group 2 drug administration

[0475] 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 to give a final concentration of 2.5 mM NMDA / 100 μM MK-801.

[0476] Group 3-6 Dosing

[0477] Combine 300 μL of 5 mM NMDA stock with 300 μL of sterile PBS and inject 1 μL on day 0 to give a final concentration of 2.5 mM NMDA.

[0478] Intravitreal injection

[0479] Based on the experimental design, mice were given 0.01-0.05 mg / kg buprenorphine subcutaneously (SQ) on day -28 or day 0. The animals were then sedated with ketamine / xylazine or inhaled isoflurane for intravitreal injection, and one drop of 0.5% proparacaine hydrochloride was applied to both eyes. The conjunctiva was gently clamped with Dumont #4 forceps and injected using a 33G needle and Hamilton syringe. After the syringe contents were dispensed, the syringe needle was slowly withdrawn. After the injection procedure, 1 drop of ofloxacin ophthalmic solution was topically applied to the ocular surface along with an ophthalmic lubricant.

[0480] During NMDA induction on Day 0, animal 409 (Group 4) was noted to have inflammation and adhesions in OD, animal 519 OD (Group 5) was noted to have cataracts, and animal 524 (Group 5) was noted to have cataracts OU. In Group 6, animals 625 and 626 were noted to have cataracts OS, and animals 629, 630, and 632 were noted to have cataracts OD. No other abnormalities were noted in the study records.

[0481] Cage side observation

[0482] Observations for morbidity and mortality were made daily, and cage-side observations were performed, with particular attention to both eyes. All animals were active, alert, and responsive at all observation time points, and no additional findings were noted in the study records. On the day of IVT injection, all animals weighed 20-22 g, and all animals generally maintained their weight ( Fig.9A ).

[0483] Euthanasia and terminal blood collection (Groups 3-6)

[0484] Animals in groups 3-6 were subjected to terminal blood collection on day 7. IBMX was used to inhibit phosphodiesterase activity during blood collection. Prior to necropsy on day 7, but after dosing on day -28, all remaining spare animals were used to ensure that the addition of IBMX to whole blood did not interfere with the coagulation and extraction of serum. In brief, each syringe used for cardiac puncture was pre-rinsed with a stock solution of 10 mM IBMX before blood was drawn. All mice were sedated to a deep sedation level under isoflurane and euthanized via exsanguination. After pre-rinsing each syringe with 10 mM IBMX, a 25G needle was inserted into the heart and the animals were exsanguinated and euthanized, and blood was collected. The volume of blood collected was recorded on paperwork 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 to obtain a final concentration of 1.5 mM IBMX in whole blood. The blood was then drained into tubes, gently mixed by inversion with IBMX, and allowed to clot at room temperature for at least 20 minutes before serum processing. The samples were centrifuged at 4,000 x g for 10 minutes at room temperature in a tabletop microcentrifuge. After centrifugation, the clarified serum was transferred to pre-labeled polypropylene tubes, snap frozen on liquid nitrogen and stored frozen at -80°C.

[0485] Ocular tissue collection and processing

[0486] After euthanasia, the eyes of the selected animals were processed for histological or immunological examinations. The OD of animals 519 (group 5) and 629 (group 6) was recorded as having very small lenses, blood in the retina, and the eyes were filled with gelatinous substances. No other abnormalities in necropsy and tissue processing were noted in the study records. For groups 1 and 2, the selected eyes were collected into 10% neutral buffered formalin immediately after euthanasia. The next day, the eyes were placed in 70% ethanol. The eyes were then processed into paraffin blocks for sectioning. Sagittal sections (5 μm thickness) of each eye were prepared for all animals. At least 3 slides containing bands of approximately 5 segments were collected sequentially. The optic nerve was included in the sections. The slides were stained for hematoxylin and eosin (H&E) and examined using an optical microscope. Fig. 9B Representative images of central retina from H&E histopathology are shown. Retinal ganglion cells (RGCs) were counted (Tables 19 and Fig. 9C The mean RGC counts in the right eyes (OD; experimental eyes) were lower than those in the left eyes (OS; control eyes), and there was no difference in the mean RGC counts in OD and OS between Group 1 and Group 2.

[0487] Table 19. RGC counts of Group 1 and Group 2

[0488]

[0489] Ocular tissue collection for retinal flat mount analysis (all groups)

[0490] The eyes were enucleated and immediately fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) and stored overnight at 4°C. The next day, the eyes were transferred to cold immunocytochemistry (ICC) buffer (PBS containing 0.5% BSA and 0.2% Tween 20) until processing. Using a dissecting microscope, extraneous tissue at the edge of the eye was carefully trimmed and the anterior chamber was removed using fine curved scissors. The retina in the eye cup was rinsed with cold ICC buffer. At 4°C, the eye cup was placed in cold ICC buffer containing 1 / 100 rat anti-CNP36 (groups 3-6 only; resuspended in PBS at 0.5 mg / mL) for 3 hours. The eye cup was then washed thoroughly 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, while groups 7 and 8 were incubated with 1 / 1,000 DAPI alone. The eye cup was washed thoroughly, but gently with cold ICC buffer. Using fine curved scissors and eyelash knife, the retina was separated from the optic nerve head and removed from the RPE / choroid. Then, radial cuts were made toward the center, and the retina was flat mounted, covered and sealed. 2D fluorescence microscopy images were acquired using an Olympus Bx63 upright fluorescence microscope. TUJ-1 signals were highly variable across groups 1 and 2 and provided little additional information, so the Cy2 channel was not imaged. Images at 20x were acquired in 4 quadrants, and the central 248.1x 325.7μm area was analyzed for RGC counting using Olympus cellSens and ImageJ software.

[0491] Groups 1 and 2 were stained with DAPI and TUJ-1 and images were captured at 20x ( Fig. 10A ). RGCs were counted in four quadrants and cells / mm2 were averaged across groups. When RGC counts were quantified across groups ( Fig. 10B ), the average count of the control group (group 1OS; PBS, no NMDA) was 11,000±700 cells / mm2. The reduced RGC count of the NMDA control group (group 1OD; PBS+NMDA) was 8,000±1,000 cells / mm2. The RGC count of group 2OD was slightly increased compared with group 1OD, which was 9,000±500 cells / mm2.

[0492] Groups 3-8 were stained for DAPI and four images were captured for each retina at 20x approximately 600 μm from the optic nerve head. RGCs were counted in the central 248.1 x 325.7 μm area and cells / mm2 were averaged across groups ( Fig. 10C When RGC counts were calculated, the mean counts in the control group (Group 7OS; PBS, no NMDA) were 12,000 ± 700 cells / mm2 ( Fig. 10D). The reduced RGC counts of the NMDA control group (Group 7 OD; PBS + NMDA) were 9,000 ± 300 cells / mm2. The RGC counts of Group 8 OD were similar to Group 7 OD, at 12,000 ± 300 cells / mm2. The RGC counts of Group 3 OD were similar to Group 7 OD, at 9,000 ± 1,000 cells / mm2. The RGC counts of Group 4 OD were 10,000 ± 1,400 cells / mm2. The RGC counts of Group 5 OD were 10,000 ± 600 cells / mm2. The RGC counts of Group 6 OD were 10,000 ± 1,500 cells / mm2.

[0493] Statistical analysis was performed on Group 3-8 RGC data (one-way ANOVA followed by multiple comparisons using Dunnett's statistical hypothesis comparison). Treatment of PBS-injected eyes with NMDA resulted in a statistically significant decrease in RGC counts (P<0.0001). Increasing the dose of MK-801 to 100 μM rescued Group 8 RGC counts 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 Groups 4-6 were compared to Group 3 NMDA / sham vehicle controls, both AMI 182 (Group 5) and AMI 088 (Group 6) reached statistical significance (P<0.0001). Fig.11A ; P = 0.0332 for Group 5 vs. Group 3; P = 0.0021 for Group 6 vs. Group 3). When RGC counts of AAV-treated groups were compared to NMDA / PBS controls (Group 7 OD), only the AMI 088 construct maintained statistical significance (P = 0.002; Fig. 11B ). In addition to DAPI, groups 3-6 were stained for CNP36 to assess the expression location and level of the AAV constructs, and images were captured (4x and 20x) ( Fig. 12A ).

