Compositions and methods for expressing therapeutic agents

Abstract

Described herein are compositions for modulating transgene expression of one or more therapeutic agents. In some aspects, an engineered polynucleotide comprises one or more expression cassettes, the one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor. Also described herein are methods of using the compositions described herein for modulating transgene expression and for treating diseases or conditions, in particular therapeutic agents for treating ocular diseases or inhibiting neovascularization.

Description

Cross-references

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 531,202, filed August 7, 2023, and U.S. Provisional Application Serial No. 63 / 665,105, filed June 27, 2024, each of which is incorporated herein by reference in its entirety. Background Technology

[0002] Angiogenesis, including angiogenesis, vascularization, and arteriosclerosis, is regulated by multiple cell signaling pathways. One of these pathways is regulated by vascular endothelial growth factor (VEGF). VEGF is a potent mitogen targeting endothelial cells, inducing proliferation, migration, vessel formation, and permeability. Therefore, an increase in VEGF signaling increases angiogenesis signaling, while a decrease or inhibition of VEGF signaling decreases it. VEGF inhibition is one of the most popular treatment options for diseases or conditions associated with angiogenesis. For example, the treatment of ocular diseases often involves the use of angiogenesis inhibitors such as VEGF inhibitors. Summary of the Invention

[0003] Current treatments using VEGF inhibitors can be cumbersome, requiring repeated monthly injections to achieve and maintain inhibition of angiogenesis due to their short half-life. Therefore, therapeutic agents for treating ocular diseases remain needed. Therapeutic agents targeting or in combination with VEGF signaling pathways to inhibit angiogenesis are also needed. Accordingly, this document describes, in some aspects, an engineered polynucleotide comprising one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor. In some embodiments, the first and second angiogenesis inhibitors are covalently linked via a linker. In some embodiments, the first angiogenesis inhibitor comprises a complement inhibitor. In some embodiments, the complement inhibitor comprises a complement C3 inhibitor or a C3 degradation fragment. In some embodiments, the complement 3 inhibitor comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 1-15. In some embodiments, the first or second angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC). In some embodiments, the MAC inhibitor comprises CD59. In some embodiments, CD59 comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 41-45, 312-319, or 325-329. In some embodiments, the second angiogenesis inhibitor comprises a natriuretic peptide. In some embodiments, the natriuretic peptide comprises a C-type natriuretic peptide (CNP). In some embodiments, the natriuretic peptide is covalently linked to an antibody or a fragment thereof. In some embodiments, the antibody or a fragment thereof comprises a crystallizable fragment (Fc) region. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 61-72. In some embodiments, the second angiogenesis inhibitor comprises endostatin or a fragment thereof. In some embodiments, a third angiogenesis inhibitor is encoded. In some embodiments, the engineered polynucleotide comprises a viral vector. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the AAV vector is an AAV2 vector. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises the amino acid sequence of either SEQ ID NO: 161-182 or SEQ ID NO: 191-210. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises CNP36. In some embodiments, the engineered polynucleotide further encodes a third angiogenesis inhibitor. In some embodiments, the third angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC), and wherein the inhibitor of MAC comprises CD59.In some embodiments, the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises a complement 3 inhibitor fused to Fc-CNP36. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises endostatin. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises Fc-CNP36, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59. In some embodiments, the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises a complement 3 inhibitor, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising Fc-CNP36. In some embodiments, the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.

[0004] This document describes, in some aspects, an engineered peptide comprising a first angiogenesis inhibitor and a second angiogenesis inhibitor. In some embodiments, the first and second angiogenesis inhibitors are covalently linked via a linker. In some embodiments, the first angiogenesis inhibitor comprises a complement inhibitor. In some embodiments, the first or second angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC), wherein the MAC inhibitor comprises CD59. In some embodiments, the second angiogenesis inhibitor comprises a natriuretic peptide. In some embodiments, the second angiogenesis inhibitor comprises endostatin or a fragment thereof. In some embodiments, the engineered peptide further encodes a third angiogenesis inhibitor.

[0005] This document describes, in some respects, a vector comprising the engineered polynucleotides or engineered peptides described herein. In some embodiments, the vector encodes an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid.

[0006] This document describes, in some respects, a viral particle comprising the engineered polynucleotides, engineered peptides, or vectors disclosed herein. In some embodiments, the viral particle comprises an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid.

[0007] This article describes, in some respects, a cell that comprises the engineered polynucleotides, engineered peptides, vectors, or viral particles disclosed herein.

[0008] This document describes, in some respects, a composition comprising: a complement 3 inhibitor or a C3 degradation fragment comprising C3a, C3b, iC3b, C3f, C3c, C3d, C3g, or a combination thereof; and a natriuretic peptide. In some embodiments, the natriuretic peptide comprises a C-type natriuretic peptide (CNP).

[0009] This document describes, in some respects, a composition comprising: a complement 3 inhibitor or a C3 degradation fragment comprising C3a, C3b, iC3b, C3f, C3c, C3d, C3g, or a combination thereof; and an inhibitor of the membrane attack complex (MAC). In some embodiments, the MAC inhibitor comprises CD59.

[0010] This document describes, in some respects, a composition comprising CD59 and a natriuretic peptide. In some embodiments, the natriuretic peptide comprises a C-type natriuretic peptide (CNP).

[0011] This document describes, in some respects, a pharmaceutical composition comprising the engineered polynucleotide, engineered peptide, vector, viral particle, cell, or composition disclosed herein.

[0012] This document describes, in some respects, a method comprising contacting cells obtained from an object with the engineered polynucleotide, engineered peptide, vector, viral particle, cell, composition, or pharmaceutical composition disclosed herein.

[0013] This document describes in some respects a method for treating a disease or condition in a subject, comprising: administering to the subject an engineered polynucleotide disclosed herein, an engineered peptide disclosed herein, a vector disclosed herein, a viral particle disclosed herein, a cell disclosed herein, a composition disclosed herein, or a pharmaceutical composition disclosed herein.

[0014] This article describes in some respects a method for treating a disease or condition in a subject, the method comprising administering an engineered polynucleotide to the subject, the engineered polynucleotide comprising one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor. Incorporation

[0015] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference. Where a publication, patent, or patent application incorporated by reference contradicts the disclosure contained in this specification, this specification is intended to supersede and / or give precedence to any such contradictory material. Attached Figure Description

[0016] The novel features of this disclosure are set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description of illustrative embodiments utilizing the principles of this disclosure, along with the accompanying drawings (also “Figures” herein), in which: Figure 1 The vector construct for expressing complement C3 inhibitors (C3i) is described.

[0017] Figure 2A and Figure 2B The vector construct for expressing C3i that is operatively coupled to a natriuretic peptide is described.

[0018] Figure 3 The vector constructs for expressing C3i, CD59, and natriuretic peptides are described.

[0019] Figure 4 Exemplary vectors for expressing the angiogenesis inhibitors described herein are illustrated.

[0020] Figures 5A-5C The AAV construct created and tested in Example 3 is described.

[0021] Figure 6 This study describes the expression of the fusion protein in cells on a 6-well plate, as detected by SDS-PAGE and Western blot using an HRP-conjugated goat anti-human IgG1 Fc antibody. Lane contents: 1. GAM vector 3d; 2. EKQ vector 3d; 3. CME vector 3d; 4. Untransfected 3d; 5. GAM vector 6d; 6. EKQ vector 6d; 7. CME vector 6d; 8. Untransfected 6d.

[0022] Figure 7 This study describes the expression of the fusion protein in cells from T125 flasks, as detected by SDS-PAGE and Western blot using HRP-conjugated goat anti-human IgG1 Fc antibody. Lane contents: 1. GAM vector 3d; 2. EKQ vector 3d; 3. CME vector 3d; 4. Untransfected 3d; 5. GAM vector 6d; 6. EKQ vector 6d; 7. CME vector 6d; 8. Untransfected 6d.

[0023] Figure 8 This study describes the expression of the fusion protein in cells on a 6-well plate, as detected by SDS-PAGE and Western blotting using rat anti-human CNP antibody. Lane contents: 1. GAM vector 3d; 2. EKQ vector 3d; 3. CME vector 3d; 4. Untransfected 3d; 5. GAM vector 6d; 6. EKQ vector 6d; 7. CME vector 6d; 8. Untransfected 6d.

[0024] Figure 9 This study describes the expression of fusion proteins in cells on T125 flasks, detected by SDS-PAGE and Western blot analysis using rat anti-human CNP antibody. Lane contents: 1. GAM vector 3d; 2. EKQ vector 3d; 3. CME vector 3d; 4. Untransfected 3d; 5. GAM vector 6d; 6. EKQ vector 6d; 7. CME vector 6d; 8. Untransfected 6d.

[0025] Figure 10 This study describes the expression of membrane-bound CD59 in transfected cells based on Western blot analysis. Lane contents: 1. Vector GTM; 2. Vector GTP; 3. Vector GTQ; 4. Vector GTR; 5. Vector GAT; 6. Vector EKQ; 7. Untransfected; 8. Purified CD59 protein.

[0026] Figure 11 This study describes the detection of soluble CD59 (sCD59) protein in day 3 cell culture supernatant using HRP-conjugated anti-human CD59 antibody based on Western blot analysis. Lane contents: 1. Vector GCK; 2. Vector GCM; 3. Vector GEM; 4. Vector GEP; 5. Vector EKQ; 6. Untransfected; 7. Purified CD59 protein.

[0027] Figure 12 This study describes the detection of soluble CD59 (sCD59) protein in day 6 cell culture supernatant using HRP-conjugated anti-human CD59 antibody based on Western blot analysis. Lane contents: 1. Vector GCK; 2. Vector GCM; 3. Vector GEM; 4. Vector GEP; 5. Vector EKQ; 6. Untransfected; 7. Purified CD59 protein.

[0028] Figure 13 The schematic AAV carrier design of Example 5 is illustrated.

[0029] Figure 14This demonstrates the large-scale production of soluble CD59 (sCD59) protein in the cell culture supernatant on day 6, and its purity was shown by SDS-PAGE gel analysis.

[0030] Figure 15A and 15B The figure illustrates the results of the cell lysis inhibition assay. Figure 15A The dose-response curves illustrate the percentage cytotoxicity of increased concentrations from normal human serum (NHS). Figure 15B This demonstrates the dose-response curves for the % maximum cytotoxicity, normalized to the BSA control, compared to the cytoprotective effects of the vectors GEM and CP40.

[0031] Figure 16 The schematic AAV carrier design of Example 6 is illustrated.

[0032] Figure 17 This describes the expression of the fusion protein in cells using HRP-conjugated mouse anti-human Fc antibody, as detected by SDS-PAGE and Western blotting. Lane contents: M. Pre-stained protein markers; 1. Vector GAM; 2. Vector GGE; 3. Vector GGG; 4. Vector GGQ; 5. Vector GKA; 6. Vector GKK; 7. Vector EKQ; 8. Vector CPE; 9. Untransfected cells.

[0033] Figure 18 This study describes the expression of a fusion protein in cells, detected by SDS-PAGE and Western blot using a biotinylated goat anti-human IgG antibody and an HRP-streptavidin conjugate. Lane contents: M. Pre-stained protein markers; 1. Vector GGE (repeat 1); 2. Vector GGE (repeat 2); 3. Vector GGG (repeat 1); 4. Vector GGG (repeat 2); 5. Vector GKA (repeat 1); 6. Vector GKA (repeat 2); PC, positive control, purified vector GGE protein.

[0034] Figure 19 This study describes the expression of the vector GGG protein in HEK293LTV cells in 6-well plates, which was confirmed to be pure by SDS-PAGE gel analysis. Lane contents: M. Protein markers; 1. Cell culture medium; 2. Flow-through; 3. Wash 1; 4. Wash 2; 5. Eluted sample; 6. Protein in PBS buffer.

[0035] Figure 20This study describes the expression of the vector GGE protein in HEK293LTV cells in 6-well plates, which was confirmed to be pure by SDS-PAGE gel analysis. Lane contents: M. Protein markers; 1. Cell culture medium; 2. Flow-through; 3. Wash 1; 4. Wash 2; 5. Eluted sample; 6. Protein in PBS buffer.

[0036] Figures 21A-21C This describes the expression of the mutant fusion protein in cells as detected by SDS-PAGE and hemolysis inhibition assay. Figure 21A This illustrates the design of mutant proteins derived from the vector GGG. Figure 21B The proteins expressed by Expi293F cells were identified, and SDS-PAGE gel analysis showed that they had high purity. Lane contents: M: protein markers; 1, vector KTP; 2, vector KTQ; 3, vector KTR; 4, vector KAT; 5, vector KAA; 6, vector KAC; 7, vector KAE; 8, vector KAG; 9, vector KAK; 10, vector KAM; 11, vector KAP; 12, vector GGG. Figure 21C The figure illustrates the results of hemolysis inhibition assays for all mutant proteins.

[0037] Figure 22A and 22B This describes the expression of fusion proteins in cells as detected by SDS-PAGE and the results from the hemolysis inhibition assay. Figure 22A This indicates that the expressed protein was pure, as shown by SDS-PAGE gel analysis. Lane contents: M. Protein markers; 1, vector GGE; 2, vector GGG; 3, vector KAG; 4, vector KKT; 5, vector KKA; 6, vector KPT; 7, vector KPC. Figure 22B The figure illustrates the results of hemolysis inhibition assays for all mutant proteins.

[0038] Figures 23A-23C The diagram illustrates the results of the binding affinity assays of the fusion protein with C3b, C3c, and C3. Figure 23A The estimated IC50 values ​​and KD-to-C3b values ​​for vectors KPT, CP40, KKT, KPC, KKA, GGE, KAG, and GGG are presented. Figure 23B The estimated IC50 values ​​and KD-to-C3c values ​​for vectors KPT, CP40, KKT, KPC, KKA, GGE, KAG, and GGG are presented. Figure 23C The estimated IC50 values ​​and KD-to-C3 values ​​for vectors KPT, CP40, KKT, KPC, KKA, GGE, KAG, and GGG are presented.

[0039] Figure 24 The schematic design of AAV carriers KMR, KKT, and KPP is illustrated.

[0040] Figures 25A-25D This demonstrates the expression of proteins secreted into the cell culture medium or remaining in the cell in ARPE-19 cells. Figure 25A This demonstrates the expression of CD59 from vectors KMR, KKT, and KPP. Figure 25B This demonstrates the expression of C3i from vectors KMR, KKT, and KPP. Figure 25C This demonstrates the expression of Fc-CNP from the vector KKT. Figure 25D This demonstrates the expression of endostatin from the vector KPP.

[0041] Figure 26 The schematic AAV carrier designs of carriers KGQ, KGR, KKT, and KKA are illustrated.

[0042] Figure 27A-27G The results and statistical analysis of AAV expression in mice were presented. Figure 27A The figure illustrates the results of CD59 expression from vectors KGQ, KGR, KKT, and KKA. Figure 27B A figure illustrating the results of Fc-CNP expression from the vector KKT. Figure 27C The figure illustrates the results of hFc expression from vectors KGQ, KGR, KKT, and KKA. Figure 27D A diagram illustrating the results of endostatin expression from the vector KKA. Figure 27E The figure illustrates the results depicting the expression of CD59 and C3i from vectors KGQ and KGR. Figure 27F The figure illustrates the results depicting the expression of CD59, C3i, and CNP from the vector KKT. Figure 27G The figure illustrates the results depicting the expression of CD59, C3i, and endostatin from the vector KKA.

[0043] Figures 28A-28C This explains the results of ligand binding assays from the C3i fusion protein. Figure 28A The results of ELISA assays of C3b bound to vectors KMR, KKT, and KPP proteins, along with estimated Kd values, are presented. Figure 28B The results of ELISA assays of C3c bound to vectors KMR, KKT, and KPP proteins, along with estimated Kd values, are presented. Figure 28CThe results of ELISA assays involving C3 and vectors KMR, KKT, and KPP proteins, along with estimated Kd values, are presented.

[0044] Figure 29 The figure illustrates the results of ligand binding assays from NPR-b and the vector KKT protein, Fc1-CNP36, and Fc4-CNP36.

[0045] Figure 30 The diagram illustrates the results of measurements of cGMP production in NIG-ETE cells triggered by the vector CME protein (Fc4-CNP36), the vector KKT protein (C3i-Fc4-CNP36), CNP-22, or Cp40.

[0046] Figure 31 The results of the endostatin ligand receptor assay from the carrier KPP protein and the estimated Kd value are illustrated in the diagram.

[0047] Figure 32 The schematic AAV carrier design of Example 12 is illustrated.

[0048] Figure 33 The results of ligand binding assays for wild-type and mutant sCD59 proteins were presented.

[0049] The novel features of this disclosure are set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description of illustrative embodiments. Detailed Implementation

[0050] Overview This document describes, in some aspects, an engineered polynucleotide comprising one or more expression cassettes encoding one or more angiogenesis inhibitors. In some embodiments, one or more angiogenesis inhibitors are operatively coupled (e.g., covalently linked). In some embodiments, one or more angiogenesis inhibitors comprise complement inhibitors, natriuretic peptides, inhibitors of the membrane attack complex (MAC), VEGF inhibitors, or combinations thereof. In some embodiments, the complement inhibitor comprises a complement 3 inhibitor (C3i) or a complement 3 (C3) degradation fragment. In some embodiments, the C3 degradation fragment comprises C3a, C3b, iC3b, C3f, C3c, C3d, C3g, or combinations thereof. In some embodiments, the engineered polynucleotide comprises a vector, such as an AAV vector. In some embodiments, the engineered polynucleotide encodes a first angiogenesis inhibitor and a second angiogenesis inhibitor. In some embodiments, the engineered polynucleotide encodes an angiogenesis inhibitor comprising a complement 3 inhibitor (C3i). For example, Figure 1The vector constructs encoding complement 3 inhibitors are described (e.g., vector GGG, vector KTP, vector KTQ, vector KTR, vector KAT, vector KAA, vector KAC, vector KAE, vector KAG, vector KAK, vector KAM, or vector KAP). In some embodiments, engineered polynucleotides encode complement 3 inhibitors and natriuretic peptides (e.g., C-type natriuretic peptides or CNPs), such as vector GGE. In some embodiments, complement 3 inhibitors and natriuretic peptides are covalently linked. For example, Figure 2A and 2B (Carrier GAM and carrier GGE) and Figure 3 (Top image: vector GKR sCD59-EVQL C3i-Fc4-CNP36; and bottom image: vector KAR sCD59-DKC3i-Fc4-CNP36) illustrates vector constructs encoding complement 3 inhibitors and CNPs. In some embodiments, the complement 3 inhibitor contains at least one modification compared to a comparable wild-type complement 3 inhibitor. In some embodiments, the complement 3 inhibitor contains an amino acid sequence having at least 80% identity with any of SEQ ID NO: 1-15.

[0051] In some embodiments, the engineered polynucleotide encodes one or more angiogenesis inhibitors, wherein one angiogenesis inhibitor comprises a CNP. In some embodiments, the CNP is covalently linked to an antibody or a fragment thereof (e.g., a crystallizable fragment region). In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 61-72. In some embodiments, the engineered polynucleotide encodes one or more angiogenesis inhibitors, wherein one angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC). In some embodiments, the MAC inhibitor comprises CD59. For example, Figure 3 The vector construct encoding CD59 is described. In some embodiments, CD59 comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 41-45. In some embodiments, the engineered polynucleotide encodes one or more angiogenesis inhibitors, wherein one angiogenesis inhibitor comprises collagen or a fragment thereof (e.g., endostatin or a fragment thereof). In some embodiments, endostatin comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 51. In some embodiments, the engineered polynucleotide encodes one or more angiogenesis inhibitors, wherein one angiogenesis inhibitor comprises a VEGF inhibitor. In some embodiments, the VEGF inhibitor comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 81-92.

[0052] In some embodiments, the engineered polynucleotide comprises a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector includes an adeno-associated virus (AAV) vector. In some embodiments, the viral vector encodes a modified viral capsid (e.g., as shown in Tables 13 and 14). In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises the amino acid sequence of any one of SEQ ID NO: 161-182 and SEQ ID NO: 191-210. In some embodiments, the engineered AAV capsid comprises the amino acid sequence of SEQ ID NO: 169. In some embodiments, the engineered AAV capsid comprises the amino acid sequence of any one of SEQ ID NO: 277-303 and SEQ ID NO: 312-319 and 325-329.

[0053] In some embodiments, the engineered polynucleotide encodes a first angiogenesis inhibitor comprising a complement 3 inhibitor and a second angiogenesis inhibitor comprising CNP36. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor and the second angiogenesis inhibitor comprises CNP36. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor and the second angiogenesis inhibitor comprises Fc-CNP36. In some embodiments, the engineered polynucleotide encodes a third angiogenesis inhibitor. In some embodiments, the third angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC).

[0054] In some embodiments, the engineered polynucleotide encodes a first angiogenesis inhibitor comprising an inhibitor of CD59 and a second angiogenesis inhibitor comprising CNP36. In some embodiments, the first angiogenesis inhibitor comprises CD59 and the second angiogenesis inhibitor comprises CNP36. In some embodiments, the first angiogenesis inhibitor comprises CD59 and the second angiogenesis inhibitor comprises Fc-CNP36. In some embodiments, the first angiogenesis inhibitor comprises CD59 and the second angiogenesis inhibitor comprises a complement 3 inhibitor fused to Fc-CNP36.

[0055] In some embodiments, the engineered polynucleotide encodes a first angiogenesis inhibitor comprising a complement 3 inhibitor and a second angiogenesis inhibitor comprising endostatin. In some embodiments, the engineered polynucleotide encodes an Fc region flanking the first and second angiogenesis inhibitors. In some embodiments, the first angiogenesis inhibitor comprises a VEGF inhibitor, and the second angiogenesis inhibitor comprises a complement 3 inhibitor. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises Fc-CNP36, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises Fc-CNP36, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59. In some embodiments, the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises a complement 3 inhibitor, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising Fc-CNP36. In some embodiments, the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor. In some embodiments, the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises a VEGF inhibitor, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59. In some embodiments, the first, second, and third angiogenesis inhibitors are not VEGF inhibitors. In some embodiments, after administration to a subject, the engineered polynucleotide inhibits angiogenesis in the subject. In some embodiments, after administration to a subject, the first or second angiogenesis inhibitor exhibits reduced inhibition of angiogenesis in the subject compared to inhibition of angiogenesis induced by a VEGF inhibitor.

[0056] This document describes, in some aspects, an engineered polypeptide comprising a first angiogenesis inhibitor and a second angiogenesis inhibitor. In some embodiments, the first and second angiogenesis inhibitors are covalently linked via a linker. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor. In some embodiments, the complement 3 inhibitor comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 1-15. In some embodiments, the first angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC). In some embodiments, the MAC inhibitor comprises CD59, which comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 41-45. In some embodiments, the MAC inhibitor comprises CD59, which comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 312-319. In some embodiments, the MAC inhibitor comprises CD59, which comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 325-329. In some embodiments, the second angiogenesis inhibitor comprises a natriuretic peptide. In some embodiments, the natriuretic peptide is covalently linked to an antibody or a fragment thereof. In some embodiments, the antibody or a fragment thereof comprises a crystallizable fragment (Fc) region. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 61-72. In some embodiments, the second angiogenesis inhibitor comprises collagen or a fragment thereof (e.g., endostatin or a fragment thereof). In some embodiments, the second angiogenesis inhibitor comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 51. In some embodiments, the second angiogenesis inhibitor comprises a VEGF inhibitor. In some embodiments, the VEGF inhibitor comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 81-92.

[0057] This document describes, in some aspects, a method for treating a disease or condition in a subject by administering an engineered polynucleotide or engineered peptide to the subject. In some embodiments, the method includes a single application to cure the disease or condition. In some embodiments, the method does not include daily application. In some embodiments, the disease or condition includes an eye disease. In some implementation schemes, ocular diseases include ocular ischemia syndrome, proliferative retinopathy, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, 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, choroidal agenesis, Leber congenital amaurosis, macular degeneration, polypoid choroidal vascular disease (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, and Oguchi disease. disease, Malattia leventinese (familial dominant drusen), blue cone monochromatic vision, or a combination thereof.

[0058] Engineered polynucleotides This document describes, in some aspects, an engineered polynucleotide comprising one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor. In some embodiments, the engineered polynucleotide comprises one or more expression cassettes for expressing one or more angiogenesis inhibitors. In some embodiments, the one or more expression cassettes encode a sequential polypeptide. In some embodiments, the sequential polypeptide comprises a protease peptide sequence. In some embodiments, the protease peptide sequence is cleavable by a protease endogenously expressed in the cell. Non-limiting examples of proteases may include serine endopeptides, aspartate endopeptides, cysteine ​​thiol endopeptides, metalloendinogens, or glutamate and threonine endopeptides. In some embodiments, the protease peptide sequence is cleavable by a serine endopeptide. In some embodiments, the protease peptide sequence is cleavable by furin. In some embodiments, the sequential polypeptide comprises a protease-cleavable sequence. In some embodiments, the protease-cleavable sequence is cleavable by any of the proteases described herein. In some embodiments, the protease-cleavable sequence is cleavable by furin. In some embodiments, the sequential polypeptide comprises a self-cleaving polypeptide sequence. In some embodiments, the self-cleaving polypeptide sequence comprises a 2A self-cleaving peptide sequence. Non-limiting examples of 2A self-cleaving peptide sequences may include T2A, P2A, E2A, F2A, or combinations thereof. In some embodiments, the self-cleaving polypeptide sequence comprises an F2A peptide sequence. In some embodiments, the sequential polypeptide comprises a protease-cleavable sequence and a self-cleaving polypeptide sequence. For example, the sequential polypeptide described herein may comprise a furin-F2A fusion polypeptide sequence. In some embodiments, the engineered polynucleotide comprises a viral vector, such as an AAV vector.

[0059] In some embodiments, the engineered polynucleotide includes one or more promoters or internal ribosome entry sites (IRES). In some embodiments, the expression cassette includes one or more promoters or IRES. In some embodiments, the expression cassette is under the expression control of a promoter. In some embodiments, the expression cassette is under the expression control of a promoter. In some embodiments, the expression cassette may further exert expression control via at least one IRES.

[0060] 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, one or more angiogenesis inhibitors each encode one of the one or more expression cassettes.

[0061] In some embodiments, the engineered polynucleotide encodes a first angiogenesis inhibitor and a second angiogenesis inhibitor. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor (C3i). In some embodiments, the engineered polynucleotide encodes a complement 3 inhibitor covalently linked to a natriuretic peptide. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any of SEQ ID NO: 1-15 (Table 10). In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any of SEQ ID NO: 1. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 2. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 3. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 4. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 5. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 5.In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 6. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 7. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 8. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 9. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 10. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 11. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 12. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 12.In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 13. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 14. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 15. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 15.

[0062] In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as specified in SEQ ID NO: 1-15. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as specified in SEQ ID NO: 1. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as specified in SEQ ID NO: 2. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as specified in SEQ ID NO: 3. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as specified in SEQ ID NO: 4. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 5 consisting of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 6 consisting of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 7 consisting of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 8 consisting of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 9 consisting of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 10 consisting of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 11 consisting of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 12 consisting of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids.In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 13 consisting of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 14 consisting of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids. In some embodiments, the complement 3 inhibitor (C3i) comprises the amino acid sequence of SEQ ID NO: 15 consisting of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids.

[0063] Table 10. Exemplary complement 3 inhibitor (C3i) amino acid sequences

[0064] In some embodiments, the engineered polynucleotide encoding the complement 3 inhibitor comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any of SEQ ID NO: 20-33 (Table 11). In some embodiments, the complement 3 inhibitor (C3i) is encoded by a nucleic acid sequence of any of SEQ ID NO: 20-33. In some embodiments, the complement 3 inhibitor (C3i) is encoded by a nucleic acid sequence of at least 50, at least 60, at least 70, or at least 50 consecutive nucleotide bases from any of SEQ ID NO: 20-33.

[0065] Table 11. Exemplary nucleic acid sequences encoding complement 3 inhibitors

[0066] In some embodiments, the engineered polynucleotide encodes a natriuretic peptide or a natriuretic peptide fusion protein (e.g., the CNP-Fc fusion protein described herein). In some embodiments, the natriuretic peptide is a CNP. In some embodiments, the CNP is covalently linked to a complement 3 inhibitor. In some embodiments, the CNP is covalently linked to a complement 3 inhibitor via a linker. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any of SEQ ID NO: 61-72 (Table 12). In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 61. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 62. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 63. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 64. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 65. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 66. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 67.In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 68. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 69. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 70. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 71. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 72. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises the amino acid sequence of any one of SEQ ID NO: 61-72.

[0067] Table 12. Exemplary amino acid sequences of natriuretic peptides or natriuretic peptide fusion proteins

[0068] In some embodiments, the engineered polynucleotide encodes an inhibitor of the membrane attack complex (MAC). In some embodiments, the MAC inhibitor comprises CD59. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any of the amino acid sequences in SEQ ID NO: 41-45 (Table 13). In some embodiments, CD59 comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 41. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 42. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 43. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 44. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 45. In some embodiments, CD59 comprises an amino acid sequence of any one of SEQ ID NO: 41-45.

[0069] Table 13. Exemplary CD59 amino acid sequences

[0070] In some embodiments, the engineered polynucleotide encodes an inhibitor of the membrane attack complex (MAC). In some embodiments, the MAC inhibitor comprises CD59. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any of SEQ ID NO: 312-319 (Table 40). In some embodiments, CD59 comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 312. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 313. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 314. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 315. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 316. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 317. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 318. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 319. In some embodiments, CD59 comprises an amino acid sequence of any one of SEQ ID NO: 312-319.

[0071] In some embodiments, the engineered polynucleotide encodes an inhibitor of the membrane attack complex (MAC). In some embodiments, the MAC inhibitor comprises CD59. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any of SEQ ID NO: 325-329 (Table 50). In some embodiments, CD59 comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 325. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 326. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 327. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 328. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 329. In some embodiments, CD59 comprises an amino acid sequence of any one of SEQ ID NO: 325-329.

[0072] Table 45. Exemplary CD59 amino acid sequences

[0073] In some embodiments, the engineered polynucleotide encodes collagen or a fragment thereof. In some embodiments, the collagen or a fragment thereof comprises endostatin or a fragment thereof. In some embodiments, endostatin or a fragment thereof comprises an amino acid sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity with SEQ ID NO: 51: MHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASK. In some embodiments, endostatin or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 51.

[0074] In some embodiments, the engineered polynucleotide encodes a VEGF inhibitor. In some embodiments, the VEGF inhibitor comprises repressive RNA for targeting and degrading VEGF transcripts. In some embodiments, the VEGF inhibitor comprises an antibody or a fragment thereof. In some embodiments, the VEGF antibody binds to VEGF to reduce angiogenesis signaling involving the VEGF signaling pathway. In some embodiments, the VEGF antibody binds to VEGF-A, VEGF-B, VEGF-C, VEGF-D, or combinations thereof. In some embodiments, the VEGF antibody binds to one or more VEGF-A isoforms, including VEGF121, VEGF145, VEGF148, VEGF162, VEGF165, VEGF165b, VEGF183, VEGF189, or VEGF206. In some embodiments, the antibody comprises a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-chain variable fragment (scFv), bis-scFv, (scFv)2, a double-chain antibody, a microantibody, a nanobody, a triple-chain antibody, a tetra-chain antibody, a disulfide-stabilized Fv protein (“dsFv”), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or a combination thereof, a binding fragment thereof, or a chemically modified derivative thereof. Non-limiting examples of VEGF antibodies include ranibizumab or bevacizumab. In some embodiments, the VEGF antibody comprises a polypeptide sequence, a combination thereof, or a fragment thereof, having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or higher identity with any of SEQ ID NO: 81-87 (Table 14).

[0075] Table 14. Exemplary amino acid sequences of VEGF antibodies

[0076] In some embodiments, the VEGF inhibitor is not an antibody. For example, the VEGF inhibitor described herein may comprise a VEGF receptor, a combination of VEGF receptors, or a fragment thereof, for binding to VEGF to inhibit or reduce the VEGF signaling pathway. The VEGF receptor may include VEGF receptor 1 (FLT1), VEGF receptor 2 (KDR / FLK1), VEGF receptor 3 (FLT4), fragments thereof, or combinations thereof. In some embodiments, the VEGF receptor may be a soluble VEGF receptor. For example, a soluble VEGF receptor may comprise soluble VEGFR1, soluble VEGFR2, soluble VEGFR3, soluble fragments thereof, or combinations thereof. In some embodiments, the non-antibody VEGF inhibitor comprises at least one of FLT1, KDR / FLK1, FLT4, fragments thereof, or combinations thereof. In some embodiments, the non-antibody VEGF inhibitor comprises at least one of soluble FLT1, soluble KDR / FLK1, soluble FLT4, fragments thereof, or combinations thereof. In some embodiments, the non-antibody VEGF inhibitor comprises a VEGF-Trap. In some embodiments, the non-antibody VEGF inhibitor comprises a polypeptide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or higher identity with any of SEQ ID NO: 88-92 (Table 15).

[0077] Table 15: Exemplary amino acid sequences of non-antibody VEGF inhibitors

[0078] In some embodiments, the engineered polynucleotide comprises a viral vector, such as an AAV vector comprising one or more expression cassettes for one or more angiogenesis inhibitors. In some embodiments, the engineered polynucleotide comprises a vector. In some embodiments, the vector is a viral vector. In some embodiments, the engineered polynucleotide comprises an AAV vector. In some embodiments, the engineered polynucleotide comprises an AAV vector encoding an engineered AAV capsid. In some embodiments, the AAV vector comprises one or more expression cassettes encoding an engineered polypeptide comprising: a peptide or a fusion protein comprising an antibody or a fragment thereof operatively coupled to the peptide.

[0079] In some embodiments, the engineered polynucleotide is a vector. In some embodiments, the engineered polynucleotide is a viral vector containing an AAV vector. In some embodiments, the engineered polynucleotide is an AAV vector containing an AAV serotype, which 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 containing the AAV2 serotype. In some embodiments, the AAV vector encodes a modified AAV capsid. In some embodiments, the engineered polynucleotide comprises a viral vector. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the AAV vector comprises an AAV serotype, which includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV vector is an AAV2 vector. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises the amino acid sequence of any one of SEQ ID NO: 161-182 and SEQ ID NO: 191-210. In some embodiments, the engineered AAV capsid comprises the amino acid sequence of SEQ ID NO: 169.

[0080] In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any of SEQ ID NOs: 101-103 and 121-129. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 101. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 102. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 103. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 121. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 122. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 123. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 124. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 126. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 127. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 128. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 129. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of any one of SEQ ID NO: 101-103 and 121-129.

[0081] In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any of SEQ ID NO: 111-113 and 43-45. In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence of any of SEQ ID NO: 111-113 and 43-45.

[0082] In some embodiments, the engineered polynucleotide encodes a first angiogenesis inhibitor and a second angiogenesis inhibitor. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises a CNP. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises CNP36. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises an Fc region and CNP36. In some embodiments, the engineered AAV further encodes a third angiogenesis inhibitor. In some embodiments, the third angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC). In some embodiments, the MAC inhibitor comprises CD59. In some embodiments, the engineered polynucleotide encodes a protease site flanking both the second and third angiogenesis inhibitors. In some embodiments, the first angiogenesis inhibitor comprises an inhibitor of CD59, and the second angiogenesis inhibitor comprises a CNP. In some embodiments, the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises CNP36. In some embodiments, the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises Fc-CNP36. In some embodiments, the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises a complement 3 inhibitor fused to Fc-CNP36. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises endostatin. In some embodiments, an engineered polynucleotide encodes the Fc region of both the first and second angiogenesis inhibitors. In some embodiments, the first angiogenesis inhibitor comprises a VEGF inhibitor, and the second angiogenesis inhibitor comprises a complement 3 inhibitor. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises Fc-CNP36, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59. In some embodiments, the engineered polynucleotide further encodes a protease site flanking both the second and third angiogenesis inhibitors. In some embodiments, the protease site comprises a furin protease site. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises Fc-CNP36, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59. In some implementations, the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises a complement 3 inhibitor, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising Fc-CNP36.In some embodiments, the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor. In some embodiments, the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises a VEGF inhibitor, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor.

[0083] In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59. In some embodiments, the first, second, and third angiogenesis inhibitors are not VEGF inhibitors. In some embodiments, after administration to a subject, the first and second angiogenesis inhibitors inhibit angiogenesis in the subject. In some embodiments, after administration to a subject, either the first or second angiogenesis inhibitor exhibits reduced inhibition of angiogenesis in the subject compared to angiogenesis inhibition induced by a VEGF inhibitor.

[0084] In some embodiments, the engineered polynucleotide encodes sCD59-Fc4-CNP36. In some embodiments, the engineered polynucleotide encodes sCD59-C3i-Fc4-CNP36. In some embodiments, the engineered polynucleotide encodes C3i-Fc4-endostatin. In some embodiments, the engineered polynucleotide encodes aflibercept (SEQ ID NO: 71)-linker-C3i. In some embodiments, the engineered polynucleotide encodes C3i-Fc4-CNP36-furin protease-sCD59. sCD59-furin protease 2A-C3i-Fc4-CNP36. In some embodiments, the engineered polynucleotide encodes sCD59-furin protease 2A-endostatin-linker-C3i. In some embodiments, the engineered polynucleotide encodes sCD59-furin protease 2A-aflibercept-linker-C3i. In some embodiments, the engineered polynucleotide encodes C3i-Fc4-CNP36-furin protease-mCD59. In some embodiments, the engineered polynucleotide encodes C3i-Fc4-endostatin-furin protease sCD59. In some embodiments, the engineered polynucleotide encodes C3i-Fc4-CNP36. In some embodiments, the engineered polynucleotide encodes Fc4-(G4S)4-CNP36. In some embodiments, the engineered polynucleotide encodes (DK)C3i-Fc4-CNP36. In some embodiments, the engineered polynucleotide encodes Fc4-C3i. In some embodiments, the engineered polynucleotide encodes aflibercept-Fc1-C3i. In some embodiments, the engineered polynucleotide encodes C3i-Fc4-endostatin. In some embodiments, the engineered polynucleotide encodes (DK)C3i-Fc4-endostatin. In some embodiments, the engineered polynucleotide encodes Fc-C3i(T14A). In some embodiments, the engineered polynucleotide encodes Fc-C3i(+2Y). In some embodiments, the engineered polynucleotide encodes Fc-C3i(+2Y, T14A). In some embodiments, the engineered polynucleotide encodes Fc-C3i(-N15). In some embodiments, the engineered polynucleotide encodes Fc-C3i(T14A, -N15). In some embodiments, the engineered polynucleotide encodes Fc-C3i(+2Y, -N15). In some embodiments, the engineered polynucleotide encodes Fc-C3i(N15Q). In some embodiments, the engineered polynucleotide encodes Fc-C3i(T14A, N15Q). In some embodiments, the engineered polynucleotide encodes Fc-C3i(+2Y, N15Q).In some embodiments, the engineered polynucleotide encodes Fc-C3i(+2Y, T14A, N15Q). In some embodiments, the engineered polynucleotide encodes Fc4-C3i(N15Q). In some embodiments, the engineered polynucleotide encodes C3i(N15Q)-Fc4-CNP36. In some embodiments, the engineered polynucleotide encodes C3i(N15Q)-Fc4-endostatin. In some embodiments, the engineered polynucleotide encodes C3i(N15Q)-Fc4-C3i(N15Q). In some embodiments, the engineered polynucleotide encodes C3i(N15Q)-Fc4-endostatin. In some embodiments, the engineered polynucleotide encodes sCD59. In some embodiments, the engineered polynucleotide encodes Vh(DK)sCD59. In some embodiments, the engineered polynucleotide encodes vsCD59-6×His. In some embodiments, the engineered polynucleotide encodes vsCD59 N18Q-6×His. In some embodiments, the engineered polynucleotide encodes vsCD59 Q34E-6×His. In some embodiments, the engineered polynucleotide encodes vsCD59 K38R-6×His. In some embodiments, the engineered polynucleotide encodes vsCD59 Q34E, K38R-6×His. In some embodiments, the engineered polynucleotide encodes vsCD59 N18Q, K38R-6×His. In some embodiments, the engineered polynucleotide encodes vsCD59 N18Q, Q33E, K38R-6×His.

[0085] In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with any of SEQ ID NO: 250-276, 304-311, and 320-324. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 250. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 251. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 252. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 253. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 254. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 255. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 256. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 257. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 258. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 259. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 260. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 261. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 262. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 263. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 264. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 265. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 266. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 267. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 268.In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 269. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 270. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 271. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 272. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 273. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 274. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 275. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 276. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 304. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 305. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 306. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 307. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 308. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 309. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 310. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 311. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 320. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 321. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 322. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 323. In some embodiments, the engineered polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 324.

[0086] In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with any of the amino acid sequences in SEQ ID NO: 277-303, 312-319, and 325-329. In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence in SEQ ID NO: 277-303, 312-319, and 325-329.

[0087] In some implementation schemes, engineered polynucleotides encode, for example, Figure 1 The sequence is shown. In some implementations, engineered polynucleotides encode, for example, Figure 2A The sequence shown in -B. In some implementations, engineered polynucleotides encode sequences such as... Figure 3 The sequence is shown. In some implementations, engineered polynucleotides encode, for example, Figure 4 The sequence is shown. In some implementations, engineered polynucleotides encode, for example, Figure 5A The sequence shown is -C. In some implementations, engineered polynucleotides encode sequences such as... Figure 16 The sequence is shown. In some implementations, engineered polynucleotides encode, for example, Figure 24 The sequence is shown. In some implementations, engineered polynucleotides encode, for example, Figure 26 The sequence is shown. In some implementations, engineered polynucleotides encode, for example, Figure 32The sequence is shown. In some cases, engineered polynucleotides include additional features. Additional features may include sequences such as tags, signal peptides, intron sequences, promoters, filler sequences, etc. In some cases, engineered polynucleotides encode signal peptides. Signal peptides, sometimes referred to as signal sequences, targeting signals, localization signals, localization sequences, transport peptides, leader sequences, or leader peptides, are short peptides present at the N-terminus of most newly synthesized proteins destined for secretion pathways. These proteins include those present in certain organelles (endoplasmic reticulum, Golgi apparatus, or endosomes), secreted from the cell, or inserted into most cell membranes. In some cases, the nucleic acids presented herein may contain signal peptides. Signal peptides can be of any length, but are typically 15-30 amino acids long. Signal peptides can 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 can be used, including but not limited to: human antibody heavy chain (Vh), human antibody light chain (Vl), and aflibercept.

[0088] In some cases, engineered polynucleotides contain intron sequences. An intron is any nucleotide 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 an RNA transcript or the DNA that encodes 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 after splicing through further processing to generate non-coding RNA molecules. Alternative splicing is widely used to generate multiple proteins from a single gene. Furthermore, some introns play crucial roles in a wide range of gene expression regulatory functions, such as nonsense-mediated degradation and mRNA export. In embodiments, intron sequences are included in the nucleic acids of this disclosure and may be selected from: hCMV intron A, adenovirus triple leader sequence introns, SV40 introns, hamster EF-1 α gene intron 1, intercalation sequence introns, human growth hormone introns, and / or human β-globin introns. Any number of intron sequences is considered. In the implementation scheme, 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.

[0089] In the embodiments, the engineered polynucleotide includes the additional feature of a promoter. A promoter is a DNA sequence that binds to a protein and initiates the transcription of a single RNA from its downstream DNA. This 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 can be approximately 100-1000 base pairs long. Various promoters are considered and may be employed in the engineered polynucleotides of this disclosure. In the implementation scheme, the promoters are: cytomegalovirus (CMV) promoter, elongation factor 1α (EF1α) 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, polyhedral promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKIIa) promoter, GAL1 promoter, GAL10 promoter, TEF1 promoter, glyceraldehyde-3-phosphate dehydrogenase (GDS) promoter, ADH1 promoter, CaMV35S promoter, Ubi promoter, human polymerase III RNA (H1) promoter, U6 promoter, their polyadenylated constructs, and any combination thereof. In some cases, the promoter is the CMV promoter.

[0090] Any of the engineered polynucleotides provided may contain a viral vector sequence. The viral vector may be, but is not limited to, lentivirus, retrovirus, or adeno-associated virus. The viral vector may 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 considered, including but 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 better suit its use for biological function, tissue, or cell selection. In some embodiments, the AAV vector is AAV2 and variants AAV2.N53 and AAV2.N54. Chimeric AAV vectors that may contain at least two AAV serotypes are also considered. In some cases, at least three, at least four, at least five, at least six, at least seven, or up to eight different serotypes are combined in the chimeric AAV vector. In some cases, only a portion of the AAV is chimeric. For example, suitable portions may include the 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 a wild-type AAV capsid protein that is otherwise comparable. In some cases, mutations may occur in VP1 and VP2, VP1 and VP3, VP2 and VP3, or VP1, VP2, and VP3. In some embodiments, at least one of VP1, VP2, and VP3 has 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, VPs may be removed. For example, in some embodiments, the mutant AAV does not include at least one of VP1, VP2, or VP3.

[0091] In some cases, AAV vectors can be modified. For example, AAV vectors can contain modifications such as insertions, deletions, chemical alterations, 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.

[0092] Codon optimization In embodiments, the engineered polynucleotides described herein contain modifications that confer increased expression of one or more angiogenesis inhibitors described herein. For example, one or more angiogenesis inhibitors are derived from natural gene sequences and contain unmodified sequences that are not optimized for introduction and expression in target cells. In embodiments, the isolated engineered polynucleotides are codon-optimized. Codon optimization can be cell-type specific to codon usage. Different organisms and cell types exhibit a tendency to use certain codons for the same amino acid rather than others. Some species are known to almost completely avoid certain codons. Similarly, some cell types tend to use certain codons for the same amino acid rather than others. In embodiments, methods for optimizing the codons of engineered polynucleotides may include redistributing codon usage based on the frequency of use of each codon in target cells. In some cases, target cells may be certain tissues or organs. In some cases, modifications are made to increase guanine and / or cytosine content.

[0093] In the implementation scheme, the engineered nucleic acid sequence may be modified to replace at least one codon with another codon encoding the same amino acid. In some cases, the codon is modified within the coding region of the sequence. In some cases, the codon is modified within the non-coding region of the sequence. In some cases, the codon is modified within approximately 100, 50, 25, 15, or 5 bases from the stop codon. E-CAI can be used to estimate the value of the codon fitness index.

[0094] This paper considers various modifications. 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-restrictive substitutions provided in Table 16 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 substituted. In one embodiment, the engineered polynucleotide contains 3 codon modifications. In another embodiment, the engineered polynucleotide contains 16 codon modifications. In the 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 the embodiments, the engineered polynucleotide comprises two codon modifications: AGA to AGG and at least one of the following: CCT to CCC, AGC to TCC, or CCC to CCG. In the embodiments, the engineered polynucleotide comprises three codon modifications: AGA to AGG and at least two of the following: CCT to CCC, AGC to TCC, or CCC to CCG. In the embodiments, the engineered polynucleotide comprises four codon modifications: AGA to AGG, CCT to CCC, AGC to TCC, and CCC to CCG. Additional modifications may include any codon modifications provided in Table 16 combined with the above codons and / or any possible additional modifications from Table 16. In one implementation, the nucleic acid is modified such that AGA is replaced with AGG and CCT is replaced with CCC. In another implementation, the nucleic acid is modified such that AGA is replaced with AGG and AGC is replaced with TCC. In yet another implementation, the nucleic acid is modified such that AGA is replaced with AGG and CCC is replaced with CCG.

[0095] Table 16. Non-restrictive exemplary codons that can be used interchangeably to modify nucleic acids. In the following codons, thymine can be replaced with uracil.

[0096] In some embodiments, the engineered polynucleotide sequence may include a viral vector sequence. In some embodiments, the viral vector sequence may be an scAAV vector sequence. In some embodiments, the AAV vector sequence may be serotypes 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 serotype AAV2. In some embodiments, the viral vector sequence may contain sequences of at least two AAV serotypes. In some embodiments, the at least two serotypes may be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV11, and AAV12.

[0097] In some cases, modifications can also include chemical modifications. Modified nucleic acids can include modifications to their backbone, sugars, or nucleobases, as well as even novel bases or base pairs. Modified nucleic acids can possess 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 increased target binding.

[0098] Exemplary chemical modifications include, but are not limited to: 2'F, 2'-fluorine; 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-ribulosinic acid; TNA, α-L-threonine nucleic acid; tPhoNA, 3'-2'-phosphonomethylthreonyl nucleic acid; dXNA, 2'-deoxyxylanic acid; PS, phosphate thiosulfate; phNA, alkylphosphonate nucleic acid; PNA and peptide nucleic acid.

[0099] Modified outer shell This article provides compositions containing modified adeno-associated virus (AAV) capsids and methods of using them. Modified AAV capsids may contain exogenous sequences compared to otherwise comparable unmodified AAV capsids. Exogenous sequences may refer to exogenous polypeptide sequences. AAV capsids can be modified to impart improved functionality to them and any compositions and / or methods utilizing them, resulting in better therapeutic agents, particularly for ocular use.

[0100] The AAV wild-type (WT) genome contains at least three genes: rep , capAnd X. The X gene is located at the 3' end of the genome (nucleotides 3929-4393 in AAV2) and appears to encode a protein that has a supporting function in genome replication. Significantly more information is available. rep and cap . rep The gene is located in the first half of the AAV WT genome and encodes a family of non-structural proteins (Rep proteins) required for viral transcriptional control and replication, as well as for packaging the viral genome into a newly generated, pre-assembled capsid. The second half of the AAV genome contains... cap The gene encodes viral proteins (VPs) VP1, VP2, and VP3, as well as the assembly activation protein (AAP). 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) reduce the presence of VP1 and VP2 to approximately 1 / 10 that of 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 (“common VP3 region”), and additionally, VP2 and VP1 share approximately 65 amino acids (“common VP1 / VP2 region”). Only VP1 contains a unique sequence (approximately 138 amino acids, unique to VP1) at its N-terminus. AAP was identified in 2010 as a substitute for... cap The ORF encodes a 23 kDa protein. It is used to stabilize newly generated VP proteins and transport them from the cytoplasm to the nucleus. Although AAV serotypes 1-3, 6-9, and rh10 failed to produce capsids in the absence of AAP, AAV4 and AAV5 have been reported to produce capsids in small quantities, but these are detectable.

[0101] In one aspect, the AAV may contain modifications. Modifications may be polypeptide sequences encoding the rep, cap, and / or X domains of the AAV. In some cases, the modification may be a cap polypeptide. The cap polypeptide may be modified in any of the VP domains, 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. Furthermore, VP2 and VP3 may be modified, or VP1, VP2, and VP3 may be modified. Other combinations, such as modifications in Rep and Cap, Cap and X, Rep and X, and / or Rep, Cap, and X, are considered. Any combination of domains may be modified, such as any of the foregoing VP modifications combined with Rep and / or X modifications. In some cases, Rep and VP1 and / or VP2 are modified. In some respects, the subject Rep is modified. Rep modification may include modifications as provided herein and may be in at least one of Rep 78, Rep 68, Rep 52, or Rep 40. In some cases, Rep is an AAV serotype different from the subject capsid.

[0102] In some cases, the AAV capsid is modified. The AAV serotype capsid is assembled from 60 VP monomers, approximately 50 copies of VP3, 5 copies of VP2, and 5 copies of VP1. The topologically prominent capsid surface structure consists of pores or “channel-like structures” for each quintuple, indentations for each duplex, and three protrusions around each triple axis of symmetry. These pores allow exchange between the capsid’s interior and exterior. The indentations, more precisely, the bottom layer of each duplex axis, are the thinnest part of the viral capsid. The protrusions around the triple axes enclose five of the nine so-called variable regions (VRs). Specifically, VR-IV, VR-V, and VR-VIII form loops (loops 1-4) at the top of the protrusions, while VR-VI and VR-VII are present at their base. VRs differ between serotypes and are responsible for serotype-specific variations in antibody and receptor binding. Due to their exposure sites and their function in receptor binding, the VRs that form protrusion loops are ideal locations for capsid modifications aimed at redirecting or extending AAV tropism (cell surface targeting). While reorientation (vector retargeting) combines the ablation of natural receptor binding (e.g., through site-directed mutagenesis) with the insertion of ligands mediated by novel non-natural AAV receptors, AAV vectors with directional extensions increase the ability to transduce cells via additional receptors while maintaining their natural receptor binding capacity.

[0103] In some respects, modification of the AAV capsid can refer to the insertion of a foreign polypeptide sequence. In other respects, modification can refer to the deletion of a polypeptide sequence. Modification can also refer to the modification of at least one amino acid residue (classical or non-classical) in the polypeptide sequence.

[0104] Insertion may include inserting at least one exogenous amino acid residue into the sequence encoding the AAV capsid. The amino acid can refer to classical or non-classical amino acids. Any number of amino acid residues can be inserted. In some cases, the insertion site may be in the GH ring or ring IV of the AAV capsid protein, for example, in the solvent-accessible portion of the GH ring or ring IV of the AAV capsid protein.

[0105] In some cases, the modification involves inserting a foreign polypeptide sequence comprising the sequence of Formula 1: X0-X1-X2-X1-X3-X1-X1-X4. 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, where X3 is E, S, or Q. In some cases, X4 is K, R, E, or A. In some cases, Formula 1 also includes X5. X5 can be proline (P) or R.

[0106] In some cases, Formula 1 comprises: LALG-X3-X1-X1-X4 (SEQ ID NO: 232), LKLG-X3-X1-X1-X4 (SEQ ID NO: 233), or VKLG-X3-X1-X1-X4 (SEQ ID NO: 234). In some cases, Formula 1 comprises VKLG-X3-X1-X1-X4 (SEQ ID NO: 235). In some cases, the exogenous polypeptide is VKLG-X3-X1-T-X4 (SEQ ID NO: 236) and / or VKLG-X3-X1-X1-K (SEQ ID NO: 237). In some cases, the exogenous polypeptide comprises LALG-X3-X1-X1-X4 (SEQ ID NO: 238). In some cases, the exogenous peptide comprises LALG-X3-X1-T-X4 (SEQ ID NO: 239) and / or LALG-X3-X1-S-X4 (SEQ ID NO: 240). In some cases, the exogenous peptide comprises LALG-X3-X1-TR (SEQ ID NO: 241), LALG-X3-X1-TK (SEQ ID NO: 242), LALG-X3-X1-TE (SEQ ID NO: 243), and / or LALG-X3-X1-TA (SEQ ID NO: 244). In some cases, the exogenous peptide comprises LALG-X3-X1-SK (SEQ ID NO: 246). In some cases, the exogenous peptide comprises LKLG-X3-X1-X1-X4 (SEQ ID NO: 247). In some cases, the exogenous peptide comprises LKLG-X3-X1-T-X4 (SEQ ID NO: 248). In some cases, the exogenous peptide contains LKLG-X3-X1-TK (SEQ ID NO: 249).