[0494] Ocular tissue collection for immunohistochemistry (Groups 1 and 2 only)

[0495] All eyes designated for immunohistochemistry (IHC) were enucleated, the approximate site of injection was marked, and the eyes were fixed in 4% paraformaldehyde in individually labeled vials at 4°C overnight. The eyes were then transferred to 0.1M phosphate buffer (PB), passed through a sequential sucrose gradient (10%-30%, 1 hour each), and then embedded in OCT medium and frozen on dry ice. The whole eye was cryosectioned (14 μm sections) and stained with the following antibodies shown in Table 20.

[0496] Table 20. Antibodies used for immunohistochemistry of ocular tissues

[0497]

[0498] Negative controls were performed on the staining mixture by using only the secondary antibody. Five slides were stained across the retina of each eye. Two images were taken of each retinal cross section using an Olympus Bx63 upright fluorescence microscope and cellSens software - one at or near the injection site and one from the central area. Qualitatively, there appeared to be no differences in the number of TUJ-1+ and cone arrestin+ cells between the groups. All groups had clear cone arrestin staining with some TUJ-1+ cells ( Fig. 12B ). No further staining or analysis was performed on the cross sections, as staining was not an informative endpoint.

[0499] Ocular tissue collection (groups 3-6)

[0500] Eyes assigned for the assay were enucleated, snap frozen, and stored at ≤-70°C until shipped on dry ice. Tissues were placed in appropriate pre-weighed labeled analytical vials, immediately re-weighed to determine sample weight, and placed on dry ice until transferred to the freezer. Samples were weighed on a balance capable of measuring 4 decimal places. Collected samples included: serum (2 mL polypropylene screw cap tubes); and whole globe with lens (2 mL polypropylene screw cap tubes).

[0501] in conclusion

[0502] The goal of this non-clinical study is 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 at a dosage of 4e+8vg / eye on day -28, and then retinal degeneration was induced with NMDA only into the right eye on day 0. Groups 1, 2, 7 and 8 received co-injections of NMDA and PBS (groups 1, 7) or MK-801 (groups 2, 8).

[0503] During the study, animals maintained normal body weight. When the RGC counts of groups 1 and 2 were measured via histopathology, the RGC counts of the experimental eyes (OD) were lower than those of the control eyes (OS). When the RGC counts were measured via immunohistochemistry of the flat slides, the RGC counts of the NMDA-treated eyes (group 1 and group 7 OD) were significantly lower than those of the control eyes (group 1 and group 7 OS). The damage from NMDA administration was alleviated by pretreatment with 100 μM MK-801 (group 8). Among AAV treatments, pretreatment with AMI 088 resulted in statistically significantly higher RGC counts compared to the NMDA control, with RGC counts approximately 20% more, but it was not as effective as 100 μM MK-801. When the AAV-treated group was compared with the sham AAV+NMDA group, both AMI 182 and AMI 088 reached statistical significance for higher RGC counts.

[0504] Overall, 100 μM MK-801 is an adequate positive control for retinal ganglion cell protection following injury caused by NMDA-induced excitotoxicity, as it fully rescued the NMDA RGC loss phenotype. Future studies could investigate different dose levels of AMI 182 and AMI 088 as well as different time points after NMDA administration.

[0505] 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 via intravitreal injection of NMDA. The number of animals, data collection time points, and parameters measured were selected based on the minimum required to meet the study objectives.

[0506] Bioanalytical work and incidental analysis were performed at the end of the study. Eye samples (e.g., whole eyeballs) from groups 3-6 were enucleated and frozen after separation. At the time of receipt, all samples were received under frozen conditions. The left eyes (OS) of all animals (n=8) from each group receiving different AAV constructs were analyzed. Table 21 shows the documentation of the tissue weight before homogenization and the volume of RIPA lysis and extraction buffer with protease inhibitors added to each sample. The samples were placed on ice and homogenized using a sonicator as follows: 20 seconds pulse followed by 20 seconds of rest for three cycles. After sonication, the samples were placed on ice.

[0507] Table 21. Tissue weights and RIPA buffer volumes

[0508]

[0509]

[0510]

[0511] 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. Therefore, the Fc open reading frame is interrupted.

[0512] CNP36 and CNP-Fc36 expression in eye (whole ball) and serum samples were quantified using commercial CNP36 ELISA kit and internal CNP36-Fc ELISA (Example 2), respectively. Commercial ELISA was performed according to the step-by-step procedure described in the user manual. For internal ELISA, 2 μg / mL anti-CNP36 antibody was coated on 96-well plates and incubated overnight at 4 ° C. The plate was washed with wash buffer and blocked with blocking buffer. Eye and serum samples were directly delivered to the designated wells without any dilution. The samples were incubated for 1 hour. The plates were washed, and the detection antibody was added to each well at a dilution of 1:20,000. After incubation for 1 hour, the plates were washed, and streptavidin-horseradish peroxidase (HRP) was added at a dilution of 1:10,000. After incubation for 45 minutes, the plates were washed, and CNP36-Fc was detected by adding 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The reaction was stopped by adding stop solution and the plate was immediately read at 450 nm with a reference wavelength of 600 nm.

[0513] Quantification of CNP36-Fc in ocular and serum samples

[0514] Animals from Group 3 (AMI189) and Group 6 (AMI088) were analyzed using CNP36-Fc ELISA. Group 3 (sham vehicle) animals served as negative controls ( Fig.13A ). Group 6 animals 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 in GraphPad Prism software using one-way ANOVA followed by Dunnett's multiple comparisons (****=<0.0001). Statistically significant differences were observed between Group 3 and Group 6. However, in samples where the Fc fusion was not present, CNP36 levels were quite low, likely due to the very short half-life of CNP36. Ocular samples from Groups 3, 4, and 5 were analyzed using the commercial ELISA kit described above ( Fig. 13B , left figure). Although compared with group 3 (false carrier; negative control), groups 4 and 5 show slightly higher levels of CNP36, but the observed differences are not significant. The low levels observed on ELISA may indicate that only a small portion of the total expressed CNP36 is quantified, because most of the protein can be proteolyzed before quantification. Due to group 4 and especially group 5 test products showing efficacy, CNP36 is expressed, but the optimal conditions for protecting from proteolysis and subsequent detection can be difficult. Neither CNP36-Fc nor CNP36 are expressed in serum samples. Fig.14The RGC protection performed by the AAV vector encoding CNP is illustrated. 28 days before NMDA injection, the AAV construct of 1 μl via IVT was injected into the mouse eye with 4E+8vg / eye. The image indicates that the RGC# induced by NMDA is reduced, which is rescued by MK-801. The sham vector shows reduced RGC in the NMDA-treated group only, but the AMI182 and AMI088 groups show higher RGC counts than the sham vector and NMDA-treated groups. Table 22 shows the expression summary of the detection of CNP36 and CNP36-Fc in the eye homogenate sample.

[0515] Table 22. CNP36 and CNP36-Fc expression in ocular homogenate samples

[0516]

[0517] AAV2 constructs show efficacy via retinal ganglion cell recovery in experimental NMDA excitotoxicity studies. CNP36-Fc is stable and therefore quantifiable in ocular samples. CNP36 without Fc fusion is difficult to quantify due to its short half-life due to rapid proteolysis. The expression of CNP36 and CNP36-Fc was not detected. For CNP36-Fc, this shows that protein is not systemic leakage, but local action, which will be an obvious therapeutic advantage. It is shown that the pseudo-vector without expression of CNP36 serves as a good negative control for research.