[0107] In some cases, the exogenous polypeptide comprises a sequence of Formula 1. In some cases, the sequence of Formula 1 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 at most about 100% identity with the sequences in Table 17. In some cases, the exogenous polypeptide is one of the residues in Table 17 with 0-2 modifications.

[0108] 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 may be inserted at the same or different positions. In one aspect, any of the exogenous polypeptide sequences provided in Table 17 may be inserted into an unmodified AAV capsid sequence, such as the wild-type sequences provided in Table 18, to generate a modified AAV capsid.

[0109] Table 17. Exemplary exogenous polypeptide sequences that can be inserted into the AAV capsid (exemplary insertion sites for AAV2 are shown, but comparable sites for other AAV serotypes are also considered).

[0110] Table 18. Exemplary wild-type AAV capsid polypeptide sequences

[0111] Similarly, deletions can include the deletion of at least one amino acid residue in the sequence encoding the AAV capsid. Any number of amino acids can be deleted. In some cases, at least 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 50 exogenous amino acid residues may be inserted and / or deleted in the polypeptide sequence encoding the AAV capsid. In some cases, at least 1-5, 5-10, 10-15, 15-20, or combinations thereof of exogenous amino acid residues may be inserted or deleted into the polypeptide sequence encoding the AAV capsid. In some cases, about 5 to 11 amino acids may be inserted at an insertion site in the GH ring or ring IV of the capsid protein relative to the corresponding unmodified AAV capsid protein. For example, the insertion site may be between amino acids 587 and 588 of AAV2, or at the corresponding position of the capsid subunit of another AAV serotype. It should be noted that insertion sites 587-588 are based on the AAV2 capsid protein. About 5 to 11 amino acids may be inserted at the corresponding site in AAV serotypes other than AAV2 (e.g., AAV5, AAV6, AAV8, AAV9, etc.).

[0112] In some embodiments, the insertion site is a single insertion site between two adjacent amino acids located between amino acids 570-614 of VP1 in any AAV serotype. For example, the insertion site is between two adjacent amino acids located between amino acids 570-610, am-600, 570-575, 575-580, 580-585, 585-590, 590-600, or 600-614 of VP1 in any AAV serotype or variant. For example, the insertion site may be between amino acids 580 and 581, 581 and 582, 583 and 584, 584 and 585, 585 and 586, 586 and 587, 587 and 588, 588 and 589, or 589 and 590. The insertion site can be between amino acids 575 and 576, 576 and 577, 577 and 578, 578 and 579, or 579 and 580. The insertion site can also be between amino acids 590 and 591, 591 and 592, 592 and 593, 593 and 594, 594 and 595, 595 and 596, 596 and 597, 597 and 598, 598 and 599, or 599 and 600.

[0113] In some respects, 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.

[0114] For example, the insertion site can be between amino acids 450 and 460 of the AAV capsid protein, as shown in Table 18. For instance, the insertion site can be (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.

[0115] In some embodiments, the subject capsid protein comprises a GH ring containing 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 with the amino acid sequences shown in Table 18. Those skilled in the art, based on comparisons of the amino acid sequences of capsid proteins of various AAV serotypes, will recognize that the insertion site “amino acids 587-588 corresponding to AAV2” is present in the capsid protein of any given AAV serotype.

[0116] In some cases, the exogenous polypeptide may have 0 to 4 spacer amino acids (Y1-Y4) at the amino terminus and / or carboxyl terminus of any of the exemplary polypeptides in Table 17 or Formula 1. Suitable spacer amino acids include, but are not limited to, leucine, alanine, glycine, and / or serine.

[0117] Modifications to the AAV capsid may include modifications to at least one amino acid residue in the polypeptide sequence. In some cases, modifications may be made at any AAV capsid position as described herein and may include any number of modifications. In some cases, modifications may include mutations. Mutations may include point mutations, missense mutations, nonsense mutations, deletions, duplications, frameshifts, and / or amplifications of repetitive sequences.

[0118] On one hand, amino acids can be nonpolar aliphatic residues, such as glycine, alanine, valine, leucine, methionine, isoleucine, or proline. On the other hand, amino acid residues are aromatic and are phenylalanine, tyrosine, or tryptophan. On the other hand, amino acid residues are polar, uncharged, and are serine, threonine, cysteine, asparagine, or glutamine. On the other hand, amino acids are positively charged and are lysine, arginine, or histidine. On the other hand, amino acids are negatively charged and are aspartic acid or glutamic acid.

[0119] In some cases, the mutation is a point mutation. A point mutation involves a change from a charged amino acid residue to a polar or nonpolar amino acid residue. In some cases, the charged amino acid carries a positive charge. In some cases, the charged amino acid carries a negative charge.

[0120] Point mutations can be conserved mutations. Non-restrictive examples of conserved mutations include: nonpolar aliphatic amino acid to nonpolar aliphatic amino acid; polar amino acid to polar amino acid; positively charged amino acid to positively charged amino acid; negatively charged amino acid to negatively charged amino acid; and aromatic amino acid to aromatic amino acid. For example, 20 naturally occurring amino acids can share similar characteristics. Aliphatic amino acids can be: glycine, alanine, valine, leucine, or isoleucine. Amino acids containing a hydroxyl group 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 glutamine. Conserved mutations can be serine to glycine, serine to alanine, serine to serine, serine to threonine, or serine to proline. Conserved mutations can be arginine to asparagine, arginine to lysine, arginine to glutamine, arginine to arginine, or arginine to histidine. Conserved mutations can be leucine to phenylalanine, leucine to isoleucine, leucine to valine, leucine to leucine, or leucine to methionine. Conserved mutations can be proline to glycine, proline to alanine, proline to serine, proline to threonine, or proline to proline. Conserved mutations can be threonine to glycine, threonine to alanine, threonine to serine, threonine to threonine, or threonine to proline. Conserved mutations can be alanine to glycine, alanine to threonine, alanine to proline, alanine to alanine, or alanine to serine. Conserved mutations can be valine to methionine, valine to phenylalanine, valine to isoleucine, valine to leucine, or valine to valine. Conserved mutations can be glycine to alanine, glycine to threonine, glycine to proline, glycine to serine, or glycine to glycine. Conserved mutations can be from isoleucine to phenylalanine, isoleucine to isoleucine, isoleucine to valine, isoleucine to leucine, or isoleucine to methionine. Conserved mutations can be from phenylalanine to tryptophan, phenylalanine to phenylalanine, or phenylalanine to tyrosine. Conserved mutations can be from tyrosine to tryptophan, tyrosine to phenylalanine, or tyrosine to tyrosine. Conserved mutations can be from cysteine ​​to serine, cysteine ​​to threonine, or cysteine ​​to cysteine. Conserved mutations can be from histidine to asparagine, histidine to lysine, histidine to glutamine, histidine to arginine, or histidine to histidine. Conserved mutations can be from glutamine to glutamate, glutamine to asparagine, glutamine to aspartic acid, or glutamine to glutamine. Conserved mutations can be from asparagine to glutamate, asparagine to asparagine, asparagine to aspartic acid, or asparagine to glutamine.Conserved mutations can be lysine to asparagine, lysine to lysine, lysine to glutamine, lysine to arginine, or lysine to histidine. Conserved mutations can also be aspartic acid to glutamate, aspartic acid to asparagine, aspartic acid to aspartic acid, or aspartic acid to glutamine. Conserved mutations can also be glutamine to glutamine, glutamine to asparagine, glutamine to aspartic acid, or glutamine to glutamine. Conserved mutations can also be methionine to phenylalanine, methionine to isoleucine, methionine to valine, methionine to leucine, or methionine to methionine. Conserved mutations can also be tryptophan to tryptophan, tryptophan to phenylalanine, or tryptophan to tyrosine.

[0121] Non-restrictive examples of additional amino acid mutations may 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 L 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 of the mutations previously described.

[0122] Any of the aforementioned modifications, insertions, deletions, and / or mutations may be performed 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 rather a Rep and / or X sequence. As previously mentioned, the sequence may be in VP1, VP2, and / or VP3. In some cases, the loops of the capsid sequence (such as loop 3 and / or loop 4) are modified. In some cases, the residues of the sequences in Table 18 are modified.

[0123] In some cases, residues of the capsid polypeptide sequence in Table 18 are modified, such as by insertion, deletion, and / or mutation. In some cases, the modification is made from residues at positions 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, or combinations thereof. In some cases, the modification is made from residues at positions 200-300, 300-400, 400-500, 500-600, or combinations thereof. In some cases, the modification is made from residues at positions 300-500, or combinations thereof. In one instance, the insertion site is in the GH ring or ring IV of the AAV capsid protein, for example, in the solvent-accessible portion of the GH ring or ring 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.

[0124] 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 positions 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 positions 452, 453, 466, 467, 468, 471, 585, 586, 587, and / or 588 of any of SEQ ID NO: 221-226. In some cases, the modification is an insertion at positions 452, 453, 466, 467, 468, 471, 585, 586, 587, and / or 588 of SEQ ID NO: 221. In some cases, the modification is a mutation, and the mutation is R585A or R588A of any of SEQ ID NO: 221-226. In some cases, the modification is a mutation, and the mutation is R585A or R588A of SEQ ID NO: 221.

[0125] In some embodiments, the subject-modified AAV capsid does not contain any other amino acid modification mutations, substitutions, insertions, or deletions, except for the insertion of about 5 to about 11 amino acids in the rings (rings 3 and / or 4) relative to the corresponding unmodified AAV capsid protein. In other embodiments, the subject-variant AAV capsid also contains 1 to about 25 amino acid insertions, deletions, or substitutions compared to the unmodified AAV capsid protein, except for the insertion of about 7 to about 10 amino acids in the GH ring or ring IV relative to the corresponding parental AAV capsid protein. In other embodiments, the subject-variant AAV capsid also contains 1 to about 25 amino acid insertions, deletions, or substitutions compared to the parental AAV capsid protein, except for the insertion of about 7 to about 10 amino acids in the GH ring or ring IV relative to the corresponding parental AAV capsid protein. For example, in some embodiments, in addition to inserting about 7 to about 10 amino acids in the GH ring or ring IV relative to the corresponding parental AAV capsid protein, the subject AAV virospinal capsid also contains 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 parental AAV capsid protein.

[0126] In some cases, this document provides a chimeric AAV capsid. The chimeric capsid contains polypeptide sequences of at least two AAV serotypes. The chimeric capsid may contain 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 serotypes are distinct between VP1, VP2, and / or VP3. In some cases, the chimeric capsid contains sequences selected from at least two serotypes: 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 and second AAV serotypes of the chimeric AAV vector may be AAV11 and AAV6. In some cases, the first and second AAV serotypes of the chimeric AAV vector can be AAV12 and AAV6. In some cases, the chimeric capsid contains the following sequences: AAV2 and AAV5 or AAV2 and AAV6. In some cases, the chimeric capsid contains the following sequences: AAV2 and AAV5, AAV2 and AAV6, AAV2 and AAV8, AAV2 and AAV9, AAV2 and AAV1, and AAV2 and AAV12.

[0127] Compared to unmodified or wild-type AAV, the modifications to the AAV presented in this paper can confer increased activity in the modified AAV. The modifications presented in this paper can improve cell transduction, orientation, and / or reduce capsid-associated immunogenicity.

[0128] In some cases, the modifications described herein enhance cell transduction. Cell transduction can refer to the ability of AAV to infect cells (in vivo or in vitro) and / or deliver transgenes into cells.

[0129] In some cases, the modifications described herein enhance tropism. Enhanced tropism refers to an increased ability to transduce cells via additional receptors compared to unmodified AAV. In some respects, enhanced tropism can improve the infectivity of ocular cells, thereby improving gene therapy by utilizing modified AAV. In some cases, the modifications described herein can improve tropism towards ocular cells selected from: bipolar cells, retinal ganglion cells, horizontal cells, amacrine cells, epithelial cells, retinal pigment cells, photoreceptors, or any combination thereof. In some cases, the modifications improve tropism towards retinal cells.

[0130] This article also provides AAV vectors. AAV vectors contain: inverted terminal repeat (ITR), Rep, Cap, AAP, and X sequences. Typically, the AAV viral genome is side-mounted with an ITR, which serves as a packaging signal and replication origin. 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 numbers indicate molecular weight. The expression of AAP, as well as the viral capsid proteins VP1 (90 kDa), VP2 (72 kDa), and VP3 (60 kDa) (all encoded in the cap gene), is controlled by the p40 promoter. The X gene is located in the 3' end of the genome, in a region shared with the cap gene, and has its own promoter (p81). Although the X protein appears to enhance viral replication, AAP is crucial for capsid assembly. Three different VPs contribute to the icosahedral AAV2 capsid in a ratio of 1 (VP1):1 (VP2):10 (VP3).

[0131] The modified capsid proteins disclosed herein can be isolated, for example, purified. In some embodiments, the modified capsids disclosed herein are contained in an AAV vector or AAV virion (e.g., recombinant AAV virion, rAAV, or AAV viral particles). In other embodiments, such modified AAV vectors and / or AAV variant virions are used in vivo or in vitro for methods of treating ocular diseases of the primate retina (e.g., the human retina).

[0132] This document also provides vectors containing a modified AAV capsid. Any of the modifications previously described may be incorporated into the vectors provided herein. In some cases, the AAV vector contains a modified capsid that includes a foreign sequence in at least two loops of the VP domain, compared to otherwise comparable AAV capsid sequences lacking the foreign sequence. In some aspects, the vectors provided herein may also contain a transgenic sequence.

[0133] engineered peptides This document describes an engineered polypeptide in several aspects. In some embodiments, the engineered polypeptide is encoded by an engineered polynucleotide as described herein. In some embodiments, the engineered polypeptide comprises a first angiogenesis inhibitor and a second angiogenesis inhibitor as described herein. In some embodiments, the engineered polypeptide comprises a third angiogenesis inhibitor. In some embodiments, the engineered polypeptide comprises two or more angiogenesis inhibitors, wherein the two or more angiogenesis inhibitors are covalently linked by an antibody (e.g., the Fc region as described herein) or a linker.

[0134] In some embodiments, the engineered peptide comprises a complement 3 inhibitor and at least one additional angiogenesis inhibitor. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any of SEQ ID NO: 1-15. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence of any of SEQ ID NO: 1-15. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence of any of SEQ ID NO: 1-15 consisting of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids.

[0135] In some embodiments, the engineered polypeptide comprises a natriuretic peptide and at least one additional angiogenesis inhibitor. In some embodiments, the natriuretic peptide is a CNP. In some embodiments, the CNP is covalently linked to a complement 3 inhibitor. In some embodiments, the CNP is covalently linked to a complement 3 inhibitor via a linker. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any of SEQ ID NO: 61-72. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence of any of SEQ ID NO: 61-72.

[0136] In some embodiments, the engineered peptide comprises an inhibitor of the membrane attack complex (MAC) and at least one additional angiogenesis inhibitor. In some embodiments, the MAC inhibitor comprises CD59. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any of SEQ ID NOs: 41-45. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any of SEQ ID NOs: 312-319. In some embodiments, CD59 comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any of SEQ ID NOs: 325-329. In some embodiments, CD59 comprises the amino acid sequence of any one of SEQ ID NO: 41-45. In some embodiments, CD59 comprises the amino acid sequence of any one of SEQ ID NO: 312-319. In some embodiments, CD59 comprises the amino acid sequence of any one of SEQ ID NO: 325-329.

[0137] In some embodiments, the engineered polypeptide comprises collagen or a fragment thereof. In some embodiments, the collagen or a fragment thereof comprises endostatin or a fragment thereof. In some embodiments, endostatin or a fragment thereof comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 51. In some embodiments, endostatin or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 51.

[0138] In some embodiments, the engineered peptide comprises a VEGF inhibitor and at least one additional angiogenesis inhibitor. In some embodiments, the VEGF antibody comprises a peptide sequence, or a combination thereof, or a fragment thereof, having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% or higher identity with any of SEQ ID NO: 81-87 or SEQ ID NO: 88-92. In some embodiments, the VEGF antibody comprises a peptide sequence, or a combination thereof, or a fragment thereof, with any of SEQ ID NO: 81-87 or SEQ ID NO: 88-92.

[0139] In some embodiments, the engineered peptide comprises a complement 3 inhibitor, a crystallizable fragment (Fc) region, and a natriuretic peptide. In some embodiments, the engineered peptide comprises a complement 3 inhibitor and a natriuretic peptide. In some embodiments, the natriuretic peptide comprises a C-type natriuretic peptide (CNP). In some embodiments, the natriuretic peptide is covalently linked to an antibody or a fragment thereof. In some embodiments, the antibody or a fragment thereof comprises a crystallizable fragment (Fc) region. In some embodiments, the engineered peptide further comprises CD59, endostatin, a VEGF inhibitor, or a combination thereof. In some embodiments, the engineered peptide further comprises CD59, endostatin, and a VEGF inhibitor. In some embodiments, the engineered peptide further comprises CD59 and endostatin. In some embodiments, the engineered peptide further comprises endostatin and a VEGF inhibitor. In some embodiments, the engineered peptide further comprises CD59 and a VEGF inhibitor. In some embodiments, the engineered peptide further comprises CD59 and endostatin. In some embodiments, the engineered peptide comprises a complement 3 inhibitor and an inhibitor of the membrane attack complex (MAC). In some embodiments, the MAC inhibitor comprises CD59. In some embodiments, the engineered peptide comprises a natriuretic peptide, endostatin, a VEGF inhibitor, or a combination thereof. In some embodiments, the engineered peptide comprises a natriuretic peptide, endostatin, and a VEGF inhibitor. In some embodiments, the engineered peptide comprises a complement 3 inhibitor and collagen or a fragment thereof. In some embodiments, collagen or a fragment thereof comprises endostatin or a fragment thereof. In some embodiments, the engineered peptide further comprises a natriuretic peptide, CD59, a VEGF inhibitor, or a combination thereof. In some embodiments, the engineered peptide further comprises a natriuretic peptide. In some embodiments, the engineered peptide further comprises CD59. In some embodiments, the engineered peptide further comprises a VEGF inhibitor. In some embodiments, the engineered peptide comprises a complement 3 inhibitor and a VEGF inhibitor.

[0140] In some embodiments, the engineered peptide comprises CD59, an Fc region, and a natriuretic peptide. In some embodiments, the natriuretic peptide comprises a C-type natriuretic peptide (CNP). In some embodiments, the natriuretic peptide is covalently linked to an antibody or a fragment thereof. In some embodiments, the antibody or a fragment thereof comprises a crystallizable fragment (Fc) region. In some embodiments, the engineered peptide comprises a complement 3 inhibitor, endostatin, a VEGF inhibitor, or a combination thereof. In some embodiments, the engineered peptide comprises a complement 3 inhibitor, endostatin, and a VEGF inhibitor.

[0141] In some embodiments, the engineered peptide comprises CD59 and collagen or a fragment thereof. In some embodiments, the collagen or a fragment thereof comprises endostatin or a fragment thereof. In some embodiments, the collagen or a fragment thereof comprises endostatin. In some embodiments, the engineered peptide comprises a natriuretic peptide, a complement 3 inhibitor, a VEGF inhibitor, or a combination thereof. In some embodiments, the engineered peptide comprises a natriuretic peptide, a complement 3 inhibitor, and a VEGF inhibitor. In some embodiments, the engineered peptide comprises a natriuretic peptide and a complement 3 inhibitor.

[0142] In some implementations, the engineered peptide contains CD59 and VEGF inhibitors.

[0143] In some embodiments, the engineered peptide comprises a natriuretic peptide, a complement 3 inhibitor, endostatin, or a combination thereof.

[0144] In some embodiments, the engineered peptide comprises sCD59-Fc4-CNP36. In some embodiments, the engineered peptide comprises sCD59-C3i-Fc4-CNP36. In some embodiments, the engineered polynucleotide encodes C3i-Fc4-endostatin. In some embodiments, the engineered peptide comprises aflibercept (SEQ ID NO: 71)-linker-C3i. In some embodiments, the engineered peptide comprises C3i-Fc4-CNP36-furin protease-sCD59. In some embodiments, the engineered peptide comprises sCD59-furin protease 2A-C3i-Fc4-CNP36. In some embodiments, the engineered peptide comprises sCD59-furin protease 2A-endostatin-linker-C3i. In some embodiments, the engineered peptide comprises sCD59-furin protease 2A-aflibercept-linker-C3i. In some embodiments, the engineered peptide comprises C3i-Fc4-CNP36-furin protease-mCD59. In some embodiments, the engineered peptide comprises C3i-Fc4-endostatin-furin protease sCD59. In some embodiments, the engineered peptide comprises, for example, C3i-Fc4-endostatin-furin protease sCD59. Figure 1 The sequence is shown. In some embodiments, the engineered peptide comprises, as shown... Figure 2A The sequence shown in -B. In some embodiments, the engineered peptide comprises, for example, the sequence shown in -B. Figure 3 The sequence is shown. In some embodiments, the engineered peptide comprises, as shown... Figure 4 The sequence is shown. In some embodiments, the engineered peptide comprises, as shown... Figure 5A The sequence shown is -C. In some implementations, engineered polynucleotides encode sequences such as... Figure 16 The sequence is shown. In some implementations, engineered polynucleotides encode, for example, Figure 24The sequence is shown. In some implementations, engineered polynucleotides encode, for example, Figure 26 The sequence is shown. In some implementations, engineered polynucleotides encode, for example, Figure 32 sequence shown.

[0145] In some embodiments, engineered peptides may be administered to a subject to treat a disease or condition. In some embodiments, engineered peptides may be formulated into pharmaceutical compositions for administration to a subject to treat a disease or condition. In some embodiments, engineered peptides may increase activity or signaling cascades associated with the complement pathway. In some embodiments, engineered peptides may increase activity or signaling cascades associated with natriuretic peptide receptors (NPRs). In some embodiments, engineered peptides may increase activity or signaling cascades associated with the cyclic GMP (cGMP) signaling pathway. In some embodiments, engineered peptides may increase activity or signaling cascades associated with CD59. In some embodiments, engineered peptides may increase activity or signaling cascades associated with endostatin. In some embodiments, engineered peptides may decrease activity or signaling cascades associated with VEGF.

[0146] In some embodiments, engineered peptides may be administered to a subject to treat a disease or condition by increasing activity or signaling cascades associated with the complement pathway. In some embodiments, engineered peptides may be administered to a subject to treat a disease or condition by increasing activity or signaling cascades associated with natriuretic peptide receptors (NPRs). In some embodiments, engineered peptides may be administered to a subject to treat a disease or condition by increasing activity or signaling cascades associated with the cGMP signaling pathway. In some embodiments, engineered peptides may be administered to a subject to treat a disease or condition by increasing activity or signaling cascades associated with CD59. In some embodiments, engineered peptides may be administered to a subject to treat a disease or condition by increasing activity or signaling cascades associated with endostatin. In some embodiments, engineered peptides may be administered to a subject to treat a disease or condition by decreasing activity or signaling cascades associated with VEGF.

[0147] Pharmaceutical Composition This document describes pharmaceutical compositions comprising engineered polynucleotides, AAV vectors comprising engineered polynucleotides, engineered peptides, cells transduced by AAV vectors comprising engineered polynucleotides, viral particles comprising engineered polynucleotides, or combinations thereof. In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable carriers, excipients, or diluents. 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 the diseases or conditions described herein. In some embodiments, the diseases or conditions include ocular diseases. In some implementations, diseases or conditions include ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, traumatic glaucoma, Bader-Bieder syndrome, Best disease, choroidal agenesis, Leber congenital amaurosis, macular degeneration, polypoid choroidal angiopathy (PCV), retinitis pigmentosa, Rifsum disease, Staggart disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, microstomia, Leventinese disease (familial dominant drusen), blue cone monochromaticism, or combinations thereof.

[0148] For in vivo delivery, engineered polynucleotides, AAV carriers containing engineered polynucleotides, engineered peptides, cells transduced by AAV carriers containing engineered polynucleotides, or combinations thereof, can be formulated into pharmaceutical compositions and can generally be administered intravitreal or parenterally (e.g., via intramuscular, subcutaneous, intratumoral, percutaneous, intrathecal, etc.). In some embodiments, the pharmaceutical composition is formulated for administration to a desired subject via intrathecal, intraocular, intravitreal, retinal, intravenous, intramuscular, intracardiac, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, subretinal, suprachoroidal, intratumoral, lung, tracheal, intraperitoneal, intrabladder, intravaginal, rectal, oral, sublingual, percutaneous, inhalation, inhalation nebulization, intraluminal-GI route, or combinations thereof.

[0149] In some respects, the pharmaceutical composition may be used to treat objects of need, such as humans or mammals. In some cases, the object may be diagnosed with a disease, such as an eye disease. In some respects, the subject pharmaceutical composition is administered in conjunction with a secondary therapy. The secondary therapy may include any therapy intended for ocular use. In some cases, the secondary therapy includes nutritional therapy, vitamins, laser therapies such as laser photocoagulation, photodynamic therapy, Visudyne, anti-VEGF therapy, eyeglasses, eye drops, numbing agents, vision correction therapies, behavioral / perceptual vision therapies, etc. In some respects, any of the biological agents previously described may be considered a secondary therapy.

[0150] In some embodiments, an effective amount of the pharmaceutical composition results in a decrease in the rate of loss of retinal function, anatomical integrity, or retinal health, such as a decrease in the rate of loss and consequently disease progression to 1 / 2, 1 / 3, 1 / 4, or 1 / 5 or lower, for example, a decrease in the rate of loss and consequently disease progression to 1 / 10 or lower.

[0151] In some embodiments, the effective amount of the pharmaceutical composition reduces angiogenesis signaling in cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more, compared to angiogenesis signaling in cells not treated with the pharmaceutical composition. In some embodiments, the effective amount of the pharmaceutical composition reduces angiogenesis in a desired subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more, compared to angiogenesis in the subject if the subject were not treated with the pharmaceutical composition. In some embodiments, the effective amount of the pharmaceutical composition reduces vascular leakage in the subject 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 were not treated with the pharmaceutical composition. In some embodiments, the effective amount of the pharmaceutical composition reduces inflammation in the subject 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 were not treated with the pharmaceutical composition.

[0152] In some embodiments, an effective amount of the subject rAAV virion results in gains in visual function, retinal function, retinal anatomy or health and / or eye movement and / or neurological function, for example, improvements of 2-fold, 3-fold, 4-fold, or 5-fold or more in retinal function, retinal anatomy or health and / or eye movement, and for example, improvements of 10-fold or more in retinal function, retinal anatomy or health and / or eye movement. As will be readily understood by those skilled in the art, the dose required to achieve the desired therapeutic effect is typically in the range of 1 x 10⁻⁶. 8 To approximately 1 x 10 15 Within the range of recombinant viral bodies, it is generally referred to by ordinary technicians as 1 x 10-1 8 To approximately 1 x 10 15 A "carrier genome".