[0518] Table 23. Amino acid sequences of CNP, CNP fusion proteins and antibodies or their fragments

[0519]

[0520]

[0521]

[0522] Example 4. Treatment of the rat model of partial optic nerve transection (pONT)

[0523] Target

[0524] Using a rat model of partial optic nerve transection (pONT), we evaluated the effects of AAV-CNP peptide (AAV-P) and protein Fc fusion (AAV-FP) on retinal ganglion cell (RGC) apoptosis / stress in vivo using the detection of apoptotic retinal cell (DARC) imaging; evaluated the effects of AAV-CNP peptide (AAV-P) and protein Fc fusion (AAV-FP) on intraocular inflammation in vivo using optical coherence tomography (OCT) imaging; evaluated the effects of AAV-CNP peptide (AAV-P) and protein Fc fusion (AAV-FP) on intraocular pressure (IOP) in vivo using the Tonolab tonometer; and evaluated the effects of AAV-CNP peptide (AAV-P) and protein Fc fusion (AAV-FP) on whole retinal preparations (whole retinal preparations). The effects of adeno-associated virus (AAV) CNP peptide (AAV-P) and protein Fc fusion (AAV-FP) on RGC survival were evaluated histologically using immunohistochemistry on whole mounts; and the effects of adeno-associated virus (AAV) CNP peptide (AAV-P) and protein Fc fusion (AAV-FP) on retinal microglial activity were evaluated histologically using immunohistochemistry on whole mounts.

[0525] Materials and methods: Test article information

[0526] Table 24 illustrates the final calculated dose per eye: 4 μL / eye.

[0527] Table 24. Final calculated dose per eye: 4 μL / eye

[0528]

[0529] * False: Contains the same capsid and transgenic cassette covering 5' ITR to ITR3' DNA content as AMI273 except for the interruption of the open reading frame (ORF). No protein product is produced upon administration to animals.

[0530] On day -21 or -28, AAV-P, AAV-FP, vehicle, negative and positive AAV controls: Thaw 1 vial (50 μL) of each diluted vial of test article above at ambient temperature for at least 24 hours and centrifuge in a mini centrifuge / Eppendorf centrifuge for 30 seconds to spin down water condensed on the walls of the vial and to precipitate potential aggregates. Thaw pure Fc-peptide protein vials at ambient temperature for approximately 60 minutes and then spin briefly in an Eppendorf centrifuge to bring down condensed water and remove aggregates prior to injection.

[0531] Eyeball homogenization procedure

[0532] Prepare a mixture of RIPA and protease inhibitors by adding one tablet of protease inhibitor to 10 mL of RIPA buffer. Measure the amount of buffer required for the eye tissue. For every 1 mg of tissue, add 10 μL of buffer. Sonicate the tube containing the tissue on ice for 20 seconds, then let it stand on ice for 20 seconds. Repeat the sonication and cooling on ice two more times for a total of 3 cycles. These cycles can be repeated as required (if the tissue is still not homogenized and clumps are visible).

[0533] After complete homogenization, the tubes were placed on an orbital shaker in a cold room for two hours. After two hours, the tubes were centrifuged at 13,000 rpm for five minutes. The supernatant was collected and stored at -80°C.

[0534] Randomized Experimental Design

[0535] A total of seventy-two male Dark Agouti (DA) rats aged 8-10 weeks were used in this study. Seventy-two animals were randomly divided into 3 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).

[0536] Each category contains 6 blocks, where each block covers a different treatment, for example, n=6 (see categories 1, 2, and 3). The rat ID for each treatment is shown in the left column of each block. L: lower dose; M: middle dose; and H: higher dose.

[0537] Depending on the category, intravitreal injection (IVT, 4 μL) was given under general anesthesia (GA) and applied only once to the left eye (pONT eye) using a 34-gauge Hamilton needle on day 0 after BL imaging. In categories 1 and 2, pONT was performed at week 3. In category 3, pONT was performed shortly after baseline imaging.

[0538] In categories 1 and 2, partial optic nerve transection (pONT) surgery was performed under GA at week 3. In category 3, pONT surgery was performed on day 0 after BL imaging. In brief, an incision was made in the superior conjunctiva and the optic nerve sheath was exposed. A longitudinal slit was made in the dura mater and a 0.2-mm cross cut was made in the dorsal optic nerve at a distance of 2 mm behind the eye. An ophthalmic scalpel with a 0.2 mm steel cutting guard was used during this process. Damage to major ocular blood vessels was avoided and verified by ophthalmoscopy at the end of the operation.

[0539] In categories 1 and 2, animals were sacrificed at week 4, and in category 3, animals were sacrificed at week 1. Prior to sacrifice, blood samples (at least 2 mL) were collected by cardiac puncture at the terminal procedure, centrifuged to collect serum, and stored at -80°C.

[0540] Table 25. Category 1. AAV-P (AMI302) and sham vector only (AMI189) (n=24)

[0541]

[0542] Table 26. Category 2: AAV-FP (AMI273), buffer only (PBS-F68), and positive control AAV (AMI088) (n=30)

[0543]

[0544] Table 27. Class 3: Pure FP-peptide protein (FP-263) (n=18)

[0545]

[0546] *Animal died during in vivo experiments (see exclusions below)

[0547] In vivo evaluation

[0548] DARC / OCT. In categories 1 and 2, under GA, DARC / OCT imaging was performed at baseline (BL), 1st, 2nd and 4th weeks. pONT was performed at week 3. In category 3, DARC / OCT was performed at baseline and week 1 before termination, and pONT was performed shortly after baseline imaging. In brief, fluorescently labeled annexin 776 (6 mg / mL, 40 μL) was intranasally administered 2 hours before DARC imaging, and then assessed with a confocal scanning laser ophthalmoscope (cSLO). OCT imaging was performed at the posterior pole centered on the optic disc using Spectralis cSLO.

[0549] IOP. Intraocular pressure (IOP) was measured at BL using a Tonolab tonometer under inhalation anesthesia and three times (Monday, Wednesday, and Friday) on weeks 1, 2, and 4 in categories 1 and 2, and on week 1 in category 3. Ten IOP readings were collected from each eye of each animal at each time point.

[0550] Histological evaluation

[0551] Three blocks (blocks 1-3) of animals from each category (36 rats in total) were used for immunohistochemical studies. After being sacrificed, each animal's eyes were enucleated and the whole retina specimen was dissected. Immunostaining was performed with anti-RBPMS and anti-Iba-1 antibodies to assess RGC survival and microglial activity, respectively. The whole retina specimens immunostained were then imaged under a fluorescent microscope.

[0552] Protein analysis

[0553] The remaining three blocks (blocks 4-6) of animals from each category (36 rats total) were used for protein analysis.After sacrifice, both eyes from each animal were enucleated and snap frozen with liquid nitrogen, cryogenically homogenized with a mortar and pestle, and stored at -80°C until protein analysis.

[0554] ELISA

[0555] ELISA can be used for the use of adeno-associated vectors for gene therapy or CNP-Fc assays to concentrate CNP-Fc in intermediates and drugs in analytical samples (cell culture supernatants), preclinical (plasma from monkeys, pigs and rodents, homogenized tissues, vitreous fluid, etc.) and clinical samples. CNP-Fc comprises a recombinant fusion protein consisting of CNP-36 fused to human IgG. CNP-Fc can be affinity purified from HEK293 cells transduced with AMI088, which is rAAV2 carrying the CNP gene of a 36 amino acid long peptide that can only be produced and fused to Fc. The serial dilution of CNP-Fc can be used in determination. The detection range of CNP-Fc concentration in this determination can be 0.78ng / mL to 50ng / mL. Materials and equipment may include: CNP-Fc stored in 1X PBS at -80°C (concentration can be determined by BCA); CNP coating / capture antibody: 100 μg / mL, antigen expressed and purified in HEK293 cells (GeneScript, catalog # Z03073); pre-adsorbed goat anti-human IgG Fc (biotin) (Abcam, catalog # ab98618); HRP-streptavidin conjugate (Abcam, catalog # ab7403); TMB substrate: 1-Step TM Ultra TMB-ELISA substrate solution (ThermoFisher, catalog #34028); 96-well microplate reader (Molecular Device: VERSAmax adjustable microplate reader); coating buffer: 3.7 g sodium bicarbonate (NaHCO 3 )、0.64g sodium carbonate (Na 2 CO 3) and 1L Milli Q water, pH 9.60, with a storage condition of one month at room temperature; 1X PBS (phosphate buffered saline): 8.0g sodium chloride, 1.3g disodium hydrogen phosphate, 0.2g sodium dihydrogen phosphate and 1.0 liter Milli Q water, pH7.4, with a storage condition of one year at room temperature; wash buffer (PBST): 1X phosphate buffered saline and 0.1% Tween 20 (v / v), with a storage condition of 30 days expiration at room temperature from the preparation date; blocking buffer (BB): 1X phosphate buffered saline (PBS) with 0.1% Tween 20 (v / v) and 1% casein, with a storage condition of 30 days at 4°C from the preparation date; dilution buffer (DB): same as blocking buffer; stop solution for TMB substrate: 2N HCl, diluted locally from HCl stock solution purchased from Millipore Sigma, catalog #1003172510; and 96-well microplate.

[0556] The ELISA procedure may include: diluting a CNP antibody (500 μg / mL) stock solution to 2 μg / mL with coating buffer (20 μL VEGF stock solution to 5 mL coating buffer); adding the coating antigen to a 96-well microplate at 50 μL / well, and covering and placing the plate at 2-8°C for approximately 12 hours or overnight; discarding the coating antigen and washing the plate three times with 300 μL / well PBS-T wash buffer; adding 300 μL / well blocking buffer, covering and incubating the plate at 37±1°C for 120 minutes; diluting a CNP-Fc standard (8.3 mg / mL) to 50 ng / mL with dilution buffer as follows: diluting 8.3 mg / mL to 1 / 83 to 100 μg / mL, dilute 100 μg / mL to 1 / 10 to 10 μg / mL, dilute 10 μg / mL to 1 / 10 to 1 μg / mL, and add 25 μL of 1 μg / mL solution in 475 μL dilution buffer as the first standard point of 50 ng / mL; prepare the remaining CNP-Fc standards in duplicate using the 1:2 serial dilution scheme in Table 28; or dilute process intermediate samples, such as cell culture supernatant HEK293 with dilution buffer to 1:2000, followed by 1:4000, 1:8000 and 1:16000. For ARPE19 cell culture supernatant, 1:250, 1:500, 1:1000 and 1:2000 dilutions can be prepared in separate plates. Table 29 is a schematic representation of a spent media quantitation plate for HEK293 expression, indicating known CNP-Fc concentrations (rows 1A-1G and rows 2A-2G) to construct a standard curve, blanks with buffer only used as a negative control (blank) and unknown samples (rows 3-10) for CNP-Fc concentration determination. White wells represent empty wells in a 96-well plate. Unknown samples were tested in duplicate, and the ratios in brackets are dilution factors.

[0557] Table 28. Exemplary CNP-Fc Standard Serial Dilutions

[0558]

[0559] Table 29. Exemplary schematic representation of a spent media quantification plate for HEK293 expression

[0560]

[0561]

[0562] Additional ELISA procedures may include: transferring diluted standard samples and unknown samples to a plate according to Table 29, with 50 μL per well in duplicate for each dilution; covering the plate and incubating the plate at 37±1°C for 60 minutes; discarding the reactants in the plate and washing 6 times with 300 μL / well of wash buffer; diluting the pre-absorbed goat anti-human IgG Fc (biotin) at 1:20,000 with dilution buffer and adding 50 μL / well; covering and incubating the plate at 37±1°C for 60 minutes; discarding the reaction mixture, washing the plate 6 times, washing 300 μL / well with wash buffer; diluting streptavidin-HRP at 1:10,000 with dilution buffer and adding 50 μL / well. Cover the plate and incubate at 37±1°C for 60 minutes; discard the reactants in the plate and wash 6 times with 300 μL / well of wash buffer; add TMB substrate, 50 μL / well; cover the plate and incubate at 37±1°C for 15 minutes; stop the reaction by adding 50 μL / well of stop solution; read the plate in a microplate reader with 600nm as the reference wavelength at a 450nm wavelength filter; or copy the data to an Excel spreadsheet and construct a standard curve for CNP-Fc. Unknown samples can be analyzed by first taking the average of duplicates. The concentration of the unknown sample can be determined by merging the obtained OD value into the equation generated by the standard curve. Finally, the concentration is adjusted for the dilution factor. The assay is considered valid when the following criteria are met: the negative control gives an A value of the TMB substrate that is substantially similar to the reading of the blank control 450 ; or the standard curve is linear, R value ≥ 0.975.

[0563] Data analysis

[0564] IOP measurements were performed three times per week at weeks 1, 2, and 4. DARC spots on in vivo images were automatically counted by an algorithm developed and validated in the Cordeiro laboratory. The DARC count was defined as the number of annexin-positive spots seen in retinal images at 120 minutes 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 the DARC spots observed at the terminal time point. A linear transformation of +22 was applied to all DARC counts for visualization purposes only and had no effect on the statistical properties of the data. Inflammation was assessed by manual counting of vitreous inflammatory cells using OCT images. RBPMS in whole-mount retinal specimens were evaluated by an algorithm recently developed and validated in the Cordeiro laboratory. + RGCs were automatically counted and analyzed. To assess whether processing was regionally different, whole-mount retina sections were split in half into upper and lower halves. The morphology of microglia in whole-mount retina sections was automatically analyzed using a machine learning approach recently developed and validated in the Cordeiro laboratory.

[0565] Statistical analysis

[0566] Statistical analysis and graphics of data were completed on IBM SPSS and GraphPad Prism 9. Data are expressed as mean ± SEM, and p < 0.05 is considered statistically significant. Where appropriate, ANOVA and t-tests were used to analyze data. In this inappropriate case, the nonparametric Mann-Whitney U test was used instead. Multiple paired comparisons were always corrected using Tukey adjustments. In order to overcome the small n number of each treatment dose, and to improve statistical power, medium and high concentration conditions were combined to evaluate the effect of treatment as a single group. Therefore, FP, AAV-P and AAV-FP consisted of only medium and high concentrations.

[0567] exclude

[0568] Four rats (R3663, R3667, R3708 and R3719) died during the in vivo experimental process. Two of them (R3663 and R3667) were treated with only vehicle, one (R3663) died before the 4th week, and the other (R3667) died just after the 4th week imaging. Therefore, there is no data from the available DARC / OCT, histology and blood samples of R3663. However, because R3667 died after the 4th week imaging (just before being killed), all data of DARC / OCT and histology are available except blood samples. A rat (R3708) treated with AAV-FP low dose died before the 4th week. Therefore, there is no data available about DARC / OCT, blood samples and protein. A rat (R6719) treated with pure FP peptide protein high dose died before the 1st week. Therefore, there is no data available about DARC / OCT, histology and blood samples. For various reasons listed in Table 30, several rats were excluded from data analysis.

[0569] Table 30. List of animals excluded from data analysis

[0570]

[0571] result

[0572] IOP analysis of both Fc4-CNP36 (FP) and AAV treatment. In order to assess whether the treatment applied intravitreally in the left eye affects the IOP situation, IOP measurements were performed three times a week at 1, 2 and 4 weeks. IOP data were analyzed by subtracting OD (right eye) from the OS (left eye) of each animal at each time point. The results show that after intravitreal injection of 2, 20 and 80 μg / eye affinity-purified FP, FP has a significant effect on IOP reduction. The separate analysis of FP group does show a significant effect (p < 0.01) of FP on IOP after pONT. Figure 16 illustrates the effect of Fc4-CNP36 (FP) on intraocular pressure (IOP) changes in rat partial optic nerve transection (pONT) model. Fig.16A The IOP of the animals monitored during the course of the study is illustrated. Fig. 16B IOP changes in animals following intravitreal (IVT) administration of Fc4-CNP36 are shown. Fig. 16CThe effect of Fc4-CNP36 concentration on IOP changes after intravitreal administration is illustrated. 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 was observed for all FP concentrations on day 1 compared to baseline (day 0) or day 7 after pONT surgery (p < 0.01). Within FP4-CNP36 (FP) of 2 μg / eye-80 μg / eye IVT, IOP reduction was not significantly affected, indicating that IPO reduction reached a maximum at 2 μg / eye. Table 31 illustrates the calculated dose delivered per eye.