[0153] In some respects, the compositions provided herein, such as pharmaceutical compositions, are applied to a subject in need. In some cases, the dose administered, including delivery of AAV, is approximately 0.5 x 10⁻⁶ carriers. 9 vg, 1.0 x 109 vg, 1.0 x 10 10 1.0x 10 11 vg, 3.0 x 10 11 vg, 6 x 10 11 vg, 8.0 x 10 11 vg, 1.0 x 10 12 vg, 1.0 x 10 13 vg, 1.0x 10 14 vg, 1.0 x 10 15 vg, 1.5 x 10 15 vg. For example, for internal injections, such as direct injection into the eye, the therapeutically effective dose could be approximately 10. 6 To about 10 15 Orders of magnitude of AAV virions, for example, approximately 10 8 Up to 10 12 One engineered AAV virus. For in vitro transduction, the effective amount of engineered AAV virus to be delivered to cells will be approximately 10. 8 To about 10 13 Orders of magnitude of engineered AAV viral particles. Other effective doses can be easily determined by those skilled in the art through routine experiments that establish dose-response curves.

[0154] Application can be repeated at any time. In some cases, it can be applied as follows: twice daily, every other day, twice a week, every two months, every three months, once a month, every other month, every six months, every year, or every two years.

[0155] Dosage therapy can be administered via a single-dose regimen or a multiple-dose regimen. Furthermore, the subject may be given as many doses as appropriate. Those skilled in the art can readily determine the appropriate number of doses. In some aspects, the pharmaceutical composition is administered via intravitreal injection, subretinal injection, microinjection, or intraocular injection.

[0156] When practicing the treatments or methods of use provided herein, a therapeutically effective amount of the pharmaceutical composition described herein is administered to a mammal suffering from a disease, symptom, or condition to be treated (e.g., cancer). In some embodiments, the mammal is a human. The therapeutically effective amount can vary considerably depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used, and other factors. The therapeutic agents described herein and, in some cases, the compositions may be used alone or as components of a mixture in combination with one or more therapeutic agents.

[0157] The pharmaceutical compositions described herein may be administered to a subject via suitable routes of administration, including but not limited to intravenous, intra-arterial, oral, parenteral, oral, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration. The compositions described herein may include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, rapidly melting formulations, tablets, capsules, pills, delayed-release formulations, extended-release formulations, pulsatile-release formulations, multi-particle formulations, and mixtures of immediate-release and controlled-release formulations.

[0158] Pharmaceutical compositions can be manufactured in conventional ways, for example (by way of example only), by means of conventional mixing, dissolving, granulating, grinding, emulsifying, encapsulating, embedding or compressing processes.

[0159] In some embodiments, the pharmaceutical compositions provided herein include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as phenylmercuric borate and thiomersal; stable chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, hexadecyltrimethylammonium bromide, and hexadecylpyridine chloride.

[0160] 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, rapidly melting formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, sugar-coated pills, capsules, delayed-release formulations, extended-release formulations, pulsatile-release formulations, multi-particle formulations, and mixtures of immediate-release and controlled-release formulations. In one aspect, therapeutic agents, such as those discussed herein, are formulated into pharmaceutical compositions suitable for intramuscular, subcutaneous, or intravenous injection. In another aspect, formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for rehydration to synthesize sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or media include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerin, cremoprolol, etc.), suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial growth can be ensured by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. In some cases, it is also desirable to include isotonic agents such as sugars, sodium chloride, etc. Absorption of injectable drug forms can be prolonged by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0161] On the other hand, dosage forms include microencapsulated formulations. In some embodiments, one or more other compatible materials are present in the microencapsulated material. Non-limiting examples of materials include pH adjusters, erosion promoters, defoamers, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0162] Liquid dosage forms for oral administration are optionally aqueous suspensions selected from, but not limited to, the group consisting of, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. In addition to therapeutic agents, the liquid dosage forms optionally include additives such as: (a) disintegrants; (b) dispersants; (c) wetting agents; (d) at least one preservative; (e) viscosity enhancers; (f) at least one sweetener; and (g) at least one flavoring agent. In some embodiments, the aqueous dispersion also includes crystal formation inhibitors.

[0163] In some embodiments, the pharmaceutical compositions described herein are self-emulsifying drug delivery systems (SEDDS). An emulsion is a dispersion of one immiscible phase in another, typically in the form of droplets. Generally, emulsions are produced through vigorous mechanical dispersion. Unlike emulsions or microemulsions, SEDDS spontaneously form an emulsion when added to excess water without any external mechanical dispersion or agitation. An advantage of SEDDS is that only gentle mixing is required to disperse the droplets throughout the solution. Additionally, the optional addition of water or an aqueous phase just before administration ensures the stability of unstable or hydrophobic active ingredients. Therefore, SEDDS provides an efficient delivery system for the oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provides improved bioavailability of the hydrophobic active ingredient.

[0164] 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(hydroxymethyl)aminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0165] Additionally, the pharmaceutical composition optionally includes one or more salts in an amount required to achieve an acceptable isotonicity and gravimetric friction of the composition. Such salts include those having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0166] Reagent test kit In some embodiments, this document discloses kits for use comprising engineered polynucleotides, AAVs containing engineered polynucleotides, engineered peptides, cells transduced by AAV vectors containing engineered polynucleotides, viral particles containing 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 kit comprises a combination of materials or components other than those comprising engineered polynucleotides, AAVs containing engineered polynucleotides, engineered peptides, cells transduced by AAV vectors containing engineered polynucleotides, or pharmaceutical compositions.

[0167] In some embodiments, the kit described herein includes components for selecting a homogeneous population of AAVs containing the engineered polynucleotides described herein. In some embodiments, the kit includes components for determining the number of units of a biomolecule (e.g., AAV) synthesized by a host cell and / or released or expressed on its surface. In some embodiments, the kit includes components for performing assays such as enzyme-linked immunosorbent assays (ELISA). The accuracy of the components configured in the kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating diseases or conditions (e.g., cancer) disclosed herein in a subject. In some embodiments, the kit is specifically configured for the purpose of treating mammalian subjects. In some embodiments, the kit is specifically configured for the purpose of treating human subjects.

[0168] Instructions for use may be included in the kit. In some embodiments, the kit includes instructions for administering engineered polynucleotides, AAV vectors containing engineered polynucleotides, engineered peptides, AAVs containing engineered polynucleotides, cells transduced with AAV vectors, pharmaceutical compositions, or combinations thereof to a subject in need. In some embodiments, the kit includes instructions for further engineering cells to express biomolecules (e.g., engineered polynucleotides, AAV vectors containing engineered polynucleotides, engineered peptides, AAVs containing engineered polynucleotides, or cells transduced with AAV vectors). In some embodiments, the kit includes instructions for thawing or otherwise restoring the bioactivity of engineered polynucleotides, AAV vectors containing engineered polynucleotides, or AAVs containing engineered polynucleotides, which may be cryopreserved or lyophilized during storage or transport. In some implementations, the kit includes instructions for measuring the viability of the restored engineered polynucleotide, the AAV vector containing the engineered polynucleotide, and the AAV containing the engineered polynucleotide to ensure efficacy for its intended purpose (e.g., therapeutic efficacy, if used for a therapeutic subject).

[0169] Optionally, the kit may also contain other available components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring instruments, bandage materials, or other available equipment. The materials or components assembled in the kit may be provided to practitioners in any convenient and suitable manner to maintain their operability and usability. For example, components may be in dissolved, dehydrated, or lyophilized form; they may be provided at room temperature, refrigerated, or frozen temperatures. Components are typically contained in suitable packaging materials.

[0170] delivery method Engineered polynucleotides can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, using any method in the art. For example, engineered polynucleotides can be transferred into host cells by physical, chemical, or biological means. In some embodiments, engineered polynucleotides can be delivered to host cells by encapsulating them in viral particles such as AAV particles. In some embodiments, engineered polynucleotides can be delivered into cells via physical methods such as calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, gene gun, electroporation, etc.

[0171] Physical methods for introducing engineered polynucleotides 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.

[0172] Chemical approaches for introducing engineered polynucleotides encoding non-natural substances into cells can include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, spherical nucleic acids (SNAs), liposomes, or lipid nanoparticles. An example colloidal system used as a delivery medium in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other state-of-the-art nucleic acid targeted delivery methods are available, such as delivering engineered polynucleotides or carriers encoding engineered polynucleotides using targeted nanoparticles.

[0173] In the case of using non-viral delivery systems, an example delivery medium is liposomes. The use of lipid formulations for introducing engineered polynucleotides or vectors encoding engineered polynucleotides into cells (in vitro, ex vivo, or in vivo) is considered. Alternatively, the vector can associate with lipids. Lipid-associated vectors can be encapsulated within the aqueous interior of liposomes, dispersed within a lipid bilayer of liposomes, attached to liposomes via linker molecules associated with both the liposome and the engineered polynucleotide, embedded in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. Lipids, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, in some embodiments, they exist as bilayer structures, micelles, or “collapsed” structures. Alternatively, they may simply be dispersed in solution, potentially forming aggregates of varying sizes or shapes. Lipids are fatty substances, and in some embodiments, they are naturally occurring or synthetic lipids. For example, lipids include fat droplets that are 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.

[0174] Lipids suitable for use are obtained from commercial sources. Lipid stock solutions in chloroform or chloroform / methanol are typically stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term encompassing a variety of monolayer and multilayer lipid mediators formed by the formation of 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 spontaneously form when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo rearrangement before forming a closed structure, embedding water and dissolved solutes between the lipid bilayers. However, compositions having a structure in solution different from a normal vesicular structure are also included. For example, in some embodiments, lipids exhibit a micellar structure or exist simply as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0175] In some cases, non-viral delivery methods include lipid transfection, nuclear transfection, microinjection, gene gun, virions, liposomes, immunoliposomes, exosomes, polycationic or lipid:cargo conjugates (or aggregates), naked peptides (e.g., recombinant peptides), naked DNA, artificial virions, and reagent-enhanced uptake of peptides or DNA. In some embodiments, delivery methods include 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(imino carbonates), polyorthoesters, polyoxyethylene esters, polyamide esters, amine-containing polyoxyethylene esters, and poly(anhydrides). Such synthetic polymers can be homopolymers or copolymers of multiple different monomers (e.g., random, block, segmented, grafted), such as two or more of lactic acid, lactide, glycolic acid, glycolide, ε-caprolactone, trimethylene carbonate, p-dioxanone, etc. In examples, the scaffold can be composed of polymers containing glycolic acid and lactic acid, such as polymers having a glycolic acid to lactic acid ratio of 90 / 10 or 5 / 95. Non-limiting examples of naturally occurring biocompatible, biodegradable polymers may include glycoproteins, proteoglycans, polysaccharides, glycosamineoglycans (GAG) and fragments derived from these components, elastin, laminin, decrorin, fibrinogen / fibrin, fibronectin, osteopontin, cohesin, hyaluronic acid, collagen, chondroitin sulfate, heparin, heparan sulfate, ORC, carboxymethyl cellulose, and chitosan.

[0176] 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 vesicles, cancerous bodies, exopher, enveloped viruses, exomere, or other very large extracellular vesicles.

[0177] In some embodiments, engineered polynucleotides can be delivered into cells via biological methods, such as the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. In some embodiments, other viral vectors are derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, and adeno-associated viruses. 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 the genome of Moloney murine leukemia virus (MoMLV, MMLV, MuLV, or MLV) or murine stem cell virus (MSCV). In some cases, retroviral vectors also include lentiviral vectors, such as lentiviral vectors derived from the genome of human immunodeficiency virus (HIV). In some cases, AAVs include serotypes, including AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or combinations thereof. Based on these initial serotypes, the AAV capsid for each serotype can be engineered to better suit its use for biological function, tissue, or cell selection. In some embodiments, the AAV is AAV2 and its variants AAV2.N53 and AAV2.N54, as used in the examples of this disclosure. Chimeric AAVs that may contain at least two AAV serotypes are also contemplated. In some cases, at least three, at least four, at least five, at least six, at least seven, or up to eight different serotypes are combined in a chimeric AAV. In some cases, only a portion of the AAV is chimeric. For example, suitable portions may include the capsid, VP1, VP2, or VP3 domains, and / or Rep. In some cases, at least one of VP1, VP2, and VP3 has at least one amino acid substitution compared to otherwise comparable wild-type AAV capsid proteins. In some cases, the mutation can occur in VP1 and VP2, VP1 and VP3, VP2 and VP3, or VP1, VP2, and VP3. In some embodiments, at least one of VP1, VP2, and VP3 has 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, the mutant AAV does not include at least one of VP1, VP2, or VP3.

[0178] Methods of modifying cells On the other hand, this article also provides methods for modifying cells to generate engineered cells. Cells can refer to primary cells, recombinant cells, or cell lines. In some cases, the cells are packaging cells. Packaging cells can be any of the following: HEK 293 cells, HeLa cells, and Vero cells (to name just a few). Engineered cells can be primary cells. In some cases, engineered cells can be ocular cells. Suitable ocular cells include, but are not limited to: photoreceptor cells, ganglion cells, RPE cells, amacrine cells, horizontal cells, Müller cells, etc.

[0179] In some cases, the cells are packaging cells used to generate viral particles. To generate AAV virions or viral particles, AAV vectors are introduced into suitable host cells using known techniques, such as transfection. In some cases, transfection techniques, such as CaPO4 transfection or electroporation, are used, and / or the cell lines, such as the human embryonic kidney cell line HEK 293 (containing a functional adenovirus E1 gene that provides the trans-acting E1 protein), are infected using hybrid adenovirus / AAV vectors. Suitable transfection methods include calcium phosphate coprecipitation, direct microinjection, electroporation, liposome-mediated gene transfer, and nucleic acid delivery using high-speed microboluses, all of which are known in the art.

[0180] To engineer cells, multiple cells can be contacted with isolated engineered polynucleotides. Contact can be of any duration and can range from approximately 5 minutes to approximately 5 days. Contact can last for approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some cases, contact can last for 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, or up to approximately 5 days.

[0181] In some cases, the supernatant of the packaging cell line is treated with PEG precipitation to concentrate the virus. In other cases, a centrifugation step can be used to concentrate the virus. For example, a column can be used to concentrate the virus during centrifugation. In some embodiments, precipitation occurs at a temperature not higher 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 the PEG precipitation by low-speed centrifugation followed by a CsCl gradient. Low-speed centrifugation can be up to about 4000 RPM, about 4500 RPM, about 5000 RPM, or about 6000 RPM for about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes. In some cases, recombinant AAV is separated from the supernatant of the PEG precipitation by centrifugation at about 5000 RPM for about 30 minutes followed by a CsCl gradient. In some cases, CsCl purification can be replaced by IDX gradient ultracentrifugation. The supernatant can be collected at approximately 12, 24, 36, 48, 72, 96, or 120 hours post-transfection, or any time between these two time points. 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.

[0182] On the other hand, multiple AAV particles (containing the engineered polynucleotides described herein) isolated from engineered cells are also provided. The viral titer can be approximately 10. 2 vp / mL, approximately 10 3 vp / mL, approximately 10 4 vp / mL, approximately 10 5 vp / mL, approximately 10 6 vp / mL, approximately 10 7 vp / mL, approximately 10 8 vp / mL or up to approximately 10 9 vp / mL. The viral titer can be approximately 10. 2 GC / mL, approximately 10 3 GC / mL, approximately 10 4 GC / mL, approximately 10 5 GC / mL, approximately 10 6 GC / mL, approximately 10 7 GC / mL, approximately 10 8 GC / mL or up to about 10 9 GC / mL. In some cases, the viral titer can be approximately 10. 2 TU / mL, approximately 10 3TU / mL, approximately 10 4 TU / mL, approximately 10 5 TU / mL, approximately 10 6 TU / mL, approximately 10 7 TU / mL, approximately 10 8 TU / mL or up to approximately 10 9 TU / mL. The optimal viral titer can vary depending on the cell type to be transfected. The viral range can be approximately 1000 MOI to approximately 2000 MOI, approximately 1500 MOI to approximately 2500 MOI, approximately 2000 MOI to approximately 3000 MOI, approximately 3000 MOI to approximately 4000 MOI, approximately 4000 MOI to approximately 5000 MOI, approximately 5000 MOI to approximately 6000 MOI, approximately 6000 MOI to approximately 7000 MOI, approximately 7000 MOI to approximately 8000 MOI, approximately 8000 MOI to approximately 9000 MOI, approximately 9000 MOI to approximately 10,000 MOI. For example, to infect 1 million cells using an MOI of 10,000, it would require 10,000 x 1,000,000 = 10 10 GC.

[0183] In some cases, multiple AAV particles can be formulated into unit dosage forms. Various formulations for adult or pediatric delivery are considered, including but not limited to: 0.5 x 10 9 vg, 1.0 x 10 9 vg, 1.0 x 10 10 1.0 x 10 11 vg, 3.0x 10 11 vg, 6 x 10 11 vg, 8.0 x 10 11 vg, 1.0 x 10 12 vg, 1.0 x 10 13 vg, 1.0 x 10 14 vg, 1.0x 10 15 vg or up to 1.5 x 10 15 vg. The composition of virus particles can be cryopreserved or otherwise stored in suitable containers.

[0184] The compositions and methods provided herein are sufficient to enhance the delivery and / or expression of the subject bioagent 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 otherwise comparable unmodified nucleic acids. In some cases, otherwise comparable unmodified nucleic acids are nucleic acids encoding VEGF-Trap. In some cases, modifications may be sufficient to enhance the delivery and / or expression of the subject biological 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. Approximately 181 times, approximately 186 times, approximately 191 times, approximately 196 times, approximately 201 times, approximately 206 times, approximately 211 times, approximately 216 times, approximately 221 times, approximately 226 times, approximately 231 times, approximately 236 times, approximately 241 times, approximately 246 times, approximately 251 times, approximately 256 times, approximately 261 times, approximately 266 times, approximately 271 times, approximately 276 times, approximately 281 times, approximately 286 times, approximately 291 times, approximately 296 times, approximately 301 times, approximately 306 times, approximately 311 times, approximately 316 times, approximately 321 times, approximately 326 times, approximately 331 times, approximately 336 times, approximately 341 times, approximately 346 times, or approximately 350 times more than otherwise comparable unmodified nucleic acids. In the implementation scheme, the increased expression includes an increase of 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, as determined by an in vitro assay. Suitable in vitro assays include ELISA, Western blot, Luminex, microscopy, imaging, and / or flow cytometry.

[0185] Compared to AAV virions containing otherwise comparable WT AAV capsid proteins, the subject AAV virions can exhibit an increased infectivity to retinal cells (photoreceptors, ganglion cells, RPE cells, amacrine cells, horizontal cells, Müller cells, etc.), at least 1, at least 6, at least 10, at least 15, at least 20, at least 25, at least 50, or more than 50 times.

[0186] Treatment This document provides methods for treating the diseases or conditions described herein. In some aspects, the methods confer protection against the disease or condition. Treatment methods may include introducing engineered polynucleotides, engineered peptides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced with AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof into a subject in need. Methods for treating diseases or conditions are also provided, comprising administering a pharmaceutical composition to a subject in need. The pharmaceutical composition may contain a sequence encoding a biological agent, including engineered polynucleotides, AAV vectors containing engineered polynucleotides, AAV vectors containing engineered polynucleotides, viral particles containing engineered polynucleotides, or combinations thereof. In some embodiments, administration is performed via any suitable mode of administration, including systemic administration (e.g., intravenous, intravitreal, subretinal, or otherwise). In some embodiments, the subject is a human.

[0187] In some embodiments, the method includes treating a disease or condition in a subject of need by administering a therapeutically effective amount of the engineered polynucleotide, engineered peptide, cell transduced with engineered polynucleotide, or pharmaceutical composition described herein. In some embodiments, the method treats a disease or condition in a single administration of the engineered polynucleotide, engineered peptide, cell transduced with engineered polynucleotide, or pharmaceutical composition described herein to cure the disease or condition. In some embodiments, the method treats a disease or condition in a manner where administration of the engineered polynucleotide, engineered peptide, cell transduced with engineered polynucleotide, or pharmaceutical composition described herein does not involve daily administration. In some embodiments, the disease or condition includes an eye disease. Non-limiting examples of eye diseases may include ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, achromatopsia, 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-Bied syndrome, Best disease, choroidal agenesis, Leber congenital amaurosis, macular degeneration, polypoid choroidal angiopathy (PCV), retinitis pigmentosa, Revesum disease, Staggart disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, microstomia, Leventinese disease (familial manifest drusen), blue cone monochromatic vision, or combinations thereof. In some embodiments, the disease or condition is neovascular glaucoma (NG). In some implementations, the disease or condition is glaucoma. In some implementations, the disease or condition is traumatic glaucoma.

[0188] In another embodiment, this document provides a pharmaceutical composition comprising the engineered polynucleotides, engineered peptides, vectors, viral particles, cells, or compositions disclosed herein. In some embodiments, the pharmaceutical composition is formulated for administration to a desired subject via intrathecal, intraocular, intravitreal, retinal, intravenous, intramuscular, intracardiac, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, subretinal, suprachoroidal, intratumoral, lung, tracheal, intraperitoneal, intrabladder, vaginal, rectal, oral, sublingual, transdermal, inhalation, inhalation nebulization, intraluminal-GI route, or combinations thereof. In some embodiments, the pharmaceutical composition is formulated for intrathecal administration. In some embodiments, the pharmaceutical composition is formulated for retinal administration. In some embodiments, the pharmaceutical composition is formulated for intraocular administration. In some embodiments, the pharmaceutical composition is formulated for intravitreal, subretinal, or suprachoroidal administration.

[0189] On the other hand, this document provides a method comprising contacting cells obtained from an object with the engineered polynucleotide, engineered peptide, vector, viral particle, cell, composition, or pharmaceutical composition disclosed herein.

[0190] On the other hand, this document provides a method for treating a disease or condition in a subject, comprising: administering to the subject an engineered polynucleotide, an engineered peptide, a vector, a viral particle, a cell, a composition, or a pharmaceutical composition disclosed herein. In some embodiments, the administration cures the disease or condition. In some embodiments, the administration does not include daily administration. In some embodiments, the administration includes weekly administration, bi-weekly administration, monthly administration, bi-monthly administration, semi-annual administration, annual administration, or a combination thereof. In some embodiments, the disease or condition includes an eye disease. In some implementations, ocular diseases include ocular ischemia syndrome, proliferative retinopathy, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, 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-Bied syndrome, Best disease, choroidal agenesis, Leber congenital amaurosis, macular degeneration, polypoid choroidal angiopathy (PCV), retinitis pigmentosa, Revesum disease, Staggart disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, microstomia, Leventinese disease (familial manifest drusen), blue cone monochromaticism, or combinations thereof. In some implementations, ocular diseases include GA or dAMD.

[0191] On the other hand, this document provides a method for treating a disease or condition in a subject, the method comprising administering an engineered polynucleotide to the subject, the engineered polynucleotide comprising one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor. In some embodiments, the first angiogenesis inhibitor and the second angiogenesis inhibitor are each encoded by one of the one or more expression cassettes. In some embodiments, the first angiogenesis inhibitor and the second angiogenesis inhibitor are operatively coupled. In some embodiments, the first angiogenesis inhibitor and the second angiogenesis inhibitor are covalently linked via a linker. In some embodiments, the first angiogenesis inhibitor comprises a complement inhibitor, such as a complement 3 inhibitor. In some embodiments, the complement 3 inhibitor comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 1-15. In some embodiments, the complement 3 inhibitor is encoded by a nucleic acid sequence having at least 80% identity with any of SEQ ID NO: 20-33. In some embodiments, the first angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC), such as CD59. In some embodiments, CD59 comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 41-45. In some embodiments, the second angiogenesis inhibitor comprises a natriuretic peptide. In some embodiments, the natriuretic peptide comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 61-72. In some embodiments, the second angiogenesis inhibitor comprises collagen or a fragment thereof. In some embodiments, the second angiogenesis inhibitor comprises endostatin or a fragment thereof. In some embodiments, the second angiogenesis inhibitor comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 51. In some embodiments, the second angiogenesis inhibitor comprises a VEGF inhibitor. In some embodiments, the VEGF inhibitor comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 81-92.

[0192] In some embodiments, the engineered polynucleotide comprises a viral vector. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the AAV vector comprises an AAV serotype, which comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV vector is an AAV2 vector. In some embodiments, the AAV vector encodes an engineered AAV capsid, wherein the engineered AAV capsid comprises the amino acid sequence of any one of SEQ ID NO: 161-182 and SEQ ID NO: 191-210. In some embodiments, the engineered AAV capsid comprises the amino acid sequence of SEQ ID NO: 169.

[0193] In some implementations, the method includes a single application to cure the disease or condition. In some implementations, the application does not include daily application. In some implementations, the application includes weekly application, bi-weekly application, monthly application, bi-monthly application, semi-annual application, annual application, or a combination thereof. In some implementations, the disease or condition includes ocular diseases such as ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, 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-Bied syndrome, Best disease, choroidal agenesis, Leber congenital amaurosis, macular degeneration, polypoid choroidal angiopathy (PCV), retinitis pigmentosa, Revesum disease, Staggart disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, microstomia, Leventinese disease (familial dominant drusen), blue cone monochromaticism, or combinations thereof. In some implementations, the ocular disease includes GA or dAMD. In some implementations, when a first angiogenesis inhibitor and a second angiogenesis inhibitor are administered to a subject, neovascularization in the subject is inhibited.

[0194] In some implementations, the first or second angiogenesis inhibitor exhibits reduced inhibition of angiogenesis in the subject after administration, compared to inhibition induced by VEGF inhibitors. In such cases, the reduced inhibition of angiogenesis induced by the first or second angiogenesis inhibitor may be more therapeutically effective in treating the disease or condition. For example, reduced inhibition of angiogenesis allows for the presence of blood vessels for the transport and delivery of the first or second angiogenesis inhibitor to sites associated with the disease or condition.

[0195] In some embodiments, administering a therapeutically effective amount of the engineered polynucleotides, engineered peptides, cells transduced with engineered polynucleotides, or pharmaceutical compositions described herein to a subject protects the subject from the effects of a disease or condition. For example, administering a therapeutically effective amount of the engineered polynucleotides, engineered peptides, cells transduced with engineered polynucleotides, or pharmaceutical compositions may protect the subject from the development of a disease or condition originating from injury. In some embodiments, administering a therapeutically effective amount of the engineered polynucleotides, engineered peptides, cells transduced with engineered polynucleotides, or pharmaceutical compositions protects or promotes cell survival in the subject. In some embodiments, administering a therapeutically effective amount of the engineered polynucleotides, engineered peptides, cells transduced with engineered polynucleotides, or pharmaceutical compositions protects or promotes the survival of ocular cells in the subject. In some embodiments, administering a therapeutically effective amount of the engineered polynucleotides, engineered peptides, cells transduced with engineered polynucleotides, or pharmaceutical compositions protects or promotes the survival of retinal ganglion cells in the subject. In some embodiments, administering a therapeutically effective amount of the engineered polynucleotides, engineered peptides, cells transduced with engineered polynucleotides, or pharmaceutical compositions reduces intraocular pressure in the subject.