[0573] Table 31. Calculated delivered dose per eye

[0574]

[0575] FP-CNP,MW:26kDa

[0576] Effect of FP on DARC counts and retinal ganglion cell (RGC) protection. DARC (apoptotic retinal cell detection) is a retinal imaging technology that has developed from basic laboratory science to Phase 2 clinical trials in the past 20 years. The higher the DARC count, the more severe the apoptosis of retinal cells. Figure 17 illustrates the effect of Fc4-CNP36 on RGC protection in the rat pONT model. Fig.17A Detection of Fc4-CNP36 concentrations that reduce apoptotic retinal cells (DARC) is illustrated. Fig. 17B Fc4-CNP36 concentrations for RGC counts are illustrated. 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 the lowest DARC counts, with FP(high) synergistically significant at 80 μg / eye. The FP condition showed the lowest DARC counts, with FP(high) synergistically significantly lower than AAV negative (p = 0.0078). DARC counts were analyzed by subtracting the baseline for each animal, and the results showed that DARC reduction was associated with the dose injected intravitreally, as 80 mg / eye IVT showed complete inhibition of DARC formation ( Fig.17A ). RGC counts increased with increasing dose ( Fig. 17B ).

[0577] The protein levels of FP4-CNP36 (FP) in the animal eyes and serum samples were analyzed by ELISA assay, and the results are shown in Fig.18, which illustrates the levels of FP4-CNP36 detected by ELISA in the treated and control right eyes of the animals. The levels of FP4-CNP36 in the treated eyes (OS) correlated with the dose.

[0578] Effect of FP on DARC counts and RGC counts. DARC count analysis revealed that AMI273 (a test article of AAV2.N54-Fc4-CNP36 (AAV-FP) encoding Fc4-CNP36) and AMI302 (a test article of AAV2.N54-CNP36 (AAV-P) encoding only CNP36 peptide) had a significant effect on the DARC counts and RGC counts compared with sham vectors after 3 weeks of intravitreal administration of AMI273 and AMI302. 9 and 10 10 vg / eye showed a decrease in DARC (Figure 19). Fig.19A -B illustrates the effect of AAV2.N54-Fc4-CNP36 and AAV2.N54-CPNP36 on retinal DARC reduction in the rat pONT model. DARC values ​​have been subtracted from baseline. Terminal DARC counts are plotted. Fig.19A : AMI273 is a vector of AAV2.N54-Fc4-CNP36, which expresses Fc4-CNP36 protein. Fig.19B :AMI302 is a vector of AAV2.N54-CNP36, which expresses CNP36 peptide. For AMI273, 10 10 Animals that received vg / eye IVT showed a significant decrease in DARC counts, whereas those that received 10 10 Animals that received AMI302 in vg / eye IVT did not show a significant reduction in DARC, indicating that the bioavailability of Fc4-CNP36 is higher than that of CNP36 (peptide only). In this model, Fc4-CNP36 (FP) protein has a significant effect on reducing the level of RGC apoptosis in vivo, as shown in Fig.18 and as seen in Figure 19. All AAV treatments showed increased DARC counts compared to FP, indicating an elevated baseline level in the AAV group. This can be related to the timing of DARC imaging after intravitreal administration of AAV, indicating that persistent inflammation in the AAV group is associated with higher DARC counts. A comparison of individual concentrations of the treatment groups is shown in Figure 19. It is important to note that FP appears to have a dose-dependent effect on reducing DARC counts, with the highest concentration of 80 μg / eye FP showing a significant reduction compared to a sham control containing the same DNA sequence but interrupting the open reading frame (ORF) and resuspended in the same buffer with the same vector AMI189.

[0579] Effect on RGC survival. In this surgical model of pONT, it has been shown that initial injury in the superior retina causes primary degeneration, while secondary effects due to secondary degeneration are seen in the inferior retina. Although primary injury is inevitable, secondary injury may be preventable if therapeutic intervention is successful. To evaluate the effects of AAV-P and AAV-FP on RGC survival, regional differences were investigated. + The density of RGC (cells / mm 2 ) were analyzed by half segmentation of the superior and inferior retina (Figs. Fig.21 ). Fig. 20A -B illustrates the effects of AAV2.N54-Fc4-CNP36 (AAV-FP) and AAV2.N54-CPNP36 (AAV-P) on RGC protection in the rat pONT model. Fig. 20A : AMI273 is a vector of AAV2.N54-Fc4-CNP36, which expresses Fc4-CNP36 protein. Fig. 20B :AMI302 is a vector of AAV2.N54-CNP36, which expresses the CNP36 peptide. The difference in cell density between the upper and lower retina was then calculated. By observing the upper RGC counts subtracted from the lower RGC counts (subtracted data) as an indication of protection against secondary degeneration, it was found that the AAV-FP condition resulted in significantly higher protection than AAV-P (p=0.002) and the sham vector (p=0.005). No other comparisons were significant. The p value was adjusted for five comparisons. By subtracting the upper from the lower, the RGC density in the AAV-FP condition was significantly higher than that in the AAV-P (p=0.002) and sham vector (AAV negative, p=0.005) conditions (Figure 20). Fig.21 Figure 2 shows RGC counts in treated eyes of animals dosed with Fc4-CNP36 protein (FP) and AAV vector in the rat pONT model, where RGC density (cells / mm 2 ) are presented in the upper and lower panels with different treatment conditions in the order of buffer, FP, sham vector (AAV negative), AAV positive, AAV-FP and AAV-P.

[0580] For AAV-treated eyes (OS) and untreated (right) eyes, Fc4-CNP36 ( Fig. 22 ) and CNP36( Fig.23 ) expression in ocular tissues and serum was detected by ELISA. Fc4-CNP36 concentrations in ocular homogenates correlated well with increasing AAV dose levels in the left eye (OS), whereas no expression of the transgene was detected in the right eye (OD). Fig. 22The concentration of Fc4-CNP36 in ocular and serum samples after IVT administration of an AAV vector (AAV-FP) is illustrated. The GOI of Fc4-CNP36 was expressed in eyes dosed with AMI273 but not in untreated eyes. No transgene product Fc4-CNP36 or CNP36 was detected in the untreated eye, the right (OD) eye. The transgene was not detected in serum samples of animals dosed independently with AAV-FP or AAV-P. Fig.23 CNP36 concentrations in ocular and serum samples following IVT administration of an AAV vector (AAV-P) are shown.

[0581] Summary and Conclusion

[0582] DARC analysis revealed that the FP condition exhibited the lowest DARC counts, followed by the AAV-FP group. RGC counts increased in eyes after treatment with FP, AAV-FP, and AAV-P alone. IOP reduction was also observed in animals after IVT injection of FP (Fc4-CNP36), and the maximum IOP lowering effect was achieved at 2 μg / eye, with no further decrease detectable with increasing FP.

[0583] However, despite the secondary neurodegenerative effects, DARC counts appear to be closely associated with RGC survival. The protective effects of AAV-FP and FP on RGC survival in the inferior retina are consistent with the DARC data. A comparison of DARC counts and RGC area density is shown in Fig.24 , where reduced DARC counts correlated with increased RGC density in FP and AAV-FP conditions (upper minus lower) in an analysis of secondary degeneration. Such data support that FP and AAV-FP promote neuroprotection, which results in secondary neurodegenerative effects.

[0584] Example 5. EC of CNP-Fc construct 50

[0585] The CNP-Fc EC described in this paper 50 Measured by ELISA. 2.5E+05 NIH3T3 cells / well were plated. After 2 days, -10M to -5M different CNP-Fc (Fc4-CNP22; Fc1-CNP36; Fc4-CNP36; or Aflibercept-Fc4-CNP36) or comparable natriuretic peptides (ANP or CNP-22 as controls) were added to the designated wells in the presence of 1.8mM IBMX (an inhibitor of cyclic nucleotide phosphodiesterase). After 30 minutes, the supernatant was collected for cGMP determination via ELISA. Fig.25A A standard curve for cGMP is illustrated. Fig.25BThe EC values ​​of various CNP fusions (e.g., CNP-Fc) and comparable natriuretic peptides described herein based on cGMP secreted by cells are described. 50 Data fitting and calculation (EC 50 value: CNP-22≡Fc4-CNP36>Aflibercept-Fc4-CNP36>Fc1-CNP36>>Fc4-CNP22).

[0586] Example 6. Evaluation of the neuroprotective and therapeutic efficacy of AAV-FP in an experimental glaucoma model

[0587] Glaucoma is the main cause of irreversible blindness all over the world, and it is characterized by degeneration and loss of retinal ganglion cells (RGC) and axons thereof via apoptosis. At present, elevated intraocular pressure (IOP) is the only modifiable risk factor, and although IOP control is effective, a part of glaucoma patients continues to lose their vision. Therefore, the risk factors that do not rely on IOP are increasingly considered to play a role in glaucoma pathology, and targeting those factors can have potential in the treatment of glaucoma. In recent years, as a method of preventing the loss of function in glaucoma, the reduction of neuroprotection and neuroinflammation has obtained substantial interest.

[0588] DARC (apoptotic retinal cell detection) is a biomarker that binds to exposed phosphatidylserine, which allows identification of morbid, stressed and apoptotic cells. DARC can be used as a platform for evaluating the neuroprotective effects and therapeutic efficacy of drugs in preclinical and clinical trials. DARC can be used to evaluate gene therapy delivered by methods described herein or engineered polynucleotides in a partial optic nerve transection model.

[0589] The goal of this study was to investigate the efficacy of 2 doses of AAV-FP relative to sham AAV and NGF positive controls in an ocular hypertension model using well established and translatable endpoints including DARC. Using a well established rat model of glaucoma (ocular hypertension or OHT), the following could be investigated: whether AAV-FP could prevent or reduce RGC (retinal ganglion cell) apoptosis in vivo by DARC imaging and assess the dose response relationship using DARC imaging; and whether AAV-FP could promote RGC survival and reduce inflammation by histological markers and by assessing microglial morphology measurements by disease induction in response to AAV intervention.

[0590] Experimental design

[0591] Treatment Groups (Table 31):

[0592] Group 1: OHT only (n=6)

[0593] Group 2: OHT + vehicle (n=6)

[0594] Group 3: OHT+IVT NGF (n=6)

[0595] Group 4: OHT+IVT sham AAV-FP (n=6)

[0596] Group 5: OHT+IVT dose 1 AAV-FP (n=6)

[0597] Group 6: OHT+IVT dose 2 AAV-FP (n=6)

[0598] Table 31. Animal groups used in OHT studies

[0599]

[0600] In vivo regimen (4 months):

[0601] Animals: 36 Dark Agouti (DA) male rats weighing 150-200 g (n=6, 6 groups).

[0602] • Treatments: Animals can be randomized into two groups (see above).

[0603] • Groups 2, 4, 5 and 6 may receive intravitreal injections 4 weeks prior to IOP elevation.

[0604] Group 3 could receive IVT NGF at the time of surgery.

[0605] • OHT surgery: Intraocular pressure (IOP) can be raised in the left ...

Claims

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

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

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

4. The engineered polynucleotide of claim 3, wherein the natriuretic peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-5.

5. The engineered polynucleotide of any one of claims 1-4, wherein the natriuretic peptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

4.

6. The engineered polynucleotide of claim 5, wherein the natriuretic peptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

4.

7. The engineered polynucleotide of claim 6, wherein the natriuretic peptide comprises the amino acid sequence of SEQ ID NO:

4.

8. The engineered polynucleotide of any one of claims 1-7, wherein the antibody or fragment thereof comprises a crystallizable fragment (Fc) region.

9. The engineered polynucleotide of any one of claims 1-8, 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.

10. The engineered polynucleotide of claim 9, wherein 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.

11. The engineered polynucleotide of claim 10, wherein the antibody or fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 6-8.

12. The engineered polynucleotide of any one of claims 1-11, wherein the natriuretic peptide is covalently linked to the N-terminus of the antibody or fragment thereof.

13. The engineered polynucleotide of any one of claims 1-11, wherein the natriuretic peptide is covalently linked to the C-terminus of the antibody or fragment thereof.

14. The engineered polynucleotide of any one of claims 1-13, wherein the natriuretic peptide is covalently linked to the antibody or fragment thereof via a peptide linker.

15. The engineered polynucleotide of claim 14, wherein the peptide linker comprises an amino acid sequence comprising (GGGGS)n, wherein n is an integer between 0-20.

16. The engineered polynucleotide of claim 14, wherein said n is the integer four.

17. The engineered polynucleotide of any one of claims 1-16, wherein the engineered polypeptide comprises a protease cleavage site.

18. The engineered polynucleotide of claim 17, wherein the protease cleavage site comprises a furin site.

19. The engineered polynucleotide of claim 1, wherein the engineered polypeptide comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 131-140.

20. The engineered polynucleotide of claim 19, wherein the engineered polypeptide comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 131-140.

21. The engineered polynucleotide of claim 20, wherein the engineered polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 131-140.

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

23. The engineered polynucleotide of claim 22, wherein the CNP or a fragment thereof comprises 22 consecutive bases of SEQ ID NO:

1.

24. The engineered polynucleotide of claim 23, wherein the CNP or a fragment thereof comprises the 22 consecutive bases at the C-terminus of SEQ ID NO:

1.

25. The engineered polynucleotide of claim 22, wherein the CNP or a fragment thereof comprises 36 consecutive bases of SEQ ID NO:

1.

26. The engineered polynucleotide of claim 25, wherein the CNP or a fragment thereof comprises the 36 consecutive bases at the C-terminus of SEQ ID NO:

1.

27. The engineered polynucleotide of claim 22, wherein the CNP or a fragment thereof comprises 53 consecutive bases of SEQ ID NO:

1.

28. The engineered polynucleotide of claim 27, wherein the CNP or a fragment thereof comprises the 53 consecutive bases at the C-terminus of SEQ ID NO:

1.

29. The engineered polynucleotide of any one of claims 1-28, wherein the viral vector is an AAV vector.

30. The engineered polynucleotide of claim 29, 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.

31. The engineered polynucleotide of claim 30, wherein the AAV serotype comprises AAV2.

32. The engineered polynucleotide of any one of claims 29-31, wherein the AAV vector encodes a modified AAV capsid.

33. The engineered polynucleotide of any one of claims 29-32, wherein the AAV vector comprises a second expression cassette.

34. The engineered polynucleotide of claim 33, wherein the second expression cassette encodes a therapeutic agent.

35. The engineered polynucleotide of claim 34, wherein the therapeutic agent comprises a hormone.

36. The engineered polynucleotide of claim 34, wherein the therapeutic agent comprises an agonist of a natriuretic peptide receptor (NPR).

37. The engineered polynucleotide of claim 34, wherein the therapeutic agent comprises an agonist of the cyclic GMP (cGMP) signaling pathway.

38. The engineered polynucleotide of claim 34, wherein the therapeutic agent comprises a VEGF inhibitor.

39. An engineered polypeptide comprising an antibody or fragment thereof operably coupled to a natriuretic 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.

40. The engineered polypeptide of claim 39, wherein the natriuretic peptide comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 1-5.

41. The engineered polypeptide of claim 40, wherein the natriuretic peptide comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 1-5.

42. The engineered polypeptide of claim 41, wherein the natriuretic peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-5.

43. The engineered polypeptide of any one of claims 39-42, wherein the natriuretic peptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

4.

44. The engineered polypeptide of claim 43, wherein the natriuretic peptide comprises an amino acid sequence at least 95% identical to SEQ ID NO:

4.

45. The engineered polypeptide of claim 44, wherein the natriuretic peptide comprises the amino acid sequence of SEQ ID NO:

4.

46. ​​The engineered polypeptide of claim 39, wherein the antibody or fragment thereof comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 6-8.

47. The engineered polypeptide of claim 46, wherein the antibody or fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 6-8.

48. The engineered polypeptide of any one of claims 39-47, wherein the natriuretic peptide is covalently linked to the N-terminus of the antibody or fragment thereof.