[0196] In some embodiments, the engineered polynucleotide, engineered peptide, AAV vector containing the engineered polynucleotide, AAV containing the engineered polynucleotide, cells transduced with the AAV vector, or 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, or once a year). In some embodiments, the composition is administered two or more times 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, or 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.

[0197] In some embodiments, the method includes administering, in therapeutically effective amounts, engineered polynucleotides, engineered peptides, AAV carriers containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells or pharmaceutical compositions transduced with AAV carriers via various forms and routes (including, for example, oral or topical administration) in various forms and routes. In some embodiments, the composition can be administered via intravitreal, subretinal, suprachoroidal, parenteral, intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, subarachnoid, intraocular, intrasternal, ocular, endothelial, local, intranasal, pulmonary, rectal, intraarterial, intrasheath, inhalation, intralesional, intradermal, epidural, intracapsular, subcapsular, intracardiac, tracheal, subepidermal, subarachnoid, or spinal administration (e.g., injection or infusion). In some embodiments, the composition can be administered via absorption through epithelial or mucosal linings (e.g., oral mucosa, rectal, and intestinal mucosa). In some embodiments, the composition is delivered via multiple routes of administration.

[0198] In some embodiments, the method includes administering engineered polynucleotides, engineered peptides, AAV vectors containing engineered polynucleotides, AAV containing engineered polynucleotides, cells transduced with AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof by intravenous infusion. In some embodiments, engineered polynucleotides, AAV vectors containing engineered polynucleotides, AAV containing engineered polynucleotides, cells transduced with AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof are administered by slow, continuous infusion over a prolonged period (e.g., more than 24 hours). In some embodiments, engineered polynucleotides, AAV vectors containing engineered polynucleotides, AAV containing engineered polynucleotides, cells transduced with AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof are administered as intravenous injection or a short infusion. In some embodiments, engineered polynucleotides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced with AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof are administered via the vitreous route. In some embodiments, engineered polynucleotides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced with AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof can be administered topically, for example, by direct injection into an organ, optionally in the form of a reservoir or slow-release formulation or implant.

[0199] In some embodiments, engineered polynucleotides, engineered peptides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced with AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof may be administered in combination with other therapies, such as antiviral therapy, chemotherapy, antibiotics, cell therapy, cytokine therapy, or anti-inflammatory agents. In some embodiments, engineered polynucleotides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced with AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof may be administered before, during, or after the onset of disease or condition, and the timing of administration of compositions containing therapeutic agents may vary. In some cases, the composition may be used as a prophylactic agent and may be administered continuously to subjects susceptible to coronaviruses or predisposed to coronavirus-related conditions or diseases (e.g., for immunization or for treatment). Prophylactic administration may reduce the likelihood of infection, disease, or condition occurring, or may reduce the severity of infection, disease, or condition.

[0200] Engineered polynucleotides, engineered peptides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced with AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof may be administered to a subject prior to symptom onset. In some embodiments, engineered polynucleotides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced with AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof may be administered to a subject (e.g., for immunization or for treatment) after test results (e.g., as soon as possible after test results), such as diagnostic test results, tests showing the presence of coronavirus in the subject (e.g., for immunization or for treatment), or tests showing disease progression (e.g., decreased blood oxygen levels). Therapeutic agents may be administered after detection or suspicion of disease or disease onset (e.g., as soon as feasible). Therapeutic agents can be administered after potential exposure to the coronavirus (e.g., as soon as practicable), such as after the subject (e.g., for immunization or for treatment) has been in contact with an infected subject, or after it is known that they have been in contact with an infected subject who may be infectious.

[0201] The actual dose level of the agents (e.g., engineered polynucleotides or pharmaceutical compositions) disclosed herein can be varied to obtain a specific amount of agent to achieve the desired therapeutic response to a particular subject, composition, and administration mode without causing toxicity to the subject (e.g., for immunization or for treatment). The selected dose level can depend on a variety of pharmacokinetic factors, including the activity of the particular composition used in this disclosure, route of administration, time of administration, excretion rate, duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors well known in the medical field.

[0202] Dosing regimens can be adjusted to provide the optimal desired response (e.g., therapeutic and / or preventative response). For example, a single bolus can be administered, several separate doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by an emergency in the treatment situation. Formulating parenteral compositions in unit dosage form is particularly advantageous for ease of administration and uniform dosage. As used herein, unit dosage form refers to physically discrete units suitable as unit doses for a subject (e.g., for immunization or for treatment); each unit contains a predetermined amount of active agent calculated to produce the desired therapeutic effect when combined with the desired drug delivery system. The specifications of the unit dosage form of this 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 individuals being treated. Dosage can be determined by reference to the plasma or local concentration of a cyclic polynucleotide or antibody or its antigen-binding fragment. Dosage can be determined by reference to the plasma or local concentration of a linear polynucleotide or antibody or its antigen-binding fragment.

[0203] The engineered polynucleotides, engineered peptides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced with AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof described herein can be presented in unit dosage forms suitable for precise single-dose administration. In unit dosage forms, the formulation can be divided into unit doses containing adequate amounts of the composition. In unit dosage forms, the formulation can be divided into unit doses containing adequate amounts of one or more linear polynucleotides, antibodies, or antigen-binding fragments thereof, and / or therapeutic agents. Unit doses can be presented in the form of packages containing discrete amounts of the formulation. Non-limiting examples are packaged injections, vials, and ampoules. The aqueous suspension compositions disclosed herein can be packaged in single-dose, non-reclosed containers. Multi-dose, reclosed containers can be used, for example, with or without preservatives. The formulations disclosed herein for injection can be presented in unit dosage forms, such as in ampoules containing preservatives or in multi-dose containers.

[0204] In some cases, the level of increase in biological agents in the subject is increased to at least 5, 10, 20, 50, 100, 200, or 500 times, as determined by diagnostic assays.

[0205] Appropriate diagnostic assays may include ophthalmic diagnostic assays. These may include ophthalmic tests such as refractive tests, ocular scans, ocular coherence tomography (OCT), Farnworth-Munsell 100 chromaticity tests, computed optic disc imaging and nerve fiber layer analysis (GDX, HRT, OCT), corneal topography, electroretinography (ERG), electrooculography (EOG), visual evoked potentials (VEP), visual evoked responses (VER), fluorescein angiography, OCT, retinal photography, fundus photography, corneal endothelial microscopy, Goldmann, Humphrey, FDT, Octopus, biostatistics / IOL calculations, A-scans, B-scans, and combinations thereof.

[0206] In some cases, retinal tests may be used. Non-limiting methods for assessing retinal function and its changes include assessing visual acuity (e.g., best-corrected visual acuity [BCVA], walking, navigation, object detection, and discrimination), assessing visual field (e.g., static and dynamic visual field tests), performing clinical examinations (e.g., slit-lamp examination of the anterior and posterior segments), and assessing electrophysiological responses to light and darkness at all wavelengths (e.g., all forms of electroretinography (ERG) [full field, multifocal, and modal], all forms of visual evoked potentials (VEP), electrooculography (EOG), color vision, dark adaptation, and / or contrast sensitivity). Non-limiting methods for assessing anatomical and retinal health and its changes include optical coherence tomography (OCT), fundus photography, adaptive optics laser confocal scanning ophthalmoscopy (AO-SLO), fluorescence and / or autofluorescence; measurement of eye movements and oculomotor activity (e.g., nystagmus, fixation preference and stability), measurement of reported outcomes (patient-reported changes in visually and non-visually guided behavior and activities, patient-reported outcomes [PRO], questionnaire-based assessments of quality of life, daily activities and neurological function measurements (e.g., functional magnetic resonance imaging (MRI)).

[0207] In some embodiments, compared to comparable cells that have not been contacted with engineered polynucleotides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, or pharmaceutical compositions, the engineered polynucleotides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced with AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof are 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.

[0208] In some implementations, the treatment methods described herein can treat ocular diseases. Relevant ocular diseases and conditions may include, but are not limited to: blindness, achromatopsia, age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma, Budd-Bied syndrome, Best disease, choroidal agenesis, Leber congenital amaurosis, macular degeneration, polypoid choroidal vasculopathy (PCV), retinitis pigmentosa, Revesum disease, Staggart disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, microstomia, Leventinese disease (familial dominant drusen), and blue cone monochromatic vision. In some implementations, the ocular disease or condition is AMD. AMD can be wet AMD or dry AMD.

[0209] In some cases, administration of a pharmaceutical composition is sufficient to at least alleviate, treat, and / or eliminate the symptoms of a disease or condition. In some cases, improvement in the disease or condition can be determined by any available diagnostic assay. In other cases, improvement can be obtained through an interview with the treated subject. For example, the subject may be able to communicate with their attending physician that their vision has improved compared to before the administration of the subject medication. In other cases, in vivo animal models can be used to determine the reduction of the disease or condition after treatment. Suitable animal models include mouse models, primate models, rat models, canine models, etc.

[0210] The use of absolute or sequential terms, such as “will,” “will not,” “should,” “should not,” “must,” “must not,” “first,” “initially,” “next,” “following,” “before,” “after,” “finally,” and “end,” is not intended to limit the scope of the embodiments disclosed herein, but is merely exemplary.

[0211] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” are intended to also include the plural forms. Furthermore, to the extent used in the Detailed Description and / or the claims, the terms “including,” “includes,” “having,” “has,” “with,” or variations thereof are intended to be inclusive in a manner similar to the term “comprising.”

[0212] As used in this article, the phrases “at least one,” “one or more,” and “and / or” are open-ended expressions that function as both conjunctions and antonymous conjunctions in practice. 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” indicates A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0213] As used herein, "or" can mean "and," "or," or "and / or," and can be used exclusively and inclusively. For example, the term "A or B" can mean "A or B," "A but not B," "B but not A," and "A and B." In some cases, the context can determine the specific meaning.

[0214] 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.

[0215] When referring to a number or range of values, the term "about" means that the number or range mentioned is an approximation within experimental variation (or within statistical experimental error), and that the number or range may vary, for example, between 1% and 15% of the stated number or range. In the example, the term "about" refers to ±10% of the stated number or value.

[0216] The terms “increased,” “increasing,” or “increase” are used herein to generally mean an increase that is statistically significant. In some embodiments, the terms “increased” or “increase” mean 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% and 100%. Other examples of “increase” include increases 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 than 1000 times compared to a reference level.

[0217] The terms “decreased,” “decreasing,” or “decrease” are used herein to generally mean a reduction in a statistically significant amount. In some embodiments, “decreased” or “decrease” means a reduction of at least 10% compared to a reference level, such as 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% reduction (e.g., a level that is not present or undetectable compared to the reference level) or any reduction between 10% and 100%. In the context of a biomarker or symptom, these terms mean that the level is statistically significant. A reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more, and is preferably a decrease to a level accepted as being within the normal range for an individual without a given disease.

[0218] The terms “AAV,” “AAV construct,” or “recombinant AAV” or simply “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 helper viruses (such as adenovirus, herpes simplex virus, vaccinia virus, and CMV) to replicate. Wild-type AAVs are common in the general population and are not associated with any known pathologies. Hybrid AAVs are AAVs containing the capsid protein of one AAV serotype and the genomic material of another AAV serotype. Chimeric AAVs comprise gene and / or protein sequences derived from two or more AAV serotypes and may include mutations in the gene sequences of those two or more AAV serotypes. Exemplary chimeric AAVs may comprise a chimeric AAV capsid, for example, a capsid protein having one or more amino acid regions derived from two or more AAV serotypes. An AAV variant is an AAV that contains one or more amino acid mutations in its genome or protein compared to its parent AAV (e.g., one or more amino acid mutations in its capsid protein compared to its parent AAV). As used herein, AAVs include avian AAVs, bovine AAVs, canine AAVs, equine AAVs, primate AAVs, non-primate AAVs, and sheep 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 birds. In some cases, wild-type AAV contains both the rep and cap genes, where the rep gene is required for viral replication and the cap gene is required for capsid protein synthesis. As used herein, the terms "recombinant AAV" and "rAAV" are used interchangeably.

[0219] The terms “recombinant AAV vector” or “AAV vector” refer to a vector derived from any of the AAV serotypes mentioned above. In some cases, an AAV vector may contain one or more AAV wild-type genes, such as the rep and / or cap genes, that are wholly or partially missing, but contain the functional elements required for packaging and using the AAV virus to achieve gene therapy. For example, functional inverted terminal repeat sequences or ITR sequences flanked by open reading frames or cloned foreign sequences are known to be important for the replication and packaging of AAV virions, but the ITR sequences can be modified from wild-type nucleotide sequences, including nucleotide insertions, deletions, or substitutions, to make the AAV suitable for the embodiments described herein, such as gene therapy or gene delivery systems. In some aspects, self-complementary vectors (sc), such as self-complementary AAV vectors, can be used, which can bypass the need for viral second-strand DNA synthesis and can result in higher expression rates of transgenic proteins. In some aspects, AAV vectors can be generated to allow for the selection of optimal serotype, promoter, and transgene. In some cases, the vector may be a targeting vector or a modified vector that selectively binds to or infects immune cells.

[0220] The term "AAV virion" or "AAV virion" refers to a viral particle containing 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 nucleic acid sequence or a transgene. The virion may be an engineered virion.

[0221] The terms “object,” “host,” “individual,” and “patient” are used interchangeably herein to refer to an animal, typically a mammal. Any suitable mammal may be administered the compositions (such as engineered guide RNA) as described herein or treated by the methods described herein. The object may be a vertebrate or an invertebrate. The object may be a laboratory animal. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domesticated 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 may be of any age or at any developmental stage (e.g., adult, adolescent, child, infant, or intrauterine mammal). The mammal may be male or female. In some embodiments, the object is a human. The object may be a patient. The object may suffer from a disease. The object may exhibit symptoms of a disease. The object may not exhibit symptoms of a disease but may still suffer from a disease. The subject may receive medical care from caregivers (e.g., the subject is hospitalized and treated by a physician).

[0222] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably and, in their broadest sense, refer to compounds containing two or more subunit amino acids, amino acid analogs, or peptide mimics. The term also covers modified amino acid polymers; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeled component. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptide mimics. Subunits may be linked by peptide bonds. In another embodiment, subunits may be linked by other bonds, such as esters, ethers, etc. Proteins or peptides must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may constitute a protein or peptide sequence. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including glycine and its D and L optical isomers, amino acid analogs, and peptide mimics. As used herein, the term "fusion protein" refers to a protein comprising domains derived from more than one naturally occurring or recombinant protein, where each domain typically has a distinct function. In this regard, the term "connector" refers to a protein fragment used to link these domains together, optionally to preserve the conformation of the fusion protein domains and / or prevent adverse interactions between the fusion protein domains that could impair their respective functions.

[0223] A certain percentage of "sequence identity" between a polynucleotide or polypeptide and another polynucleotide or polypeptide means that the percentages of bases or amino acids are the same when comparing the two sequences during alignment. Sequence similarity can be determined in several different ways. To determine sequence identity, methods and computer programs can be used to align sequences, including BLAST, which is available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, which is available in the Genetics Computing Group (GCG) software package.

[0224] Although preferred embodiments of the 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 invention is not intended to be limited to the specific embodiments provided herein. While the invention has been described with reference to the foregoing detailed description, the description and illustration of embodiments herein are not intended to be construed as limiting. Various modifications, alterations, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific depictions, configurations, or relative proportions described herein, and are subject to various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. Therefore, any such alternatives, modifications, variations, or equivalents should also be considered in relation to the invention. The following claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

[0225] Implementation Plan Implementation Scheme 1: An engineered polynucleotide comprising one or more expression cassettes, the one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor.

[0226] Implementation Scheme 2: An engineered polynucleotide comprising one or more expression cassettes encoding: a complement 3 (C3) inhibitor or a C3 degradation fragment; and a natriuretic peptide.

[0227] Implementation Scheme 3: The engineered polynucleotide according to Implementation Scheme 2, wherein the complement 3 inhibitor or the C3 degradation fragment and the natriuretic peptide are covalently linked by a linker.

[0228] Implementation Scheme 4: The engineered polynucleotide according to Implementation Scheme 2 or 3, wherein the complement 3 inhibitor comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 1-15.

[0229] Implementation Scheme 5: An engineered polynucleotide according to any one of Implementation Schemes 2-4, wherein the complement 3 inhibitor comprises an amino acid sequence containing any one of SEQ ID NO: 1-15.

[0230] Implementation Scheme 6: An engineered polynucleotide according to any one of Implementation Schemes 2-5, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence having at least 80% identity with any one of SEQ ID NO: 20-23.

[0231] Implementation Scheme 7: An engineered polynucleotide according to any one of Implementation Schemes 2-6, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence comprising any one of SEQ ID NO: 20-23.

[0232] Implementation Scheme 8: An engineered polynucleotide according to any one of Implementation Schemes 2-7, wherein the natriuretic peptide comprises a C-type natriuretic peptide (CNP).

[0233] Implementation Scheme 9: An engineered polynucleotide according to any one of Implementation Schemes 2-8, wherein the natriuretic peptide is covalently linked to an antibody or a fragment thereof.

[0234] Implementation Scheme 10: The engineered polynucleotide according to Implementation Scheme 9, wherein the antibody or fragment thereof comprises a crystallizable fragment (Fc) region.

[0235] Implementation Scheme 11: An engineered polynucleotide according to any one of Implementation Schemes 8-10, wherein the natriuretic peptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 61-72.

[0236] Implementation Scheme 12: An engineered polynucleotide according to any one of Implementation Schemes 8-11, wherein the natriuretic peptide comprises the amino acid sequence of any one of SEQ ID NO: 61-72.

[0237] Implementation Scheme 13: The engineered polynucleotide according to Implementation Scheme 1, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are each encoded by one of the one or more expression cassettes.

[0238] Implementation Scheme 14: The engineered polynucleotide according to Implementation Scheme 1 or 13, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are encoded by one of the one or more expression cassettes.

[0239] Implementation Scheme 15: An engineered polynucleotide according to any one of Implementation Scheme 1 or 13-14, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are operatively coupled.

[0240] Implementation Scheme 16: An engineered polynucleotide according to Implementation Scheme 1 or any one of 13-15, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are covalently linked by a linker.

[0241] Implementation Scheme 17: An engineered polynucleotide according to Implementation Scheme 1 or any one of 13-16, wherein the first angiogenesis inhibitor comprises a complement inhibitor.

[0242] Implementation Scheme 18: An engineered polynucleotide according to Implementation Scheme 1 or any one of 13-17, wherein the complement inhibitor comprises a complement 3 inhibitor or a C3 degradation fragment.

[0243] Implementation Scheme 19: An engineered polynucleotide according to any one of Implementation Scheme 1 or 13-18, wherein the complement inhibitor comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 1-15.

[0244] Implementation Scheme 20: An engineered polynucleotide according to any one of Implementation Scheme 1 or 13-19, wherein the complement 3 inhibitor comprises an amino acid sequence containing any one of SEQ ID NO: 1-15.

[0245] Implementation Scheme 21: An engineered polynucleotide according to any one of Implementation Scheme 1 or 13-20, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence having at least 80% identity with any one of SEQ ID NO: 20-33.

[0246] Implementation Scheme 22: An engineered polynucleotide according to any one of Implementation Scheme 1 or 13-21, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence comprising any one of SEQ ID NO: 20-33.

[0247] Implementation Scheme 23: An engineered polynucleotide according to any one of Implementation Scheme 1 or 13-22, wherein the first angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC).

[0248] Implementation Scheme 24: An engineered polynucleotide according to any one of Implementation Scheme 1 or 13-23, wherein the inhibitor of said MAC comprises CD59.

[0249] Implementation Scheme 25: An engineered polynucleotide according to any one of Implementation Scheme 1 or 13-24, wherein the CD59 comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 41-45.

[0250] Implementation Scheme 26: An engineered polynucleotide according to any one of Implementation Scheme 1 or 13-25, wherein the CD59 comprises the amino acid sequence of any one of SEQ ID NO: 41-45.

[0251] Implementation Scheme 27: An engineered polynucleotide according to any one of Implementation Scheme 1 or 13-26, wherein the second angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC).

[0252] Implementation Scheme 28: An engineered polynucleotide according to any one of Implementation Scheme 1 or 13-27, wherein the inhibitor of said MAC comprises CD59.

[0253] Implementation Scheme 29: An engineered polynucleotide according to any one of Implementation Scheme 1 or 13-28, wherein the CD59 comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 41-45.

[0254] Implementation Scheme 30: An engineered polynucleotide according to any one of Implementation Scheme 1 or 13-29, wherein the CD59 comprises the amino acid sequence of any one of SEQ ID NO: 41-45.

[0255] Implementation Scheme 31: An engineered polynucleotide according to any one of Implementation Scheme 1 or 13-30, wherein the second angiogenesis inhibitor comprises a natriuretic peptide.

[0256] Implementation Scheme 32: The engineered polynucleotide according to Implementation Scheme 31, wherein the natriuretic peptide comprises a C-type natriuretic peptide (CNP).

[0257] Implementation Scheme 33: The engineered polynucleotide according to Implementation Scheme 31 or 32, wherein the natriuretic peptide is covalently linked to an antibody or a fragment thereof.

[0258] Implementation Scheme 34: The engineered polynucleotide according to Implementation Scheme 33, wherein the antibody or fragment thereof comprises a crystallizable fragment (Fc) region.

[0259] Implementation Scheme 35: An engineered polynucleotide according to any one of Implementation Schemes 31-34, wherein the natriuretic peptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 61-72.

[0260] Implementation Scheme 36: An engineered polynucleotide according to any one of Implementation Schemes 31-35, wherein the natriuretic peptide comprises the amino acid sequence of any one of SEQ ID NO: 61-72.

[0261] Implementation Scheme 37: An engineered polynucleotide according to any one of Implementation Schemes 1, 13-36, wherein the second angiogenesis inhibitor comprises collagen or a fragment thereof.

[0262] Implementation Scheme 38: An engineered polynucleotide according to any one of Implementation Schemes 1, 13-37, wherein the second angiogenesis inhibitor comprises endostatin or a fragment thereof.

[0263] Implementation Scheme 39: An engineered polynucleotide according to any one of Implementation Schemes 1, 13-38, wherein the second angiogenesis inhibitor comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 51.

[0264] Implementation Scheme 40: An engineered polynucleotide according to any one of Implementation Schemes 1, 13-39, wherein the second angiogenesis inhibitor comprises the amino acid sequence of SEQ ID NO: 51.

[0265] Implementation Scheme 41: An engineered polynucleotide according to any one of Implementation Schemes 1, 13-40, wherein the second angiogenesis inhibitor comprises a VEGF inhibitor.

[0266] Implementation Scheme 42: The engineered polynucleotide according to Implementation Scheme 41, wherein the VEGF inhibitor comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO: 81-92.

[0267] Implementation Scheme 43: The engineered polynucleotide according to Implementation Scheme 41 or 42, wherein the VEGF inhibitor comprises the amino acid sequence of any one of SEQ ID NO: 81-92.

[0268] Implementation Scheme 44: An engineered polynucleotide according to any one of Implementation Schemes 3-43, wherein the adapter comprises a cuttable adapter.

[0269] Implementation Scheme 45: The engineered polynucleotide according to Implementation Scheme 44, wherein the cleavable adapter comprises a furin protease adapter.

[0270] Implementation Scheme 46: The engineered polynucleotide according to any one of the preceding implementation schemes further encodes a third angiogenesis inhibitor.

[0271] Implementation Scheme 47: An engineered polynucleotide according to any one of Implementation Schemes 2-46, wherein the C3 degradation fragment comprises C3a, C3b, iC3b, C3f, C3c, C3d, C3g or a combination thereof.

[0272] Implementation Scheme 48: An engineered polynucleotide according to any one of the preceding implementation schemes, wherein the engineered polynucleotide comprises a viral vector.

[0273] Implementation Scheme 49: The engineered polynucleotide according to Implementation Scheme 48, wherein the viral vector includes an AAV vector.

[0274] Implementation Scheme 50: The engineered polynucleotide according to Implementation Scheme 49, wherein the AAV vector comprises an AAV serotype, the AAV serotype comprising AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or any combination thereof.

[0275] Implementation Scheme 51: The engineered polynucleotide according to Implementation Scheme 49 or 50, wherein the AAV vector is an AAV2 vector.

[0276] Implementation Scheme 52: An engineered polynucleotide according to any one of Implementation Schemes 49-51, wherein the AAV vector encodes an engineered AAV capsid.

[0277] Implementation Scheme 53: The engineered polynucleotide according to Implementation Scheme 52, wherein the engineered AAV capsid comprises the amino acid sequence of any one of SEQ ID NO: 161-182 and SEQ ID NO: 191-210.

[0278] Implementation Scheme 54: The engineered polynucleotide according to Implementation Scheme 52 or 53, wherein the engineered AAV capsid comprises the amino acid sequence of SEQ ID NO: 169.

[0279] Implementation Scheme 55: An engineered polynucleotide according to any one of Implementation Schemes 1-54, wherein the first angiogenesis inhibitor comprises: a complement 3 inhibitor or a C3 degradation fragment comprising C3a, C3b, iC3b, C3f, C3c, C3d, C3g or a combination thereof; and the second angiogenesis inhibitor comprises CNP.

[0280] Implementation Scheme 56: An engineered polynucleotide according to any one of Implementation Schemes 1-55, wherein the first angiogenesis inhibitor comprises the complement 3 inhibitor, and the second angiogenesis inhibitor comprises CNP36.

[0281] Implementation Scheme 57: An engineered polynucleotide according to any one of Implementation Schemes 1-56, wherein the first angiogenesis inhibitor comprises the complement 3 inhibitor, and the second angiogenesis inhibitor comprises Fc-CNP36.

[0282] Implementation Scheme 58: The engineered polynucleotide according to any one of Implementation Schemes 1-57 further encodes a third angiogenesis inhibitor.

[0283] Implementation Scheme 59: The engineered polynucleotide according to Implementation Scheme 58, wherein the third angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC).

[0284] Implementation Scheme 60: The engineered polynucleotide according to Implementation Scheme 59, wherein the inhibitor of the MAC comprises CD59.

[0285] Implementation Scheme 61: An engineered polynucleotide according to any one of Implementation Schemes 58-60, wherein the engineered polynucleotide encodes a protease site side-attached to the second angiogenesis inhibitor and the third angiogenesis inhibitor.

[0286] Implementation Scheme 62: An engineered polynucleotide according to any one of Implementation Schemes 1-61, wherein the first angiogenesis inhibitor comprises an inhibitor of CD59, and the second angiogenesis inhibitor comprises CNP.

[0287] Implementation Scheme 63: An engineered polynucleotide according to any one of Implementation Schemes 1-62, wherein the first angiogenesis inhibitor comprises CD59 and the second angiogenesis inhibitor comprises CNP36.

[0288] Implementation Scheme 64: An engineered polynucleotide according to any one of Implementation Schemes 1-63, wherein the first angiogenesis inhibitor comprises CD59 and the second angiogenesis inhibitor comprises Fc-CNP36.

[0289] Implementation Scheme 65: An engineered polynucleotide according to any one of Implementation Schemes 1-64, wherein the first angiogenesis inhibitor comprises the CD59, and the second angiogenesis inhibitor comprises a complement 3 inhibitor fused with Fc-CNP36.

[0290] Implementation Scheme 66: An engineered polynucleotide according to any one of Implementation Schemes 1-65, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor and the second angiogenesis inhibitor comprises endostatin.

[0291] Implementation Scheme 67: An engineered polynucleotide according to any one of Implementation Schemes 1-66, wherein the engineered polynucleotide encodes the Fc region of the first angiogenesis inhibitor and the second angiogenesis inhibitor.

[0292] Implementation Scheme 68: An engineered polynucleotide according to any one of Implementation Schemes 1-67, wherein the first angiogenesis inhibitor comprises a VEGF inhibitor and the second angiogenesis inhibitor comprises a complement 3 inhibitor.

[0293] Implementation Scheme 69: An engineered polynucleotide according to any one of Implementation Schemes 1-68, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor and the second angiogenesis inhibitor comprises Fc-CNP36, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.

[0294] Implementation Scheme 70: An engineered polynucleotide according to any one of Implementation Schemes 1-69, wherein the engineered polynucleotide further encodes a protease site side-attached to the second angiogenesis inhibitor and the third angiogenesis inhibitor.

[0295] Implementation Scheme 71: An engineered polynucleotide according to any one of Implementation Schemes 1-70, wherein the protease site comprises a furin protease site.

[0296] Implementation Scheme 72: An engineered polynucleotide according to any one of Implementation Schemes 1-71, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor and the second angiogenesis inhibitor comprises Fc-CNP36, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.

[0297] Implementation Scheme 73: An engineered polynucleotide according to any one of Implementation Schemes 1-72, wherein the first angiogenesis inhibitor comprises CD59 and the second angiogenesis inhibitor comprises a complement 3 inhibitor, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising Fc-CNP36.

[0298] Implementation Scheme 74: An engineered polynucleotide according to any one of Implementation Schemes 1-73, wherein the first angiogenesis inhibitor comprises CD59 and the second angiogenesis inhibitor comprises endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor.

[0299] Implementation Scheme 75: An engineered polynucleotide according to any one of Implementation Schemes 1-74, wherein the first angiogenesis inhibitor comprises CD59 and the second angiogenesis inhibitor comprises a VEGF inhibitor, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor.

[0300] Implementation Scheme 76: An engineered polynucleotide according to any one of Implementation Schemes 1-75, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor and the second angiogenesis inhibitor comprises endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.

[0301] Implementation Scheme 77: The engineered polynucleotide according to any one of Implementation Schemes 1-76, wherein the first angiogenesis inhibitor, the second angiogenesis inhibitor and the third angiogenesis inhibitor are not VEGF inhibitors.

[0302] Implementation Scheme 78: An engineered polynucleotide according to any one of Implementation Schemes 1-77, wherein, after administration to a subject, the first angiogenesis inhibitor and the second angiogenesis inhibitor inhibit neovascularization in the subject.

[0303] Implementation Scheme 79: An engineered polynucleotide according to any one of Implementation Schemes 1-78, wherein, after administration to a subject, the first angiogenesis inhibitor or the second angiogenesis inhibitor exhibits reduced inhibition of angiogenesis in the subject compared to inhibition of angiogenesis induced by VEGF inhibitors.

[0304] Implementation Scheme 80: An engineered polypeptide comprising a first angiogenesis inhibitor and a second angiogenesis inhibitor.

[0305] Implementation Scheme 81: The engineered polypeptide according to Implementation Scheme 80, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are covalently linked by a connector.

[0306] Implementation Scheme 82: An engineered polypeptide according to any one of Implementation Schemes 80-81, wherein the first angiogenesis inhibitor comprises a complement inhibitor.

[0307] Implementation Scheme 83: An engineered polypeptide according to any one of Implementation Schemes 80-82, wherein the complement inhibitor comprises a complement 3 inhibitor or a C3 degradation fragment comprising C3a, C3b, iC3b, C3f, C3c, C3d, C3g or a combination thereof.

[0308] Implementation Scheme 84: An engineered polypeptide according to any one of Implementation Schemes 80-83, wherein the complement 3 inhibitor comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 1-15.

[0309] Implementation Scheme 85: An engineered polypeptide according to any one of Implementation Schemes 80-84, wherein the complement 3 inhibitor comprises an amino acid sequence containing any one of SEQ ID NO: 1-15.

[0310] Implementation Scheme 86: An engineered polypeptide according to any one of Implementation Schemes 80-85, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence having at least 80% identity with any one of SEQ ID NO: 20-33.

[0311] Implementation Scheme 87: An engineered polypeptide according to any one of Implementation Schemes 80-86, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence comprising any one of SEQ ID NO: 20-33.

[0312] Implementation Scheme 88: An engineered polypeptide according to any one of Implementation Schemes 80-87, wherein the first angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC).

[0313] Implementation Scheme 89: An engineered polypeptide according to any one of Implementation Schemes 80-88, wherein the inhibitor of the MAC comprises CD59.

[0314] Implementation Scheme 90: An engineered polypeptide according to any one of Implementation Schemes 80-89, wherein the CD59 comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 41-45.

[0315] Implementation Scheme 91: An engineered polypeptide according to any one of Implementation Schemes 80-90, wherein the CD59 comprises the amino acid sequence of any one of SEQ ID NO: 41-45.

[0316] Implementation Scheme 92: An engineered polypeptide according to any one of Implementation Schemes 80-91, wherein the second angiogenesis inhibitor comprises a natriuretic peptide.

[0317] Implementation Scheme 93: An engineered polypeptide according to any one of Implementation Schemes 80-92, wherein the natriuretic peptide is covalently linked to an antibody or a fragment thereof.

[0318] Implementation Scheme 94: An engineered polypeptide according to any one of Implementation Schemes 80-93, wherein the antibody or a fragment thereof comprises a crystallizable fragment (Fc) region.

[0319] Implementation Scheme 95: An engineered polypeptide according to any one of Implementation Schemes 80-94, wherein the natriuretic peptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 61-72.

[0320] Implementation Scheme 96: An engineered polypeptide according to any one of Implementation Schemes 80-95, wherein the natriuretic peptide comprises the amino acid sequence of any one of SEQ ID NO: 61-72.

[0321] Implementation Scheme 97: An engineered polypeptide according to any one of Implementation Schemes 80-96, wherein the second angiogenesis inhibitor comprises collagen or a fragment thereof.

[0322] Implementation Scheme 98: An engineered polypeptide according to any one of Implementation Schemes 80-97, wherein the second angiogenesis inhibitor comprises endostatin or a fragment thereof.

[0323] Implementation Scheme 99: An engineered polypeptide according to any one of Implementation Schemes 80-98, wherein the second angiogenesis inhibitor comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 51.

[0324] Implementation Scheme 100: An engineered polypeptide according to any one of Implementation Schemes 80-99, wherein the second angiogenesis inhibitor comprises the amino acid sequence of SEQ ID NO: 51.

[0325] Implementation Scheme 101: An engineered polypeptide according to any one of Implementation Schemes 80-100, wherein the second angiogenesis inhibitor comprises a VEGF inhibitor.

[0326] Implementation Scheme 102: An engineered polypeptide according to any one of Implementation Schemes 80-101, wherein the VEGF inhibitor comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 81-92.

[0327] Implementation Scheme 103: An engineered polypeptide according to any one of Implementation Schemes 80-102, wherein the VEGF inhibitor comprises the amino acid sequence of any one of SEQ ID NO: 81-92.

[0328] Implementation Scheme 104: An engineered polypeptide according to any one of Implementation Schemes 80-103, wherein the connector comprises a cuttable connector.

[0329] Implementation Scheme 105: An engineered polypeptide according to any one of Implementation Schemes 80-104, wherein the cleavable adapter comprises a furin protease adapter.

[0330] Implementation Scheme 106: The engineered polypeptide according to any one of the aforementioned Implementation Schemes 80-105 further encodes a third angiogenesis inhibitor.

[0331] Implementation Scheme 107: The engineered polypeptide according to Implementation Scheme 106, wherein the third angiogenesis inhibitor is different from the first angiogenesis inhibitor and the second angiogenesis inhibitor.

[0332] Implementation Scheme 108: A carrier comprising an engineered polynucleotide according to any one of Implementation Schemes 1-79.

[0333] Implementation Scheme 109: A carrier encoding an engineered polypeptide according to any one of Implementation Schemes 80-107.

[0334] Implementation Scheme 110: The carrier according to Implementation Scheme 108 or 109, wherein the carrier encodes an AAV capsid.

[0335] Implementation Scheme 111: The carrier according to any one of Implementation Schemes 108-110, wherein the AAV capsid comprises an engineered AAV capsid.

[0336] Implementation Scheme 112: The carrier according to any one of Implementation Schemes 108-111, wherein the modified AAV capping comprises an engineered AAV capping.

[0337] Implementation Scheme 113: A viral particle comprising an engineered polynucleotide according to any one of Implementation Schemes 1-79 or a vector according to any one of Implementation Schemes 108-112.

[0338] Implementation Scheme 114: The viral particle according to Implementation Scheme 113, wherein the viral particle comprises an AAV capsid.

[0339] Implementation Scheme 115: A viral particle according to any one of Implementation Schemes 113-114, wherein the AAV capsid comprises an engineered AAV capsid.

[0340] Implementation Scheme 116: Virus particle according to any one of Implementation Schemes 113-115, wherein the modified AAV capsid comprises an engineered AAV capsid.

[0341] Implementation Scheme 117: A cell comprising a cell of engineered polynucleotide according to any one of Implementation Schemes 1-79, an engineered polypeptide according to any one of Implementation Schemes 80-107, a vector according to any one of Implementation Schemes 108-112, or viral particles according to any one of Implementation Schemes 113-116.

[0342] Implementation Scheme 118: A composition comprising: a complement 3 inhibitor or a C3 degradation fragment comprising C3a, C3b, iC3b, C3f, C3c, C3d, C3g or a combination thereof; and a natriuretic peptide.

[0343] Implementation Scheme 119: The composition according to Implementation Scheme 118, wherein the natriuretic peptide comprises a C-type natriuretic peptide (CNP).

[0344] Implementation Scheme 120: The composition according to any one of Implementation Schemes 118-119, wherein the natriuretic peptide is covalently linked to an antibody or a fragment thereof.

[0345] Implementation Scheme 121: The composition according to any one of Implementation Schemes 118-120, wherein the antibody or fragment thereof comprises a crystallizable fragment (Fc) region.

[0346] Implementation Scheme 122: The composition according to any one of Implementation Schemes 118-121, wherein the natriuretic peptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 61-72.

[0347] Embodiment 123: The composition according to any one of Embodiments 118-122, wherein the natriuretic peptide comprises the amino acid sequence of any one of SEQ ID NO: 61-72.

[0348] Implementation Scheme 124: The composition according to any one of Implementation Schemes 118-123 further comprises CD59, endostatin, VEGF inhibitor or a combination thereof.

[0349] Implementation Scheme 125: A composition comprising: a complement 3 inhibitor or a C3 degradation fragment comprising C3a, C3b, iC3b, C3f, C3c, C3d, C3g or a combination thereof; and an inhibitor of the membrane attack complex (MAC).

[0350] Implementation Scheme 126: The composition according to any one of Implementation Schemes 118-125, wherein the inhibitor of the MAC comprises CD59.

[0351] Implementation Scheme 127: The composition according to any one of Implementation Schemes 118-126, wherein the CD59 comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 41-45.

[0352] Embodiment 128: The composition according to any one of embodiments 118-127, wherein the CD59 comprises the amino acid sequence of any one of SEQ ID NO: 41-45.

[0353] Implementation Scheme 129: The composition according to any one of Implementation Schemes 118-128 further comprises a natriuretic peptide, endostatin, a VEGF inhibitor, or a combination thereof.

[0354] Implementation Scheme 130: A composition comprising: a complement 3 inhibitor or a C3 degradation fragment comprising C3a, C3b, iC3b, C3f, C3c, C3d, C3g or a combination thereof; and collagen or a fragment thereof.

[0355] Implementation Scheme 131: The composition according to any one of Implementation Schemes 118-130, wherein the collagen or a fragment thereof comprises endostatin or a fragment thereof.

[0356] Implementation Scheme 132: The composition according to any one of Implementation Schemes 118-131, wherein the endostatin or a fragment thereof comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 51.

[0357] Implementation Scheme 133: The composition according to any one of Implementation Schemes 118-132, wherein the endostatin or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 51.

[0358] Implementation Scheme 134: The composition according to any one of Implementation Schemes 118-133 further comprises a natriuretic peptide, CD59, a VEGF inhibitor, or a combination thereof.

[0359] Implementation Scheme 135: A composition comprising: a complement 3 inhibitor or a C3 degradation fragment comprising C3a, C3b, iC3b, C3f, C3c, C3d, C3g or a combination thereof; and a VEGF inhibitor.

[0360] Implementation Scheme 136: The composition according to any one of Implementation Schemes 118-135, wherein the VEGF inhibitor comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 81-92.

[0361] Embodiment 137: The composition according to any one of Embodiments 118-136, wherein the VEGF inhibitor comprises the amino acid sequence of any one of SEQ ID NO: 81-92.

[0362] Implementation Scheme 138: The composition according to any one of Implementation Schemes 118-137 further comprises natriuretic peptide, CD59, endostatin, or a combination thereof.

[0363] Implementation Scheme 139: The composition according to any one of Implementation Schemes 118-138, wherein the complement 3 inhibitor comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 1-15.

[0364] Implementation Scheme 140: The composition according to any one of Implementation Schemes 118-139, wherein the complement 3 inhibitor comprises an amino acid sequence containing any one of SEQ ID NO: 1-15.

[0365] Implementation Scheme 141: The composition according to any one of Implementation Schemes 118-140, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence having at least 80% identity with any one of SEQ ID NO: 20-33.

[0366] Implementation Scheme 142: The composition according to any one of Implementation Schemes 118-141, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence comprising any one of SEQ ID NO: 20-33.

[0367] Implementation Scheme 143: A composition comprising CD59 and natriuretic peptide.

[0368] Implementation Scheme 144: The composition according to any one of Implementation Schemes 118-143, wherein the natriuretic peptide comprises a C-type natriuretic peptide (CNP).

[0369] Implementation Scheme 145: The composition according to any one of Implementation Schemes 118-144, wherein the natriuretic peptide is covalently linked to an antibody or a fragment thereof.

[0370] Implementation Scheme 146: The composition according to any one of Implementation Schemes 118-145, wherein the antibody or fragment thereof comprises a crystallizable fragment (Fc) region.

[0371] Implementation Scheme 147: The composition according to any one of Implementation Schemes 118-146, wherein the natriuretic peptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 61-72.

[0372] Embodiment 148: The composition according to any one of Embodiments 118-147, wherein the natriuretic peptide comprises the amino acid sequence of any one of SEQ ID NO: 61-72.

[0373] Implementation Scheme 149: The composition according to any one of Implementation Schemes 143-148 further comprises a complement 3 inhibitor, endostatin, a VEGF inhibitor, or a combination thereof.

[0374] Implementation Scheme 150: A composition comprising CD59 and collagen or fragments thereof.

[0375] Embodiment 151: The composition according to any one of Embodiments 118-150, wherein the collagen or a fragment thereof comprises endostatin or a fragment thereof.

[0376] Implementation Scheme 152: The composition according to any one of Implementation Schemes 118-151, wherein the endostatin or a fragment thereof comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 51.

[0377] Embodiment 153: The composition according to any one of Embodiments 118-152, wherein the endostatin or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 51.

[0378] Implementation Scheme 154: The composition according to any one of Implementation Schemes 118-153 further comprises a natriuretic peptide, a complement 3 inhibitor, a VEGF inhibitor, or a combination thereof.

[0379] Implementation Scheme 155: A composition comprising CD59 and VEGF inhibitors.

[0380] Implementation Scheme 156: The composition according to any one of Implementation Schemes 118-155, wherein the VEGF inhibitor comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 81-92.

[0381] Embodiment 157: The composition according to any one of Embodiments 118-156, wherein the VEGF inhibitor comprises the amino acid sequence of any one of SEQ ID NO: 81-92.

[0382] Implementation Scheme 158: The composition according to any one of Implementation Schemes 118-157 further comprises a natriuretic peptide, a complement 3 inhibitor, endostatin, or a combination thereof.

[0383] Embodiment 159: The composition according to any one of embodiments 118-158, wherein the CD59 comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 41-45.

[0384] Embodiment 160: The composition according to any one of embodiments 118-159, wherein the CD59 comprises the amino acid sequence of any one of SEQ ID NO: 41-45.

[0385] Embodiment 161: A pharmaceutical composition comprising an engineered polynucleotide according to any one of embodiments 1-79, an engineered polypeptide according to any one of embodiments 80-107, a carrier according to any one of embodiments 108-112, viral particles according to any one of embodiments 113-116, a cell according to embodiment 117, or a composition according to any one of embodiments 118-160.

[0386] Implementation Scheme 162: The pharmaceutical composition according to Implementation Scheme 161, wherein the pharmaceutical composition is formulated for administration to a subject in need via intrathecal, intraocular, intravitreal, retinal, intravenous, intramuscular, intracardiac, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, subretinal, suprachoroidal, intratumoral, pulmonary, tracheal, intraperitoneal, intrabladder, intravaginal, rectal, oral, sublingual, transdermal, inhalation, inhalation nebulization, intraluminal-GI route, or a combination thereof.

[0387] Implementation Scheme 163: The pharmaceutical composition according to any one of Implementation Schemes 160-162, wherein the pharmaceutical composition is formulated for intravitreal, subretinal or choroidal administration.

[0388] Implementation Scheme 164: A method comprising contacting cells obtained from an object with an engineered polynucleotide according to any one of embodiments 1-79, an engineered polypeptide according to any one of embodiments 80-107, a vector according to any one of embodiments 108-112, a viral particle according to any one of embodiments 113-116, a cell according to embodiment 117, a composition according to any one of embodiments 118-160, or a pharmaceutical composition according to any one of embodiments 161-163.

[0389] Implementation Scheme 165: A method of treating a disease or condition in a subject, comprising: administering to the subject an engineered polynucleotide according to any one of Implementation Schemes 1-79, an engineered polypeptide according to any one of Implementation Schemes 80-107, a carrier according to any one of Implementation Schemes 108-112, viral particles according to any one of Implementation Schemes 113-116, cells according to Implementation Scheme 117, a composition according to any one of Implementation Schemes 118-160, or a pharmaceutical composition according to any one of Implementation Schemes 161-163.

[0390] Implementation Scheme 166: The method according to any one of Implementation Schemes 164 or 165, wherein a single application cures the disease or condition.

[0391] Implementation Scheme 167: The method according to any one of Implementation Schemes 164-166, wherein the application does not include daily application.

[0392] Implementation Scheme 168: The method according to any one of Implementation Schemes 164-167, wherein the application includes weekly application, bi-weekly application, monthly application, bi-monthly application, semi-annual application, annual application, or a combination thereof.

[0393] Implementation Scheme 169: The method according to any one of Implementation Schemes 164-168, wherein the disease or condition includes an eye disease.

[0394] Implementation Scheme 170. The method described in Implementation Schemes 164-169, wherein the ocular diseases include ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, 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-Bied syndrome, Best disease, choroidal agenesis, Leber congenital amaurosis, macular degeneration, polypoid choroidal angiopathy (PCV), retinitis pigmentosa, Revesum disease, Staggart disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, microstomia, Leventinese disease (familial dominant drusen), blue cone monochromatic vision, or combinations thereof.

[0395] Implementation Scheme 171: The method according to any one of Implementation Schemes 164-170, wherein the eye disease includes GA or dAMD.

[0396] Implementation Scheme 172: A method for treating a disease or condition in a subject, the method comprising administering an engineered polynucleotide to the subject, the engineered polynucleotide comprising one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor.

[0397] Implementation Scheme 173: The method according to any one of Implementation Schemes 164-172, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are each encoded by one of the one or more expression cassettes.

[0398] Implementation Scheme 174: The method according to any one of Implementation Schemes 164-173, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are operatively coupled.

[0399] Implementation Scheme 175: The method according to any one of Implementation Schemes 164-174, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are covalently connected via a connector.

[0400] Implementation Scheme 176: The method according to any one of Implementation Schemes 164-175, wherein the first angiogenesis inhibitor comprises a complement inhibitor.

[0401] Implementation Scheme 177: The method according to any one of Implementation Schemes 164-176, wherein the complement inhibitor comprises a complement 3 inhibitor or a C3 degradation fragment comprising C3a, C3b, iC3b, C3f, C3c, C3d, C3g or a combination thereof.

[0402] Implementation Scheme 178: The method according to any one of Implementation Schemes 164-177, wherein the complement 3 inhibitor comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 1-15.

[0403] Implementation Scheme 179: The method according to any one of Implementation Schemes 164-178, wherein the complement 3 inhibitor comprises an amino acid sequence containing any one of SEQ ID NO: 1-15.

[0404] Implementation Scheme 180: The method according to any one of Implementation Schemes 164-179, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence having at least 80% identity with any one of SEQ ID NO: 20-33.

[0405] Implementation Scheme 181: The method according to any one of Implementation Schemes 164-180, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence comprising any one of SEQ ID NO: 20-33.

[0406] Implementation Scheme 182: The method according to any one of Implementation Schemes 164-181, wherein the first angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC).

[0407] Implementation Scheme 183: The method according to any one of Implementation Schemes 164-182, wherein the inhibitor of the MAC comprises CD59.

[0408] Implementation Scheme 184: The method according to any one of Implementation Schemes 164-183, wherein the CD59 comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 41-45.

[0409] Implementation Scheme 185: The method according to any one of Implementation Schemes 164-184, wherein the CD59 comprises the amino acid sequence of any one of SEQ ID NO: 41-45.

[0410] Implementation Scheme 186: The method according to any one of Implementation Schemes 164-185, wherein the second angiogenesis inhibitor comprises a natriuretic peptide.

[0411] Implementation Scheme 187: The method according to any one of Implementation Schemes 164-186, wherein the natriuretic peptide is covalently linked to an antibody or a fragment thereof.

[0412] Implementation Scheme 188: The method according to any one of Implementation Schemes 164-187, wherein the antibody or fragment thereof comprises a crystallizable fragment (Fc) region.

[0413] Implementation Scheme 189: The method according to any one of Implementation Schemes 164-188, wherein the natriuretic peptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 61-72.

[0414] Implementation Scheme 190: The method according to any one of Implementation Schemes 164-189, wherein the natriuretic peptide comprises the amino acid sequence of any one of SEQ ID NO: 61-72.

[0415] Implementation Scheme 191: The method according to any one of Implementation Schemes 164-190, wherein the second angiogenesis inhibitor comprises collagen or a fragment thereof.

[0416] Implementation Scheme 192: The method according to any one of Implementation Schemes 164-191, wherein the second angiogenesis inhibitor comprises endostatin or a fragment thereof.

[0417] Implementation Scheme 193: The method according to any one of Implementation Schemes 164-192, wherein the second angiogenesis inhibitor comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 51.

[0418] Implementation Scheme 194: The method according to any one of Implementation Schemes 164-193, wherein the second angiogenesis inhibitor comprises the amino acid sequence of SEQ ID NO: 51.

[0419] Implementation Scheme 195: The method according to any one of Implementation Schemes 164-194, wherein the second angiogenesis inhibitor comprises a VEGF inhibitor.

[0420] Implementation Scheme 196: The method according to any one of Implementation Schemes 164-195, wherein the VEGF inhibitor comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 81-92.

[0421] Implementation Scheme 197: The method according to any one of Implementation Schemes 164-196, wherein the VEGF inhibitor comprises the amino acid sequence of any one of SEQ ID NO: 81-92.

[0422] Implementation Scheme 198: The method according to any one of Implementation Schemes 164-197, wherein the joint comprises a cuttable joint.

[0423] Implementation Scheme 199: The method according to any one of Implementation Schemes 164-198, wherein the cuttable adapter comprises a furin protease adapter.

[0424] Implementation Scheme 200: The method according to any one of Implementation Schemes 164-199, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor.

[0425] Implementation Scheme 201: The method according to any one of Implementation Schemes 164-200, wherein the third angiogenesis inhibitor is different from the first angiogenesis inhibitor and the second angiogenesis inhibitor.

[0426] Implementation Scheme 202: The method according to any one of Implementation Schemes 164-201, wherein the engineered polynucleotide comprises a viral vector.

[0427] Implementation Scheme 203: The method according to any one of Implementation Schemes 164-202, wherein the viral vector comprises an AAV vector.

[0428] Implementation Scheme 204: The method according to any one of Implementation Schemes 164-203, 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.

[0429] Implementation Scheme 205: The method according to any one of Implementation Schemes 164-204, wherein the AAV vector is an AAV2 vector.

[0430] Implementation Scheme 206: The method according to any one of Implementation Schemes 164-205, wherein the AAV carrier is encoded with an engineered AAV coat.

[0431] Implementation Scheme 207: The method according to any one of Implementation Schemes 164-206, wherein the engineered AAV capsid comprises the amino acid sequence of any one of SEQ ID NO: 161-182 and SEQ ID NO: 191-210.

[0432] Implementation Scheme 208: The method according to any one of Implementation Schemes 164-207, wherein the engineered AAV capsid comprises the amino acid sequence of SEQ ID NO: 169.

[0433] Implementation Scheme 209: The method according to any one of Implementation Schemes 164-208, wherein a single application cures the disease or condition.

[0434] Implementation Scheme 210: The method according to any one of Implementation Schemes 164-209, wherein the application does not include daily application.

[0435] Implementation Scheme 211: The method according to any one of Implementation Schemes 164-210, wherein the application includes weekly application, bi-weekly application, monthly application, bi-monthly application, semi-annual application, annual application, or a combination thereof.

[0436] Implementation Scheme 212: The method according to any one of Implementation Schemes 164-211, wherein the disease or condition includes an eye disease.

[0437] Implementation Scheme 213: The method according to any one of Implementation Schemes 164-212, wherein the ocular disease includes ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, 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), Bader-Bide syndrome, Best disease, choroidal agenesis, Leber congenital amaurosis, macular degeneration, polypoid choroidal angiopathy (PCV), retinitis pigmentosa, Revesum disease, Staggart disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, microstomia, Leventinese disease (familial dominant drusen), blue cone monochromatic vision, or combinations thereof.

[0438] Implementation Scheme 214: The method according to any one of Implementation Schemes 164-213, wherein the ocular disease includes GA or dAMD.

[0439] Implementation Scheme 215: The method according to any one of Implementation Schemes 164-214, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.

[0440] Implementation Scheme 216: The method according to any one of Implementation Schemes 164-215, wherein the first angiogenesis inhibitor, the second angiogenesis inhibitor, and the third angiogenesis inhibitor are not VEGF inhibitors.

[0441] Implementation Scheme 217: The method according to any one of Implementation Schemes 164-216, wherein after administration to a subject, the first angiogenesis inhibitor and the second angiogenesis inhibitor inhibit neovascularization in the subject.

[0442] Implementation Scheme 218: The method according to any one of Implementation Schemes 164-217, wherein, after administration to the subject, the first angiogenesis inhibitor or the second angiogenesis inhibitor exhibits reduced inhibition of angiogenesis in the subject compared to the inhibition of angiogenesis caused by the VEGF inhibitor.