49. The engineered polypeptide of any one of claims 39-47, wherein the natriuretic peptide is covalently linked to the C-terminus of the antibody or fragment thereof.

50. The engineered polypeptide of any one of claims 39-49, wherein the natriuretic peptide is covalently linked to the antibody or fragment thereof via a peptide linker.

51. The engineered polypeptide of claim 50, wherein the peptide linker comprises an amino acid sequence comprising (GGGGS)n, wherein n is an integer between 0-20.

52. The engineered polypeptide of claim 51, wherein said n is the integer four.

53. The engineered polypeptide of any one of claims 39-52, wherein the engineered polypeptide comprises a protease cleavage site.

54. The engineered polypeptide of claim 53, wherein the protease cleavage site comprises a furin site.

55. The engineered polypeptide of claim 39, wherein the engineered polypeptide comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 131-140.

56. The engineered polypeptide of claim 55, wherein the engineered polypeptide comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 131-140.

57. The engineered polypeptide of claim 56, wherein the engineered polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 131-140.

58. The engineered polypeptide of claim 39, wherein the natriuretic peptide comprises C-type natriuretic peptide (CNP) or a fragment thereof.

59. The engineered polypeptide of claim 58, wherein the CNP or a fragment thereof comprises 22 consecutive bases of SEQ ID NO:

1.

60. The engineered polypeptide of claim 59, wherein the CNP or fragment thereof comprises the 22 consecutive bases at the C-terminus of SEQ ID NO:

1.

61. The engineered polypeptide of claim 39, wherein the CNP or a fragment thereof comprises 36 consecutive bases of SEQ ID NO:

1.

62. The engineered polypeptide of claim 61, wherein the CNP or fragment thereof comprises the 36 consecutive bases at the C-terminus of SEQ ID NO:

1.

63. The engineered polypeptide of claim 39, wherein the CNP or a fragment thereof comprises 53 consecutive bases of SEQ ID NO:

1.

64. The engineered polypeptide of claim 63, wherein the CNP or fragment thereof comprises the 53 consecutive bases at the C-terminus of SEQ ID NO:

1.

65. A cell comprising the engineered polynucleotide of any one of claims 1-64.

66. The cell of claim 65, wherein at least a fragment of the engineered polynucleotide is integrated into the genome of the cell.

67. A cell comprising the engineered polypeptide of any one of claims 1-64.

68. A viral particle comprising the engineered polynucleotide of any one of claims 1-64.

69. The viral particle of claim 68, wherein the viral particle comprises an AAV capsid.

70. The viral particle of claim 69, wherein the AAV capsid comprises a modified AAV capsid.

71. The viral particle of claim 70, wherein the modified AAV capsid comprises a modified AAV2 capsid.

72. A pharmaceutical composition comprising the engineered polynucleotide of any one of claims 1-38, the engineered polypeptide of any one of claims 39-64, the cell of any one of claims 65-68, or the viral particle of any one of claims 68-71.

73. The pharmaceutical composition of claim 72, wherein the pharmaceutical composition is formulated for administration to a subject in need thereof intrathecally, intraocularly, intravitreally, retinaly, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymalally, subcutaneously, subretinally, suprachoroidally, intratumorally, intrapulmonaryly, intratracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhalation of an aerosolized form, by an intraluminal-GI route, or a combination thereof.

74. The pharmaceutical composition of claim 73, wherein the pharmaceutical composition is formulated for intravitreal, subretinal, or suprachoroidal administration.

75. A pharmaceutical composition as described in any one of claims 73-74, wherein the pharmaceutical composition is used to treat an ocular disease or condition.

76. The pharmaceutical composition of any one of claims 73-75, wherein 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 thereof.

77. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide as described in any one of claims 1-38, an engineered polypeptide as described in any one of claims 39-64, a cell as described in any one of claims 65-68, a viral particle as described in any one of claims 68-71, or a pharmaceutical composition as described in any one of claims 72-76.

78. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide as described in any one of claims 1-38, an engineered polypeptide as described in any one of claims 39-64, a cell as described in any one of claims 65-68, a viral particle as described in any one of claims 68-71, or a pharmaceutical composition as described in any one of claims 72-76, wherein one administration cures the disease or condition.

79. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide as described in any one of claims 1-38, an engineered polypeptide as described in any one of claims 39-64, a cell as described in any one of claims 65-68, a viral particle as described in any one of claims 68-71, or a pharmaceutical composition as described in any one of claims 72-76, wherein the administration does not comprise daily administration.

80. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an engineered polynucleotide as described in any one of claims 1-38, an engineered polypeptide as described in any one of claims 39-64, a cell as described in any one of claims 65-68, a viral particle as described in any one of claims 68-71, or a pharmaceutical composition as described in any one of claims 72-76, wherein the administration comprises weekly administration, biweekly administration, monthly administration, bimonthly administration, semi-annual administration, annual administration, or a combination thereof.

81. The method of any one of claims 77-80, wherein the disease or condition comprises an ocular disease.

82. The method of claim 81, wherein the ocular disease comprises ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, color blindness, 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), Budd-Bieder syndrome, Best disease, choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, O'Sullivan's disease, Leventinese disease (familial dominant drusen), blue cone monochromacy, or a combination thereof.

83. The method of any one of claims 77-82, wherein the engineered polynucleotide of any one of claims 1-38, the engineered polypeptide of any one of claims 39-64, the cell of any one of claims 65-68, the viral particle of any one of claims 68-71, or the pharmaceutical composition of any one of claims 72-76 increases natriuretic peptide receptor B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof in the subject, thereby treating the disease or condition.

84. The method of any one of claims 77-82, wherein the engineered polynucleotide of any one of claims 1-38, the engineered polypeptide of any one of claims 39-64, the cell of any one of claims 65-68, the viral particle of any one of claims 68-71, or the pharmaceutical composition of any one of claims 72-76 increases the half-life of an agonist for natriuretic peptide receptor B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof in the subject, thereby treating the disease or condition.

85. The method of claim 84, wherein the half-life is increased in the subject by at least two-fold, at least five-fold, at least ten-fold, at least twenty-fold, at least fifty-fold, at least one hundred-fold, or more compared to the half-life of an endogenous agonist for the natriuretic peptide receptor B signaling, the guanylyl cyclase signaling, the cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof.

86. A method for 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 an engineered polypeptide, the engineered polypeptide comprising an antibody or fragment thereof operably coupled to a natriuretic peptide in cells of the subject, and wherein the cells express the engineered polypeptide, thereby treating the disease or condition.

87. The method of claim 86, wherein the natriuretic peptide comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 1-5.

88. The method of claim 87, wherein the natriuretic peptide comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 1-5.

89. The method of claim 88, wherein the natriuretic peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-5.

90. The method of any one of claims 86-89, wherein the natriuretic peptide comprises an amino acid sequence at least 80% identical to SEQ ID NO:

4.

91. The method of claim 90, wherein the natriuretic peptide comprises an amino acid sequence at least 95% identical to SEQ ID NO:

4.

92. The method of claim 91, wherein the natriuretic peptide comprises the amino acid sequence of SEQ ID NO:

4.

93. The method of any one of claims 86-92, wherein the antibody or fragment thereof comprises a crystallizable fragment (Fc) region.

94. The method of any one of claims 86-93, 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.

95. The method of claim 94, wherein 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.

96. The method of claim 95, wherein the antibody or fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 6-8.

97. The method of any one of claims 86-96, wherein the natriuretic peptide is covalently linked to the N-terminus of the antibody or fragment thereof.

98. The method of any one of claims 86-96, wherein the natriuretic peptide is covalently linked to the C-terminus of the antibody or fragment thereof.

99. The method of any one of claims 86-98, wherein the natriuretic peptide is covalently linked to the antibody or fragment thereof via a peptide linker.

100. The method of claim 99, wherein the peptide linker comprises an amino acid sequence comprising (GGGGS)n, wherein n is an integer between 0-20.

101. The method of claim 100, wherein said n is an integer of four.

102. The method of any one of claims 86-101, wherein the engineered polypeptide comprises a protease cleavage site.

103. The method of claim 102, wherein the protease cleavage site comprises a furin site.

104. The method of claim 86, wherein the engineered polypeptide comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 131-140.