[0443] Example The following illustrative examples represent implementations of the stimuli, systems, and methods described herein and are not intended to be limiting in any way.

[0444] Example 1. A vector for expressing multiple angiogenesis inhibitors Vectors can be designed to express the various angiogenesis inhibitors described in this paper. Figure 1 A vector map (top) illustrates the expression of complement 3 inhibitors (C3i) that are operatively coupled to a leader sequence (LS). Figure 1 The bottom vector map illustrates complement 3 inhibitors (C3i) operably coupled to the Fc region of human IgG. Complement 3 inhibitors (C3i) can be modified to include at least one amino acid substitution compared to wild-type complement 3 inhibitors (C3i). Table 1 illustrates exemplary C3i amino acid sequences that can be encoded by the vectors described herein. Table 2 illustrates exemplary nucleic acid sequences used to encode exemplary C3i.

[0445] Table 1. Exemplary C3i amino acid sequences

[0446] Table 2. Exemplary nucleic acid sequences encoding C3i

[0447] Figure 2A -B describes complement 3 inhibitors (C3i) (e.g., the GAM or GGE constructs described herein) that are operatively coupled to natriuretic peptides (e.g., CNP36) via the Fc region of human IgG. Figure 2A As shown, the clonal 15-mer-C3i was linked between the heavy chain leader sequence and Fc4 using 2xGGGS in the vector CPE (SEQ ID NO: 15). Figure 2B Similar vectors were described, in which C3i further contains an EVQL peptide or a DK peptide at the N-terminus of C3i. Figure 3Vectors for expressing CD59, C3i, and the natriuretic peptide (CNP36) were depicted. Figure 3 The C3i described herein can be any C3i, including Figure 2B C3i is shown in the figure. Figure 4 Additional exemplary vectors for expressing the angiogenesis inhibitors described herein are illustrated. Table 3 illustrates exemplary clone identification and construct names of the vectors described herein.

[0448] Table 3. Exemplary Carrier Constructions

[0449] The expression levels of vectors encoding multiple angiogenesis inhibitors can be measured. For example, enzyme-linked immunosorbent assay (ELISA), Western blotting, or SDS-PAGE can quantify the expression levels of angiogenesis inhibitors in cell lysates or cell culture supernatants. Once the expression of multiple angiogenesis inhibitors is confirmed, their therapeutic efficacy can be determined in cell or animal models. For example, the therapeutic efficacy of co-expression of multiple angiogenesis inhibitors from vectors can be evaluated in mouse models of retinal degeneration N-methyl-D-aspartate (NMDA) excitotoxicity, rat models of partial optic nerve transection (pONT), or glaucoma models demonstrating neuroprotective and therapeutic effects.

[0450] Example 2. rBV amplification and AAV generation Cell culture maintenance SF-RVF cells were cultured in ESF AF medium (Expression Systems, Davis, CA) contained in Corning flasks with gentle shaking at 160 RPM and 28°C. Cells were cultured until the cell density reached approximately 1 x 10⁻⁶ cells / mL. 7 When the number of cells / mL is 1:2 to 1:8, they are isolated with fresh culture medium and continuously cultured to maintain their condition.

[0451] Baculovirus shuttle vector (Bacmid) transfection and rBV amplification Recombinant baculoviruses expressing capsid proteins (AAV2.N54 or AAV6.N54) and GOP were generated using the Bac-to-Bac baculovirus expression system (Thermo Fisher Scientific, Fremont, CA) and GenJet DNA transfection reagent. The recombinant baculoviruses were amplified by infecting 50–100 mL of SF-RVF cell culture in 250 mL Corning flasks at a 1:200 (v / v) ratio with shaking at 180 RPM and 28 °C for three days. Baculovirus titers were detected by qPCR.

[0452] AAV generation and purification Co-infection with 5 × 10⁵ rBV at an MOI of 400–800 was performed using a mixture of rBV-AAV2.N54 (AAV6.N54) and rBV-GOI, based on rBV titers measured by qPCR analysis. 6 AAV was generated by incubating 200 mL of SF-RVF cell culture at 100 cells / mL. The rBV-infected culture was incubated for three days with shaking at 180 RPM and 28°C. The cell pellet was harvested and lysed at 37°C in lysis buffer (1% (w / v) Sarkosyl, 1% (v / v) Triton X-100, 10 mM Tris∙HCl pH 8.0, 2 M urea, 2 mM MgCl2, and 25 IU / mL Benzonase). TM The lysate was incubated at 300 RPM / min for 1 hour with shaking. At the end of the incubation, NaCl stock solution (5M) was added to the lysate to a final concentration of 0.5–1 M, and the lysate was centrifuged at 8000 RPM for 20 minutes. The clarified lysate was purified by two rounds of CsCl gradient ultracentrifugation. The intact AAV band was pulled out with a needle and replaced with PD-10 desalting column buffer in ACI formulation buffer 1a. The aseptically filtered AAV was quantified by ddPCR analysis using GOI-specific primers / probes.

[0453] Example 3. Cloning and expression of the C3i-Fc4-CNP36 fusion gene The C3i-Fc4-CNP36 fusion gene, which contains the coding sequence for the human antibody heavy chain secretion signal peptide at its 5' end, was constructed into a single-chain AAV vector, vector ETP, and named vector GAM. Figure 5A , Figure 5B or Figure 5C Using Jcat programs () Braunschweig, Lower Saxony, Germany, reverse-translated a designed human antibody heavy chain secretion signal peptide-C3i-3xGGGGS fusion protein into a protein with Homo sapiens characteristics. homo sapiens After the DNA sequence output by the codons was obtained, some of the fusion genes were further manually modified to adjust the GC content and sent to Integrated DNA Technologies, Inc. (Coralville, Iowa) for DNA synthesis. When the synthesized DNA fragment was successfully cloned into the vector ETP, the plasmid DNA was identified by restriction digestion and the sequence between the two ITRs of the plasmid DNA from the correct colony was verified by full sequencing (List 4).

[0454] Transient expression of vectors in mammalian cell culture systems Human HEK293LTV cells were used for fusion gene expression in this study. They were cultured in DMEM medium (Thermo Fisher Scientific) with 10% FBS (ATCC, Manassas, VA) at 37°C in a CO2 incubator. To maintain passage, cells were separated twice weekly at a 1:10 ratio. For transfection, cells were cultured at 0.75 x 10⁻⁶ cells / mL. 6 2 cells / well Inoculate cells with 1 mL of medium onto 6-well plates (Corning, NY) overnight. Replace the medium with DMEM containing 2% FBS 1 or 2 hours before transfection. For each well, dilute 2 µg of plasmid DNA in 300 µL of 150 mM NaCl, then add 8 µL of PEImax (Polysciences, Germany). After incubating the mixture at room temperature for 20 minutes, add it dropwise to the cells and incubate at 37°C in a CO2 incubator for 3 days. Then, harvest the medium for further experiments and add 3 mL of fresh medium to the cells. Again, harvest the medium after 3 days. Additionally, when relatively large amounts of protein are required, use a T125 flask and increase the amount of plasmid DNA and reagents accordingly.

[0455] When the GAM vector plasmid was prepared using Xtra Maxi Plus EF (Macherey-Nagel SAS, France), it was confirmed by restriction digestion with four different enzymes. In this study, the EKQ vector plasmid containing the EGFP gene was used as a transfection control, and the CPE vector plasmid containing the Fc4-CNP36 gene was used as a protein expression control. HEK293LTV cells were seeded into 6-well plates one day prior to transient transfection, and each transfection was performed using PEImax at 2 µg / well DNA. To assess transfection efficiency, cells transfected with the EKQ vector were observed under a fluorescence microscope (data not shown). Cell culture supernatants were collected at 3 and 6 days post-transfection for protein expression analysis.

[0456] Detection and quantification of C3i-Fc4-CNP36 fusion protein The C3i-Fc4-CNP36 fusion protein was detected by SDS-PAGE and Western blot analysis (Table 5). HEK293LTV cell culture medium (supernatant) was collected on days 3 and 6 after transfecting plasmid DNA into cells in 6-well plates or T125 flasks. A total volume of 26 μL of supernatant was mixed with 10 μL of 4x loading buffer and 4 μL of 10x reducing buffer, and then loaded onto Novex. TM WedgeWell TM Tris-glycine gel (Thermo Fisher Scientific) was used for electrophoresis. After running the protein gel at 150 V for approximately 1.5 hours, a piece of gel was then coated with SimplyBlue. TM SafeStain (Thermo Fisher Scientific) staining, destaining with water, and imaging using a digital camera ( Figure 6 , Figure 7 , Figure 8 and Figure 9 On the other hand, another gel was subsequently transferred to a PVDF membrane using a Trans-Blot rapid transfer system (Bio-Rad, Hercules, CA, USA). The membranes were treated with casein blocking agent in PBS (Thermo Scientific, Waltham, MA, USA) for 1 hour at room temperature, and then immediately probed with either HRP-conjugated goat anti-human IgG1 Fc antibody (A01854-200) (Genscript, Nanjing, China) or rat anti-human CNP antibody (MAB31271) (Biotechne, Minneapolis, MN), followed by detection with goat-HRP-conjugated goat anti-rat IgG secondary antibody (1:10,000) (31470) (Thermo Fisher Scientific). Supersignal was used. TM Proteins were detected using the West Atto high-sensitivity reagent kit (Thermo Fisher Scientific) and via Gel Doc. TM XR+ using Image Lab TM Software (Bio-Rad, Hercules, CA) captures images ( Figure 6 , Figure 7 , Figure 8 and Figure 9SDS-PAGE and Western blot results showed that the C3i-Fc4-CNP36 fusion protein was detected in the culture supernatant of cells transfected with the GAM vector on days 3 or 6, but not in the culture supernatant of cells transfected with the EKQ vector or untransfected cells. However, in 6-well plates or T125 flasks using HRP-conjugated goat anti-human IgG1 Fc antibody or rat anti-human CNP antibody, the expression level of the C3i-Fc4-CNP36 fusion protein of the GAM vector plasmid was much lower than that of the Fc4-CNP36 fusion protein expressed by the CPE vector plasmid.

[0457] In addition, the concentration of C3i-Fc4-CNP36 fusion protein in the supernatant was measured by sandwich ELISA. 96-well microplates were coated with anti-human CNP antibody (MAB31271) (Biotechne, Minneapolis, MN) and incubated overnight at 4°C, followed by treatment with casein blocking agent in PBS at room temperature for 1 hour. CNP protein standards or supernatant samples were then added to the microplates. After three washes, biotin-labeled goat anti-human IgG Fc (ab98618) (Abcam, Waltham, MA) was applied to the plates, followed by HRP-streptavidin conjugate (ab7403) (Abcam, Waltham, MA). A 1-step ELISA was used. TM The colorimetric reaction was developed using Ultra TMB-ELISA substrate solution (ab171523) (Abcam, Waltham, MA) and terminated by the addition of 2 M HCl. Finally, the microplates were read and recorded using a SpectaMax iD3 (Molecular Devices, San Jose, CA). ELISA data were plotted and analyzed using GraphPad Prism 9.5.1 (Dotmatics, Boston, Massachusetts). Table 6 shows the concentrations of the C3i-Fc4-CNP36 fusion protein in the supernatant at 3 days and 6 days as 4.87 and 8.97 μg / mL, respectively. Similarly, when using T125 flasks, the fusion protein concentrations in the supernatant at 3 days and 6 days were 29.02 and 16.09 μg / mL, respectively (Table 7). The fusion protein concentrations measured by ELISA were significantly higher than those measured by SDS-PAGE and Western blot.

[0458] The C3i-Fc4-CNP36 fusion protein was detected in the culture supernatant of cells transfected with vector GAM on days 3 or 6 of a 6-well plate using an HRP-conjugated goat anti-human IgG1 Fc antibody, but not in the culture supernatant of cells transfected with vector EKQ or untransfected cells. However, the expression level of the C3i-Fc4-CNP36 fusion protein in the GAM vector plasmid was significantly lower than that in the Fc4-CNP36 fusion protein expressed in the CPE vector plasmid. The obtained Western blot results are as follows: Figure 6 As shown. Lane contents: 1. GAM vector 3d; 2. EKQ vector 3d; 3. CME vector 3d; 4. Untransfected 3d; 5. GAM vector 6d; 6. EKQ vector 6d; 7. CME vector 6d; 8. Untransfected 6d.

[0459] The C3i-Fc4-CNP36 fusion protein was detected in the supernatant of cells transfected with vector GAM on days 3 or 6 in T125 flasks using an HRP-conjugated goat anti-human IgG1 Fc antibody, but not in the supernatant of cells transfected with vector EKQ or untransfected cells. However, the expression level of the C3i-Fc4-CNP36 fusion protein in the GAM vector was significantly lower than that in the CPE vector. The resulting Western blot results are shown below. Figure 7 As shown. Lane contents: 1. GAM vector 3d; 2. EKQ vector 3d; 3. CME vector 3d; 4. Untransfected 3d; 5. GAM vector 6d; 6. EKQ vector 6d; 7. CME vector 6d; 8. Untransfected 6d.

[0460] Rat anti-human CNP antibody detected the C3i-Fc4-CNP36 fusion protein in the culture supernatant of cells transfected with vector GAM on days 3 or 6 in 6-well plates, but not in the culture supernatant of cells transfected with vector EKQ or untransfected cells. However, the expression level of the C3i-Fc4-CNP36 fusion protein in the GAM vector plasmid was much lower than that of the Fc4-CNP36 fusion protein expressed in the CPE vector plasmid. The obtained Western blot results are as follows... Figure 8 As shown. Lane contents: 1. GAM vector 3d; 2. EKQ vector 3d; 3. CME vector 3d; 4. Untransfected 3d; 5. GAM vector 6d; 6. EKQ vector 6d; 7. CME vector 6d; 8. Untransfected 6d.

[0461] The C3i-Fc4-CNP36 fusion protein was detected in the culture supernatant of cells transfected with vector GAM in T125 flasks on days 3 or 6 using rat anti-human CNP antibody, but not in the culture supernatant of cells transfected with vector EKQ or untransfected cells. However, the expression level of the C3i-Fc4-CNP36 fusion protein in the GAM vector plasmid was much lower than that of the Fc4-CNP36 fusion protein expressed in the CPE vector plasmid. The obtained Western blot results are as follows... Figure 9 As shown. Lane contents: 1. GAM vector 3d; 2. EKQ vector 3d; 3. CME vector 3d; 4. Untransfected 3d; 5. GAM vector 6d; 6. EKQ vector 6d; 7. CME vector 6d; 8. Table 4. DNA sequences of the plasmids used in Example 3

[0462] Table 5. Peptide and / or fusion protein sequences

[0463] Table 6. Concentration of fusion protein in 6-well plates of HEK293 cells as measured by sandwich ELISA

[0464] Table 7. Concentration of fusion protein in T125 flasks of HEK293 cells as measured by sandwich ELISA

[0465] Example 4. Cloning and expression of membrane-bound and soluble CD59 genes Design and molecular cloning of membrane-bound and soluble CD59 (sCD59) genes A membrane-bound CD59 (mCD59) with a natural signal peptide coding sequence at the 5' end was constructed into a self-complementary AAV vector, CPE. After reverse translating the mCD59 protein into DNA sequences with Homo sapiens codon outputs using four different programs from Snapgene, Jcat, Geneart, and Genscript, the full-length mCD59 genes with different codon optimizations were further manually modified to adjust GC content and sent to Twist Bioscience (South San Francisco, CA) for DNA synthesis. After successful cloning of the synthesized DNA fragments into the CPE vector, all plasmid DNAs were identified and named by restriction digestion: vector GTM for wild-type mCD59, vector GTP for Snapgene-optimized mCD59, vector GTQ for Jcat-optimized mCD59, vector GTR for Geneart-optimized mCD59, and vector GAT for Genscript-optimized mCD59. Figure 10 (The sequences from the two ITRs in all these plasmids were verified by full sequencing, as shown in Table 8. Additional information about the sequence information is shown in Table 9.)

[0466] Because wild-type and Snapgene-optimized mCD59 showed higher expression levels in these genes, they were selected for soluble CD59 expression. After removing the GPI anchor peptide (GGTSLSEKTVLLLVTPFLAAAWSLHP) coding sequence from the full-length CD59 gene, wild-type and Snapgene-optimized sCD59 subclones with native or human antibody heavy chain secretion signal peptide coding sequences at the 5' end were inserted into single-chain AAV vectors, vector ETP. Four different plasmids were obtained: vector GCK containing the native secretion signal peptide coding sequence and the sCD59 sequence; vector GCM containing the human antibody heavy chain secretion signal peptide coding sequence and the wild-type sCD59 sequence; vector GEM containing the native secretion signal peptide coding sequence and the Snapgene-optimized sCD59 gene; and vector GEP containing the human antibody heavy chain secretion signal peptide coding sequence and the Snapgene-optimized sCD59 gene. All of these plasmids were identified by restriction digestion and their sequences between the two ITRs in all plasmids were verified by full sequencing (Table 8).

[0467] Transient expression of the mCD59 gene in mammalian cell culture systems Human EXPI293 suspension cells were used for mCD59 expression. Cells were placed in a solution containing 5 mM glutamax. TMCells were cultured in BalanCD medium (ThermoFisher Scientific) at 37°C in an orbital shaker (Fujifilm Irvine Scientific, Santa Ana, CA). Immediately before transfection with Mirus transfection reagent (San Francisco, CA), cells were diluted to 2.0 x 10⁻⁶ cells / mL in shake flasks (Corning, NY). 6 1 cell / mL in 25 mL of culture medium. For each shake flask, dilute 50 µg of plasmid DNA to 2.5 mL in a sterile tube. SELECT AAV complex formation solution and enhancer, then add 75 µl TransIT-VirusGEN to the diluted DNA. SELECT reagent. Incubate the mixture at room temperature for 15–30 minutes without additional agitation to allow the transfection complex to form. Then, add it dropwise to the cells and incubate at 37°C in an orbital shaker for 2 days. Harvest the cells for further experiments. After washing the cells once with 1xPBS, transfer them to 10 x 10⁻⁶ cells / cells. 6 Add 1 mL of RIPA buffer (89901) (Thermo Fisher Scientific) containing a 1x protease inhibitor mixture (1183670001) (Millipore Sigma, Burlington, MA) to each cell. After sonicating the cells three times at 10-second intervals, the sample is incubated at 12000 x 1000 μL. g Centrifuge for 15 minutes 。 Cell lysates were collected and processed via Pierce. TM The BCA Protein Assay Kit (23225) (Thermo Fisher Scientific) measures protein concentration.

[0468] Vectors GTM, GTP, GTQ, GTR, and GAT were prepared using Xtra Maxi Plus EF (Macherey-Nagel SAS, France) and confirmed by restriction digestion. Vector EKQ, containing the EGFP gene, was used as a transfection control. EXPI293 suspension cells were diluted to 2.0 x 10⁻⁶. 6 After adding cells / mL to 25 mL of culture medium, each transfection was performed using Mirus transfection reagent with 50 µg of DNA. To assess transfection efficiency, cells transfected with the EKQ vector were observed under a fluorescence microscope. Cell lysates were prepared, collected, and analyzed for protein expression.

[0469] mCD59 protein in cell lysates was detected by SDS-PAGE and Western blot analysis. EXPI293 cells were collected on day 2 after plasmid DNA transfection. A total volume of 26 μL of cell lysates was mixed with 10 μL of 4x loading buffer and 4 μL of 10x reducing buffer and loaded into NuPAGE. TM 4-12% Bis-Tris gel (Thermo Fisher Scientific) was used for electrophoresis. Proteins were run at 150 V for approximately 1 hour, and a piece of gel was then coated with SimplyBlue. TM SafeStain (Thermo Fisher Scientific) staining, destaining with water, and imaging with a digital camera. Figure 11 Then, another gel was transferred onto a PVDF membrane using a Trans-Blot rapid transfer system (Bio-Rad, Hercules, CA, USA). The membranes were treated with casein blocking agent in PBS (Thermo Scientific, Waltham, MA, USA) for 1 hour at room temperature, and then immediately probed with HRP-conjugated mouse anti-human CD59 monoclonal antibody (sc-133170) (Santa Cruz Biotechnology, Dallas, TX). Supersignal was used. TM Proteins were detected using the West Atto Ultrasensor Kit (ThermoFisher Scientific) and Image Labs via Gel Doc™ XR+. TM Images were captured using software (Bio-Rad, Hercules, CA). SDS-PAGE and Western blot results showed that mCD59 was expressed in cells transfected with vectors GTM, GTP, GTQ, GTR, and GAT, but not in cells transfected with vector EKQ or untransfected cells. Figure 11 Furthermore, Snapgene-optimized mCD59 showed the highest expression level, followed by wild-type mCD59, Genscript-optimized, Jcat-optimized, and Geneart-optimized mCD59 showed the lowest expression level.

[0470] Transient expression of the sCD59 gene in mammalian cell culture systems Human HEK293LTV cells were used for sCD59 gene expression analysis and were cultured in DMEM medium (Thermo Fisher Scientific) with 10% FBS (ATCC, Manassas, VA) at 37°C in a CO2 incubator. To maintain passage, cells were separated twice weekly at a 1:10 ratio. For transfection, cells were cultured at 0.6 x 10⁻⁶ cells / mL. 6 One cell / well was seeded in 2 mL of medium onto a 6-well plate (Corning, NY) and incubated overnight. One or two hours before transfection, the medium was replaced with DMEM containing 2% FBS. For each well, 2 µg of plasmid DNA was diluted in 300 µL of 150 mM NaCl, followed by the addition of 8 µL of PEImax (Polysciences, Germany). After incubating the mixture at room temperature for 20 minutes, it was added dropwise to the cells and incubated at 37°C in a CO2 incubator. After three days, all medium from each well was harvested for further experiments, and 3 mL of fresh medium was added to the cells. The medium was also harvested again within three days.

[0471] When the GCK, GCM, GEM, and GEP vectors were prepared using Xtra Maxi Plus EF (Macherey-Nagel SAS, France), they were confirmed by restriction digestion. In this study, the EKQ vector containing the EGFP gene was used as a transfection control. HEK293LTV cells were seeded into 6-well plates one day prior to transient transfection, and each transfection was performed using PEImax with 2 µg / well DNA. To assess transfection efficiency, cells transfected with the EKQ vector were observed under a fluorescence microscope. Cell culture supernatants were collected at 3 and 6 days post-transfection for protein expression analysis.

[0472] sCD59 protein was detected by SDS-PAGE and Western blot analysis. Cell culture supernatant was collected on days 3 and 6 after plasmid DNA transfection. Each 26 μL aliquot of supernatant was mixed with 10 μL of 4x loading buffer and 4 μL of 10x reducing buffer and loaded onto NuPAGE. TM 4-12% Bis-Tris gel (Thermo Fisher Scientific) was used for electrophoresis. After running the protein gel at 150 V for approximately 1 hour, a piece of gel was then coated with SimplyBlue. TM SafeStain (Thermo Fisher Scientific) staining, destaining with water, and imaging with a digital camera. Figure 11 and Figure 12On the other hand, another gel was subsequently transferred onto a PVDF membrane using a Trans-Blot rapid transfer system (Bio-Rad, Hercules, CA, USA). The membranes were treated with casein blocking agent in PBS (Thermo Scientific, Waltham, MA, USA) for 1 hour at room temperature, and then immediately probed with an HRP-conjugated mouse anti-human CD59 monoclonal antibody (sc-133170) (Santa Cruz Biotechnology, Dallas, TX). Supersignal was used. TM West Atto Ultrasensor Kit (Thermo Fisher Scientific) for Protein Detection and Image Lab Detection via Gel Doc™ XR+ TM Images were captured using software (Bio-Rad, Hercules, CA). Western blot results showed that sCD59 was expressed in cells transfected with vectors GCK, 426, 436, and 437, but not in cells transfected with vector EKQ or untransfected cells. Figure 11 and Figure 12 Furthermore, the sCD59 gene from vectors GCK and GEM showed much higher expression levels than that from vectors GCM and GEP.

[0473] Membrane-bound CD59 (mCD59) was detected in cell lysates using an HRP-conjugated anti-human CD59 antibody, but not in EKQ vector or untransfected cells. The resulting Western blot results are as follows: Figure 10 As shown. Lane contents: 1. Vector GTM; 2. Vector GTP; 3. Vector GTQ; 4. Vector GTR; 5. Vector GAT; 6. Vector EKQ; 7. Untransfected; 8. Purified CD59 protein.

[0474] Soluble CD59 (sCD59) protein was detected in the cell culture supernatant on day 3 using an HRP-conjugated anti-human CD59 antibody. Soluble CD59 (sCD59) was detected in the cell culture supernatant on day 3 using an HRP-conjugated anti-human CD59 antibody, but not in EKQ vector or untransfected cells. The obtained Western blot results are as follows: Figure 11 As shown. Lane contents: 1. Vector GCK; 2. Vector GCM; 3. Vector GEM; 4. Vector GEP; 5. Vector EKQ; 6. Untransfected; 7. Purified CD59 protein.

[0475] Soluble CD59 (sCD59) was detected in the 6-day cell culture supernatant using HRP-conjugated anti-human CD59 antibody, but not in EKQ vector or untransfected cells. The resulting Western blot images are shown below. Figure 12 As shown. Lane contents: 1. Vector GCK; 2. Vector GCM; 3. Vector GEM; 4. Vector GEP; 5. Vector EKQ; 6. Untransfected; 7. Purified CD59 protein.

[0476] sCD59 protein purification Large-scale cell cultures were prepared for vector GEM protein purification. sCD59 protein was purified using an AKTA Explorer 100 in four steps. First, sample clarification was performed by centrifugation followed by 0.2 µm filtration (Nalgene, Rochester, NY) to remove cell debris. The clarified harvest was directly transferred to downstream purification. Second, gel filtration column chromatography using a Sephadex G-25 (Cytiva, Marlboro, MA) was used to remove cell culture medium from the cell culture supernatant containing CD59 protein. After equilibrating the G-25 desalting column with Tris buffer (20 mM Tris pH 7.3, 100 mM NaCl, 0.005% F-68), the cell culture supernatant containing sCD59 protein was loaded. sCD59 protein was collected when UV A280 increased to 5 mAU and collection was stopped when UV A280 decreased to 5 mAU. Third, strong anion exchange column chromatography was performed on a G-25 desalting column using HQ resin (ThermoFisher, Walthan, MA). The G-25 desalting product was diluted with XQ column equilibration buffer (20 mM Tris pH 7.3, 0.005% F-68) to a conductivity < 3 mS / cm and loaded onto an HQ column equilibrated with equilibration buffer (20 mM Tris pH 7.3, 10 mM NaCl, 0.005% F-68). After loading and washing with the equilibration buffer, a 10-500 mM NaCl gradient elution column was used. The elution peak was collected when A280 increased to 5 mAU and collection was stopped when UV A280 decreased to 5 mAU. Fourth, size exclusion column (SEC) chromatography was performed on a Superdex 75 (Cytiva, Marlborugh, MA) to further remove impurities from the HQ purified collect. After equilibrating the SEC column with PBS buffer, the sample was loaded onto an HQ column for chromatography to collect the elution peak. The CD59 protein elution peak was collected when A280 increased to 5 mAU, and collection was stopped when UV A280 decreased to 5 mAU.

[0477] Use 4-12% Bis-Tris gradient gel (Invitrogen) according to the manufacturer's instructions. TM The CD59 sample was isolated and purified by SDS-PAGE using Novex. Protein bands were visualized by Coomassie blue staining. In the gel, the protein size was estimated to be approximately 14 kDa, which is about 5 kDa higher than the theoretical molecular weight. Figure 14 The results showed that sCD59 had high purity. The increased size may be due to protein glycosylation. Lane contents: 1. Carrier GEM.

[0478] sCD59 N-terminal sequencing Purified sCD59 protein was sent for N-terminal sequencing for protein identification. The sCD59 protein was transferred to a PVDF membrane and stained with SimplyBlue SafeStain (Thermo Fisher Scientific, catalog #465034). A single band of approximately 8 µg of protein was excised and stored in a 5 mL Eppendorf tube (data not shown). The band was thoroughly destained sequentially with 50%, 60%, and 70% methanol for 15 minutes each. The blot was then washed with milliQ water for 15 minutes. The blot was air-dried overnight. The destained membrane was transferred to a new tube and stored at 4°C until the sample was transported on ice to Creative Proteomics (Shirley, NY, USA) for identification of the first 5 amino acids at the N-terminus. N-terminal sequencing results showed that the first 4 of the 5 amino acids were identical to the prediction (Table 42). The results indicate that the sCD59 protein was correctly translated and processed for secretion.

[0479] Table 42. N-terminal sequencing results of vector GEM proteins

[0480] sCD59 Functional Measurement To test the biological function of sCD59, a cell lysis inhibition assay was performed using Hep1c1c7 cells. Before performing the cell lysis inhibition assay with purified sCD59 protein, a serum dose that resulted in approximately 70% cell lysis in neonatal normal human serum (NHS) was determined. Three hours before the experiment, 1 × 10⁻⁶ cells were injected with the purified sCD59 protein. 4 Cells / well were seeded at 1000 cells / well in a 96-well plate. Different doses of NHS were mixed with GVB2++ buffer (containing Ca2+). 2+ and Mg 2+(Complement Technology, Catalog B100) Mix. Add 100 µL of the mixture to each well. Measure background lysis using only GVB2++ buffer and maximum lysis by lysing all cells using the maximum lysis buffer (GVB2++ plus 1% SDS). After incubating the cells with the mixture for 1 hour, add 10 µL of WST-8 to each well of the plate. Incubate the cells with WST-8 again for 1–4 hours. Measure the absorbance at 450 nm using a microplate reader. To calculate the percentage of viable cells, subtract the maximum lysis from the absorbance, divide by the difference between background lysis and maximum lysis, and multiply by 100; this equals the percentage of viable cells. The percentage of dead cells is calculated by subtracting the percentage of viable cells from 100. In GraphPad Prism, plot the percentage of cell death against the NHS dose and determine the NHS doses that result in 50% and 70% hemolysis, respectively. Figure 15A ).

[0481] Purified sCD59 protein was used for cell lysis inhibition assays using an NHS dose that resulted in 70% cell lysis. sCD59 protein was serially diluted 2× and mixed with GVB2++ buffer in the presence of NHS, resulting in final concentrations of 30.11, 15.06, 7.53, 3.76, 1.88, and 0.94 µM. CP40 peptide was also used as a baseline in this study. Mixtures of each sample were added to four wells. After subtracting the readings from the maximum lysis four times and averaging, this was divided by 70% cell lysis minus the maximum lysis and multiplied by 100. The percentage of cell lysis inhibition was considered as the percentage of cell lysis inhibition. The percentage of cell lysis inhibition against the molecular concentration of sCD59 protein was plotted using GraphPad Prism software, and the IC50 value was estimated to be approximately 20 µM based on the data plot. Figure 15B The results showed that purified sCD59 could dramatically inhibit cell lysis at increased concentrations, and CP40 also showed slight inhibition, but the BSA control had no inhibitory effect at all.

[0482] Table 8. DNA sequence of the plasmid used in Example 4

[0483] Table 9. Peptide and fusion protein sequences of Example 4

[0484] Example 5. Development of a macular-selective adeno-associated virus (AAV) vector carrying multiple genes of interest targeting dry age-related macular degeneration. Age-related macular degeneration (AMD) is the leading cause of blindness in individuals over 60 years of age, affecting 19.8 million people in the United States and 198 million people globally. Currently, there are no effective treatments to stop geographic atrophy (GA) and improve vision. Adeno-associated virus (AAV) vectors can be a gene delivery method. AAV capsids can be engineered to enhance tissue tropism. In this embodiment, a capsid (AAV2.N54) with improved tropism to the macular retina was identified through multi-species screening in mice, pigs, rabbits, and monkeys. AAV2.N54 showed improved tropism compared to wild-type AAV2 and AAV2.7m8 in all four species, delivering efficiently to the macular retina while detargeting from retinal ganglion cells (RGCs). The AAV2.N54 vector carrying multiple genes of interest (GOIs) encodes: complement 3 repressor protein (C3IP), an engineered C3 / C3b repressor peptide fused to a human IgG Fc fragment (C3ip), a C3IP fused to its terminal C3ip (C3IP-C3ip), a C3IP fused to its C-terminus with a 36-amino acid peptide containing a C-type natriuretic peptide (CNP36) (C3IP-CNP36) or endostatin (C3IP-Endo), and soluble CD59 (sCD59). The resulting AAV vector for systemic analysis is as follows: Figure 13 As shown: AAV2.N54-C3IP-C3ip, sCD59, AAV2.N54-C3IP-CNP36, sCD59, and AAV2.N54-C3IP-endo, sCD59. C3IP showed comparable affinity (KD) for C3b to the positive control peptide CP40, as determined by Biacore and ELISA. In hemolysis assays using human erythrocytes (RBCs), C3IP-C3ip showed an IC50 as low as half that of CP40. 50 Both C3IP-CNP36 and C3IP-Endo exhibited hemolysis inhibition and VEGF-A165-stimulated HUVEC cell proliferation inhibition. C3IP-CNP36 also induced the formation of cyclic guanidine monophosphate (cGMP), indicating its binding to natriuretic peptide receptor B (NPR-B). Following a single dose of AAV intravitreal (IVT) injection, Fc-CNP induced dose-dependent protection of RGCs in a rat model of partial optic nerve transection (pONT) and a mouse model of N-methyl-D-aspartate (NMDA) excitotoxicity. AAV2.N54-C3IP, sCD59, AAV2.N54-C3IP-CNP36, sCD59, and AAV2.N54-C3IP-Endo, sCD59 showed good expression in aRPE-19 and HEK293 cells in vitro and in mice after IVT injection. These AAV vectors were evaluated using a sodium iodate retinal damage model in cynomolgus monkeys.

[0485] Example 6. Optimization of fusion protein expression Five different strategies, as shown in Table 5, were applied to further optimize the C3i-Fc4-CNP36 fusion gene expression in the vector GAM, such as: 1) changing the promoter from CBA to CAG (vector GGQ); 2) replacing the recognition site of the EVQL leader sequence with DK (vector GGE); 3) swapping the C3i position from the C-terminus to the N-terminus (vector GGG); 4) replacing the Fc fragment of IgG4 with the Fc fragment of IgG1 (vector GKK); 5) changing GOI from CNP36 to endostatin at the 3' end (vector GKA). The ORF sequences of GOI and their encoded protein sequences are listed in Tables 19 and 20, respectively. Additionally, Figure 16 The following structures are shown: 1. Vector GGE, 2. Vector GGG, 3. Vector GGQ, 4. Vector GKA and 5. Vector GKK.

[0486] When plasmids were prepared using Xtra Maxi Plus EF (Macherey-Nagel SAS, France), they were confirmed by restriction digestion and partial plasmid sequencing. They were then used to transfect HEK293LTV cells according to the protocol described in Example 3. In this study, the vector EKQ plasmid containing the EGFP gene was used as a transfection control, and the vector CPE plasmid containing the Fc4-CNP36 gene was used as a protein expression control. To assess transfection efficiency, cells transfected with vector EKQ were observed under a fluorescence microscope (data not shown). Cell culture supernatant was collected 5 days post-transfection and analyzed for protein expression. The fusion protein was detected using a mouse anti-human Fc antibody conjugated with HRP (catalog #A01854-200, GenScript, Nanjing, China) as described in Example 3. Lane contents: M. Pre-stained protein markers; 1. Vector GAM; 2. Vector GGE; 3. Vector GGG; 4. Vector GGQ; 5. Vector GKA; 6. Vector GKK; 7. Vector EKQ; 8. Vector CPE; 9. Untransfected cells ( Figure 17 ).

[0487] Western blot results showed that the C3i fusion protein was highly expressed in the GGE and GGG vectors, but weakly expressed in the GGQ, GKA, and GKK vectors. Figure 17The results indicated that changes to the promoter, Fc fragment, or GOI did not contribute to the improvement of C3i fusion protein expression. However, alterations to the Vh leader sequence recognition site (EVQL→DK) or placing Fc4 at the 5' end of the C3i fusion gene dramatically increased gene expression. Notably, the Fc4 fragment in the construct vector GGG uses the same recognition site (DK) as the vector GGE. Therefore, the Vh leader sequence recognition site plays a crucial role in optimizing C3i fusion gene expression in this study.

[0488] (DK) C3i-Fc-endostatin protein expression The C3i-Fc-endostatin fusion gene (vector GKA) exhibits low expression levels. Since altering the recognition site of the Vh leader sequence can enhance the expression of the C3i-Fc4-CNP36 fusion protein, the vector GKA was changed to DK in the new vector GKC plasmid. The C3i-related GOI sequences in the vector GKC plasmid are shown in Table 19, and the encoded protein sequences are shown in Table 20.

[0489] Protein expression in the GKC vector was not tested. Instead, a baculovirus shuttle vector was prepared, packaged with AAV, and used directly for protein expression assays. Figure 18 The results showed that vector GKC AAV exhibited similar C3i fusion protein expression levels to vectors GGE and GGG AAV, suggesting that alteration of the recognition site in the Vh leader sequence also enhanced the expression of the C3i-Fc4-endostatin fusion gene. Lane contents: M. Pre-stained protein markers; 1. Vector GGE (repeat 1); 2. Vector GGE (repeat 2); 3. Vector GGG (repeat 1); 4. Vector GGG (repeat 2); 5. Vector GKA (repeat 1); 6. Vector GKA (repeat 2); PC, positive control, purified vector GGE protein. Furthermore, when using the Vh leader sequence to drive the expression of genes other than C3i-related fusion proteins (data not shown), the recognition site DK also provided significantly higher expression than EVQL. Therefore, in general, optimization of the leader sequence recognition site appears to be an important consideration regarding the improvement of secretory protein gene expression.

[0490] Table 19. DNA sequence of the plasmid used in Example 6

[0491] Table 20. Peptide and fusion protein sequences of Example 6

[0492] Example 7. Purification and N-terminal sequencing of C3i fusion protein In the previous experiment, vectors GGE and GGG showed high expression of the C3i fusion gene. In this experiment, adherent HEK293LTV cells were seeded in 6-well plates, and a large number of plasmids were prepared and transfected into suspension Expi293F cells for large-scale protein purification. Expi293F cells were cultured in BalanCD HEP293 medium (catalog, #91165, Fujifim, Tokyo, Japan) with 25 mM L-glutamine (catalog, #35050-61, ThermoFisher Scientific, Waltham, MA, USA). 1–2 hours before transfection, cells were sputtered at 1 × 10⁻⁶ cells / well. 6 Dilute the cells at a density of 100 cells / ml into transfection culture vessels containing 150 mL of culture medium. In separate sterile tubes, mix 600 μg of plasmid DNA with 15 mL of the dilution and vortex briefly. Add 1200 μL of PEImax to the diluted DNA and vortex the mixture for 5 minutes, then incubate at room temperature for 20 minutes without additional agitation to allow the transfection complex to form. After gently mixing by pipetting up and down, add the entire solution to 150 mL of suspended cell culture. Shake the flasks in an incubator for 2–3 hours, then add 150 mL of fresh culture medium to each flask. Harvest the cells after 5 days of incubation.

[0493] The C3i fusion protein was detected in an SDS-PAGE gel. All cell culture supernatant was collected and purified using Protein A resin (catalog # L00210, GenScript USA, Piscataway, NJ, USA). After complete resuspension of the resin, 2 ml of resin was transferred to a new tube. The bead rotation speed was slowed and the supernatant was discarded. 12 ml of binding / washing buffer (150 mM NaCl, 20 mM Na2HPO4, pH 7.0) was added to the beads, and the mixture was vortexed for 20 seconds. The bead rotation speed was slowed again and the supernatant was discarded. The beads were diluted with a binding / washing buffer (1:1) [300 ml + 300 mL], and the pH was adjusted to 7.0. The washed beads were then added to the diluted sample and incubated overnight at 4°C. The resin / diluted sample mixture was centrifuged and the supernatant was discarded. The resin, along with the remaining supernatant, was loaded onto a column, and the flow-through sample was collected for analysis. Add 90 mL of washing buffer to the resin in the column and collect the washed sample for analysis. Repeat the washing process again. Finally, elute the protein three times with 3 mL of elution buffer (0.1 M glycine, pH 3.0). Neutralize the eluent to pH 7.4 with 300 μL of neutralization buffer (1 M Tris·Cl, pH 8.5). Finally, use 3 kDa-retained Amicon... Ultra-thin centrifugal filter columns (catalog # UFC900308, Millipore-Sigma, Burlington, MA, USA) concentrate samples and perform buffer exchange.

[0494] After collecting flow-through, washing, and elution samples of vectors GGE and GGG, they were analyzed by SDS-PAGE gel electrophoresis. Most Fc-fusion proteins were able to bind to protein A resin and were eluted by a low-pH glycine buffer. Purified vector GGE ( Figure 19 ) and carrier GGG ( Figure 20 Both proteins exhibited high purity, and dimer bands were observed in each. The contents of the two gel lanes are as follows: M. Protein marker; 1. Cell culture medium; 2. Flow-through; 3. Wash 1; 4. Wash 2; 5. Elution sample; 6. Protein in PBS buffer. The results show that both vectors GGE and GGG were highly purified, and some dimers formed even in reducing loading buffer.

[0495] After exchanging the elution buffer with the PBS buffer, the purified protein was quantified using the BCA Protein Assay Kit (catalog # 23225, Thermo Scientific, Waltham, MA, USA) and stored at ≤ -60°C until testing.

[0496] C3i fusion protein N-terminal sequencing To further identify the purified vectors GGE and GGG proteins, the purified proteins were sent for N-terminal sequencing. Proteins were blotted onto PVDF membranes and stained with SimplyBlue SafeStain (catalog # 465034, Thermo Fisher Scientific). Each sample containing approximately 8 µg of protein was individually excised and stored in 5 mL Eppendorf tubes. The bands were thoroughly destained sequentially with 50%, 60%, and 70% methanol for 1 minute each. The blots were then washed with milliQ water for 15 minutes. The blots were air-dried overnight.

[0497] Transfer the decolorized membrane to a new tube and store at 4°C until the sample is transported on an ice pack to Creative Proteomics (Shirley, NY, USA) for identification of the first 5 amino acids at the N-terminus.

[0498] N-terminal sequencing results showed that the first four amino acids of vector GGE (Table 43) and the first five amino acids of vector GGG (Table 44) were identical to the expected sequences. Since the fifth amino acid of vector GGE was cysteine, it could not be identified using conventional methods. These results demonstrate that both vector GGE and vector GGG proteins were correctly translated, cleaved from the signal peptide, and secreted extracellularly.

[0499] Table 43. N-terminal sequencing results of vector GGE proteins

[0500] Table 44. Sequencing results of the N-terminus of the vector GGG protein

[0501] Example 8. Optimization of C3i fusion protein engineering Previous studies have shown that when the compstatin sequence (GI-[CVW--QDWGAHRC]-TN) is mutated using non-natural amino acid mutations, it exhibits a higher binding affinity for C3b than wild-type compstatin. Therefore, four mutations were designed, such as... Figure 21AAs shown, and introduced into the vector GGG. Some designed mutants involved inserting glycine (Y) between the first and second amino acids (+2Y), mutating threonine (T) to alanine (A) at the 14th amino acid (T14A), deleting asparagine (N) at the 15th amino acid (-N15), and mutating asparagine (N) to glutamine (Q) at the 15th amino acid (N15Q). Different combinations of these mutants resulted in 11 mutants, each carrying one, two, or three different mutations.

[0502] The ORF sequences of GOIs and their encoding protein sequences are listed in Tables 26 and 27, respectively. New vectors KTP, KTQ, KTR, KAP, KAA, KAC, KAE, KAG, KAK, KAM, and KAP plasmids were generated using the mutations, cloned, and confirmed by Sanger sequencing. They were transfected into suspension Expi293F cells according to the procedure in Example 7. After all mutant proteins were expressed and detected by SDS-PAGE gel (data not shown), they were purified using protein A resin according to the procedure in Example 7. Figure 21B The results showed that all mutant proteins were successfully purified from the culture medium with high purity. Lane contents: M: protein markers; 1, vector KTP; 2, vector KTQ; 3, vector KTR; 4, vector KAT; 5, vector KAA; 6, vector KAC; 7, vector KAE; 8, vector KAG; 9, vector KAK; 10, vector KAM; 11, vector KAP; 12, vector GGG. Vectors KTP, KTQ, KTR, KAP, KAA, and KAC showed only one band of approximately 29 kDa, while vectors KAE, KAG, and KAK showed two bands, approximately 29 kDa and 58 kDa (suspected dimerization). However, only vector KAM showed a 58 kDa band, suggesting that all proteins dimerized even in the presence of reducing agents. After quantification by BCA, the purified proteins were stored at ≤-60°C for hemolysis inhibition assays.

[0503] In parallel, the serum dose that would induce approximately 70% hemolysis in neonormal human serum (NHS) was determined prior to hemolysis inhibition assays using purified proteins. Different doses of NHS were mixed with rabbit erythrocytes (Catalogue B300, Complement Technology) in the presence of 0.1 M Mg2+ EGTA (Catalogue B106, Complement Technology). Background lysis was measured using GVBE (GVBo + 20 mM EDTA, pH 7.4), and maximal lysis was determined by lysing all cells with a maximal lysis buffer (GVBo + 0.2% NP40). After mixing all samples on ice, they were transferred to a 37 °C water bath. The samples were vortexed every 5 minutes for a total incubation of 30 minutes. 1 mL of cold GVBE was added to each tube to terminate the reaction. The samples were then incubated at approximately 1000 x ... g After centrifugation for 3 minutes to precipitate the cells, the supernatant was transferred to a cuvette and read at 412 nm using a SpectaMax iD3 microplate reader. Each reading was normalized to background, divided by the maximum hemolysis minus the background, and multiplied by 100, which equals the maximum hemolysis %. Using GraphPad Prism software, a graph of maximum hemolysis % versus NHS μL was plotted, and the NHS doses resulting in 50% and 70% hemolysis were determined.

[0504] Using an NHS dose that induces 70% hemolysis, purified mutant proteins were used for hemolysis inhibition assays. All mutant proteins were serially diluted 2× and mixed with rabbit erythrocytes in the presence of NHS, resulting in final concentrations of 32.77, 16.38, 8.9, 4.10, 2.05, and 1.02 µM for each mutant protein. A baseline CP40 peptide was also used in this study. Each reading was subtracted from the background value, divided by 70% hemolysis minus the background value, and multiplied by 100. This value was considered the percentage of hemolysis inhibition. The percentage of hemolysis inhibition against mutant protein molecular concentration was plotted using GraphPad Prism software, and IC50 values ​​were estimated based on the data plots.

[0505] Hemolytic inhibition assays for all mutant proteins (vectors KTP, KTQ, KTR, KAP, KAA, KAC, KAE, KAG, KAK, KAM, and KAP) showed that compared to the parental vector GGG, the IC50 of vectors KAT, KAG, and KAK was decreased; the IC50 of vectors KTP and KAA remained the same; and the IC50 of vectors KAC, KAM, and KAP was significantly increased. Furthermore, vectors KTQ, KTR, and KAE almost completely lost their hemolytic inhibitory activity. BSA was used as a negative control, and CP40 was included as a control. Figure 21C (Table 21). Interestingly, IC50 variatio...

Claims

1. An engineered polynucleotide comprising one or more expression cassettes, said one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor.

2. The engineered polynucleotide according to claim 1, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are covalently linked by a linker.

3. The engineered polynucleotide according to any one of claims 1 or 2, wherein the first angiogenesis inhibitor comprises a complement inhibitor.

4. The engineered polynucleotide of claim 3, wherein the complement inhibitor comprises a complement 3 inhibitor or a C3 degradation fragment.

5. The engineered polynucleotide of claim 4, wherein the complement 3 inhibitor comprises an amino acid sequence having at least 80% identity with any of SEQ ID NO:1-15.

6. The engineered polynucleotide according to any one of claims 1-5, wherein the first angiogenesis inhibitor or the second angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC).

7. The engineered polynucleotide of claim 6, wherein the inhibitor of the MAC comprises CD59.

8. The engineered polynucleotide of claim 7, wherein the CD59 comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 41-45, 312-319 or 325-329.

9. The engineered polynucleotide according to any one of claims 1-8, wherein the second angiogenesis inhibitor comprises a natriuretic peptide.

10. The engineered polynucleotide of claim 9, wherein the natriuretic peptide comprises a C-type natriuretic peptide (CNP).

11. The engineered polynucleotide according to any one of claims 9-10, wherein the natriuretic peptide is covalently linked to an antibody or a fragment thereof.

12. The engineered polynucleotide of claim 11, wherein the antibody or fragment thereof comprises a crystallizable fragment (Fc) region.

13. The engineered polynucleotide according to any one of claims 9-12, wherein the natriuretic peptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 61-72.

14. The engineered polynucleotide according to any one of claims 1-8, wherein the second angiogenesis inhibitor comprises endostatin or a fragment thereof.

15. The engineered polynucleotide according to any one of the preceding claims, further encoding a third angiogenesis inhibitor.

16. The engineered polynucleotide according to any one of the preceding claims, wherein the engineered polynucleotide comprises a viral vector.

17. The engineered polynucleotide of claim 16, wherein the viral vector comprises an AAV vector.

18. The engineered polynucleotide of claim 17, wherein the AAV vector is an AAV2 vector.

19. The engineered polynucleotide of claim 17, wherein the AAV vector encodes an engineered AAV capsid.

20. The engineered polynucleotide of claim 19, wherein the engineered AAV capsid comprises the amino acid sequence of any one of SEQ ID NO: 161-182 and SEQ ID NO: 191-210.

21. The engineered polynucleotide according to any one of the preceding claims, wherein the first angiogenesis inhibitor comprises the complement 3 inhibitor, and the second angiogenesis inhibitor comprises CNP36.

22. The engineered polynucleotide of claim 21, further encoding a third angiogenesis inhibitor.

23. The engineered polynucleotide of claim 22, wherein the third angiogenesis inhibitor comprises an inhibitor of the membrane attack complex (MAC), and wherein the inhibitor of the MAC comprises CD59.

24. The engineered polynucleotide of claim 1, wherein the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises a complement 3 inhibitor fused to Fc-CNP36.

25. The engineered polynucleotide according to any one of the preceding claims, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises endostatin.

26. The engineered polynucleotide according to any one of the preceding claims, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises Fc-CNP36, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.

27. The engineered polynucleotide according to any one of the preceding claims, wherein the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises a complement 3 inhibitor, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising Fc-CNP36.

28. The engineered polynucleotide according to any one of the preceding claims, wherein the first angiogenesis inhibitor comprises CD59 and the second angiogenesis inhibitor comprises endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor.

29. The engineered polynucleotide according to any one of the preceding claims, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.

30. An engineered polypeptide comprising a first angiogenesis inhibitor and a second angiogenesis inhibitor.

31. The engineered polypeptide of claim 30, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are covalently linked by a connector.

32. The engineered polypeptide according to any one of claims 30-31, wherein the first angiogenesis inhibitor comprises a complement inhibitor.

33. The engineered polypeptide according to any one of claims 30-32, wherein the first angiogenesis inhibitor or the second angiogenesis inhibitor comprises an inhibitor of a membrane attack complex (MAC), and wherein the inhibitor of MAC comprises CD59.

34. The engineered polypeptide according to any one of claims 30-33, wherein the second angiogenesis inhibitor comprises a natriuretic peptide.

35. The engineered polypeptide according to any one of claims 30-33, wherein the second angiogenesis inhibitor comprises endostatin or a fragment thereof.

36. The engineered polypeptide according to any one of the preceding claims, further encoding a third angiogenesis inhibitor.

37. A vector comprising an engineered polynucleotide according to any one of claims 1-29 or an engineered polypeptide according to any one of claims 30-36.

38. The carrier of claim 37, wherein the carrier encodes an AAV shell, and wherein the AAV shell comprises an engineered AAV shell.

39. A viral particle comprising an engineered polynucleotide according to any one of claims 1-29, an engineered polypeptide according to any one of claims 30-36, or a vector according to any one of claims 37-38.

40. The viral particle of claim 39, wherein the viral particle comprises an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid.

41. A cell comprising an engineered polynucleotide according to any one of claims 1-29, an engineered polypeptide according to any one of claims 30-36, a vector according to any one of claims 37-38, or a viral particle according to any one of claims 39-40.

42. A composition comprising: a complement 3 inhibitor or a C3 degradation fragment comprising C3a, C3b, iC3b, C3f, C3c, C3d, C3g or a combination thereof; and a natriuretic peptide.

43. The composition of claim 42, wherein the natriuretic peptide comprises C-type natriuretic peptide (CNP).

44. A composition comprising: a complement 3 inhibitor or a C3 degradation fragment comprising C3a, C3b, iC3b, C3f, C3c, C3d, C3g or a combination thereof; and an inhibitor of the membrane attack complex (MAC).

45. The composition of claim 44, wherein the inhibitor of the MAC comprises CD59.

46. ​​A composition comprising CD59 and natriuretic peptide.

47. The composition of claim 46, wherein the natriuretic peptide comprises C-type natriuretic peptide (CNP).

48. A pharmaceutical composition comprising an engineered polynucleotide according to any one of claims 1-29, an engineered polypeptide according to any one of claims 30-36, a carrier according to any one of claims 37-38, a viral particle according to any one of claims 39-40, a cell according to claim 41, or a composition according to any one of claims 42-47.

49. A method comprising contacting cells obtained from an object with an engineered polynucleotide according to any one of claims 1-29, an engineered polypeptide according to any one of claims 30-36, a carrier according to any one of claims 37-38, a viral particle according to any one of claims 39-40, a cell according to claim 41, a composition according to any one of claims 42-47, or a pharmaceutical composition according to claim 48.

50. A method for treating a disease or condition, comprising: The engineered polynucleotide according to any one of claims 1-29, the engineered polypeptide according to any one of claims 30-36, the carrier according to any one of claims 37-38, the viral particle according to any one of claims 39-40, the cell according to claim 41, or the composition according to any one of claims 42-47, or the pharmaceutical composition according to claim 48, are administered to the object.

51. A method of treating a disease or condition in a subject, the method comprising administering an engineered polynucleotide to the subject, the engineered polynucleotide comprising one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor.

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