105. The method of claim 104, wherein the engineered polypeptide comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 131-140.

106. The method of claim 105, wherein the engineered polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 131-140.

107. The method of claim 86, wherein the natriuretic peptide comprises C-type natriuretic peptide (CNP) or a fragment thereof.

108. The method of claim 107, wherein the CNP or a fragment thereof comprises 22 consecutive bases of SEQ ID NO:

1.

109. The method of claim 108, wherein the CNP or fragment thereof comprises the 22 consecutive bases at the C-terminus of SEQ ID NO:

1.

110. The method of claim 107, wherein the CNP or a fragment thereof comprises 36 consecutive bases of SEQ ID NO:

1.

111. The method of claim 110, wherein the CNP or fragment thereof comprises the 36 consecutive bases at the C-terminus of SEQ ID NO:

1.

112. The method of claim 107, wherein the CNP or a fragment thereof comprises 53 consecutive bases of SEQ ID NO:

1.

113. The method of claim 112, wherein the CNP or fragment thereof comprises the 53 consecutive bases at the C-terminus of SEQ ID NO:

1.

114. The method of any one of claims 86-113, wherein the engineered polynucleotide comprises an AAV vector.

115. The method of claim 114, 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.

116. The method of claim 115, wherein the AAV serotype comprises AAV2.

117. The method of any one of claims 114-116, wherein the AAV vector encodes a modified AAV capsid.

118. The method of any one of claims 86-117, wherein the engineered polynucleotide is encapsulated in a viral particle.

119. The method of claim 118, wherein the viral particle comprises an AAV capsid.

120. The method of claim 119, wherein the AAV capsid is a modified AAV capsid.

121. The method of any one of claims 86-120, wherein the viral vector comprises a second expression cassette.

122. The method of claim 121, wherein the second expression cassette encodes a therapeutic agent.

123. The method of claim 122, wherein the therapeutic agent comprises a hormone.

124. The method of claim 122, wherein the therapeutic agent comprises an agonist of a natriuretic peptide receptor (NPR).

125. The method of claim 122, wherein the therapeutic agent comprises an agonist of the cyclic GMP (cGMP) signaling pathway.

126. The method of claim 122, wherein the therapeutic agent comprises a VEGF inhibitor.

127. The method of any one of claims 86-126, wherein the engineered polynucleotide is administered to the subject intrathecally, intraocularly, intravitreally, retinaly, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymalally, subcutaneously, subretinally, suprachoroidally, intratumorally, intrapulmonaryly, intratracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhalation of an aerosolized form, by an intraluminal-GI route, or a combination thereof.

128. The method of any one of claims 86-127, wherein the disease or condition comprises an ocular disease.

129. The method of claim 128, wherein the ocular disease comprises ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color blindness, 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, Budd-Bieder syndrome, Best disease, choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, O'Sullivan's disease, Leventinese disease (familial dominant drusen), blue cone monochromacy, or a combination thereof.

130. A method of treating glaucoma in a subject, the method comprising administering to the subject an AAV2 vector, wherein the AAV2 vector encodes a natriuretic peptide covalently linked to an antibody or fragment thereof.

131. The method of claim 130, wherein the natriuretic peptide comprises an amino acid sequence at least 80% identical to SEQ ID NO:

4.

132. The method of claim 131, wherein the natriuretic peptide comprises an amino acid sequence at least 95% identical to SEQ ID NO:

4.

133. The method of claim 132, wherein the natriuretic peptide comprises the amino acid sequence of SEQ ID NO:

4.

134. The method of any one of claims 130-133, wherein the antibody or fragment thereof comprises a crystallizable fragment (Fc) region.

135. The method of any one of claims 130-134, 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.

136. The method of claim 135, wherein 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.

137. The method of claim 136, wherein the antibody or fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 6-8.

138. The method of any one of claims 130-137, wherein the natriuretic peptide is covalently linked to the N-terminus of the antibody or fragment thereof.

139. The method of any one of claims 130-137, wherein the natriuretic peptide is covalently linked to the C-terminus of the antibody or fragment thereof.

140. The method of any one of claims 130-139, wherein the natriuretic peptide is covalently linked to the antibody or fragment thereof via a peptide linker.

141. The method of claim 140, wherein the peptide linker comprises an amino acid sequence comprising (GGGGS)n, wherein n is an integer between 0-20.

142. The method of claim 141, wherein said n is an integer of four.

143. The method of any one of claims 130-142, wherein a protease cleavage site flanks the natriuretic peptide and the antibody or fragment thereof.

144. The method of claim 143, wherein the protease cleavage site comprises a furin site.

145. The method of claim 130, wherein the natriuretic peptide comprises C-type natriuretic peptide (CNP) or a fragment thereof.

146. The method of claim 145, wherein the CNP or fragment thereof comprises 22 consecutive bases of SEQ ID NO:

1.

147. The method of claim 146, wherein the CNP or fragment thereof comprises the 22 consecutive bases at the C-terminus of SEQ ID NO:

1.

148. The method of claim 145, wherein the CNP or a fragment thereof comprises 36 consecutive bases of SEQ ID NO:

1.

149. The method of claim 148, wherein the CNP or fragment thereof comprises the 36 consecutive bases at the C-terminus of SEQ ID NO:

1.

150. The method of claim 145, wherein the CNP or fragment thereof comprises 53 consecutive bases of SEQ ID NO:

1.

151. The method of claim 150, wherein the CNP or fragment thereof comprises the 53 consecutive bases at the C-terminus of SEQ ID NO:

1.

152. The method of any one of claims 130-151, wherein the AAV2 vector encodes a modified AAV capsid.

153. The method of any one of claims 130-152, wherein the AAV2 vector comprises a second expression cassette.

154. The method of claim 153, wherein the second expression cassette encodes a therapeutic agent.

155. The method of claim 154, wherein the therapeutic agent comprises a hormone.

156. The method of claim 154, wherein the therapeutic agent comprises an agonist of a natriuretic peptide receptor (NPR).

157. The method of claim 154, wherein the therapeutic agent comprises an agonist of the cyclic GMP (cGMP) signaling pathway.

158. The method of claim 154, wherein the therapeutic agent comprises a VEGF inhibitor.

159. The method of any one of claims 130-158, wherein the AAV2 vector is encapsulated in an AAV viral particle prior to said administering.

160. The method of any one of claims 130-159, wherein the AAV2 vector is administered to a subject in need thereof intrathecally, intraocularly, intravitreally, retinaly, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymalally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonary, intratracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhalation of an aerosolized form, by an intraluminal-GI route, or a combination thereof.

161. The method of claim 160, wherein the AAV2 vector is administered intravitreally, subretinally, or suprachoroidally.

162. The method of any one of claims 130-161, wherein the administration of the AAV2 vector increases natriuretic peptide receptor B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof in the subject.

163. The method of any one of claims 130-162, wherein one said administration of said AAV2 vector cures said glaucoma.

164. The method of any one of claims 130-162, wherein said administration of said AAV2 vector does not comprise daily administration.

165. The method of any one of claims 130-162, wherein the administration of the AAV2 vector comprises weekly administration, biweekly administration, monthly administration, bimonthly administration, semi-annual administration, annual administration, or a combination thereof.

166. The method of any one of claims 130-165, wherein the administration of the AAV2 vector increases the half-life of an agonist for natriuretic peptide receptor B signaling, guanylyl cyclase signaling, cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof in the subject.

167. The method of claim 166, wherein the half-life is increased in the subject by at least two-fold, at least five-fold, at least ten-fold, at least twenty-fold, at least fifty-fold, at least one hundred-fold, or more compared to the half-life of an endogenous agonist for the natriuretic peptide receptor B signaling, the guanylyl cyclase signaling, the cyclic guanosine monophosphate (cGMP) signaling, or a combination thereof.

168. The method of any one of claims 130-167, wherein the glaucoma comprises neovascular glaucoma (NG), glaucoma, traumatic glaucoma, or a combination thereof.

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