Compositions and methods for generating therapeutic agents

Abstract

This specification describes compositions for modulating the transgene expression of one or more therapeutic agents. In some embodiments, engineered polynucleotides comprising one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor are described. Similarly, this specification describes methods of using the compositions described herein for modulating transgene expression and for treating diseases or conditions, particularly for treating eye diseases or inhibiting angiogenesis.

Description

Technical Field

[0001] Cross-reference

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

Background Art

[0002]

[0002] Angiogenesis, including vasculogenesis, angiogenesis, and arteriogenesis, is regulated by a wide variety of cell signaling pathways. One of the signaling pathways is regulated by vascular endothelial growth factor (VEGF). VEGF is a potent mitogen for endothelial cells and induces proliferation, migration, vascular tube formation, and permeability. Therefore, an increase in the VEGF signaling pathway increases angiogenic signals, while a decrease or inhibition of the VEGF signaling pathway decreases angiogenic signals. VEGF inhibition is one of the most common treatment options for diseases or conditions associated with angiogenesis. For example, the treatment of eye diseases often involves the use of angiogenesis inhibitors such as VEGF inhibitors.

Summary of the Invention

Means for Solving the Problems

[0003]

[0003] Current treatments using VEGF inhibitors can be cumbersome because, due to the short half-life of VEGF inhibitors, repeated monthly injections are required to achieve and maintain suppression of angiogenesis. Therefore, therapeutic agents for treating eye diseases are still needed. Similarly, therapeutic agents for inhibiting angiogenesis are still needed by targeting signaling pathways other than the VEGF signaling pathway, or in combination with the VEGF signaling pathway. Accordingly, in some embodiments herein, engineered polynucleotides comprising one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor are described. In some embodiments, the first angiogenesis inhibitor and the second angiogenesis inhibitor are covalently linked by a linker. In some embodiments, the first angiogenesis inhibitor comprises a complement inhibitor. In some embodiments, the complement inhibitor comprises a complement 3 inhibitor or a C3 degradation fragment. In some embodiments, the complement 3 inhibitor comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1 to 15. In some embodiments, the first or second angiogenesis inhibitor comprises an inhibitor of the membrane invasion complex (MAC). In some embodiments, the MAC inhibitor comprises CD59. In some embodiments, CD59 comprises an amino acid sequence that is at least 80% identical to one of SEQ ID NOs. 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 fragment crystallizable (Fc) region. In some embodiments, the natriuretic peptide comprises an amino acid sequence that is at least 80% identical to one of SEQ ID NOs. 61-72. In some embodiments, the second angiogenesis inhibitor comprises an 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 includes 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 includes one amino acid sequence from SEQ ID NOs: 161-182 and SEQ ID NOs: 191-210. In some embodiments, the first angiogenesis inhibitor includes a complement 3 inhibitor, and the second angiogenesis inhibitor includes CNP36. In some embodiments, the engineered polynucleotide further encodes a third angiogenesis inhibitor. In some embodiments, the third angiogenesis inhibitor includes a membrane invasion complex (MAC) inhibitor, and the MAC inhibitor includes CD59. In some embodiments, the first angiogenesis inhibitor includes CD59, and the second angiogenesis inhibitor includes a complement 3 inhibitor fused to Fc-CNP36. In some embodiments, the first angiogenesis inhibitor includes a complement 3 inhibitor, and the second angiogenesis inhibitor includes endostatin. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises Fc-CNP36, and the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.

[0004]

[0004] In this specification, in some embodiments, an engineered polypeptide comprising a first angiogenic inhibitor and a second angiogenic inhibitor is described. In some embodiments, the first angiogenic inhibitor and the second angiogenic inhibitor are covalently linked by a linker. In some embodiments, the first angiogenic inhibitor comprises a complement inhibitor. In some embodiments, the first or second angiogenic inhibitor comprises an inhibitor of a membrane invasion complex (MAC), and the MAC inhibitor comprises CD59. In some embodiments, the second angiogenic inhibitor comprises a natriuretic peptide. In some embodiments, the second angiogenic inhibitor comprises an endostatin or a fragment thereof. In some embodiments, the engineered polypeptide further encodes a third angiogenic inhibitor.

[0005]

[0005] In some embodiments of this specification, vectors comprising engineered polynucleotides or engineered polypeptides as described herein are described. In some embodiments, the vector encodes an AAV capsid, and the AAV capsid comprises an engineered AAV capsid.

[0006]

[0006] In some embodiments, viral particles comprising an engineered polynucleotide, an engineered polypeptide, or a vector disclosed herein are described. In some embodiments, the viral particle comprises an AAV capsid, and the AAV capsid comprises an engineered AAV capsid.

[0007]

[0007] In some aspects of this specification, cells containing an engineered polynucleotide, an engineered polypeptide, a vector, or a viral particle as disclosed herein are described.

[0008]

[0008] In some embodiments of this specification, compositions 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]

[0009] In this specification, in some embodiments, compositions are described that include 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 invasion complex (MAC). In some embodiments, the MAC inhibitor comprises CD59.

[0010]

[0010] In this specification, in some embodiments, compositions comprising CD59 and a natriuretic peptide are described. In some embodiments, the natriuretic peptide comprises C-type natriuretic peptide (CNP).

[0011]

[0011] In some embodiments of this specification, pharmaceutical compositions comprising an engineered polynucleotide, an engineered polypeptide, a vector, a viral particle, a cell, or a composition disclosed herein are described.

[0012]

[0012] In some embodiments of this specification, a method is described that includes the step of contacting cells obtained from a subject with an engineered polynucleotide, an engineered polypeptide, a vector, a viral particle, a cell, a composition, or a pharmaceutical composition as disclosed herein.

[0013]

[0013] In some embodiments of this specification, a method for treating a disease or condition in a subject is described, comprising the step of administering to the subject an engineered polynucleotide, an engineered polypeptide, a vector, a viral particle, a cell, a composition, or a pharmaceutical composition as disclosed herein.

[0014]

[0014] In some embodiments of this specification, a method for treating a disease or condition in a subject is described, comprising the step of administering to the subject an engineered polynucleotide comprising one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor. Inclusion by reference

[0015] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. If any publication or patent or patent application incorporated by reference conflicts with any disclosure contained herein, this specification is intended to supersede and / or take precedence over any such conflicting material.

[0015]

[0016] Novel features of this disclosure are described in detail in the attached claims. A better understanding of the features and advantages of this disclosure can be obtained by referring to the following detailed description, which includes exemplary embodiments utilizing the principles of this disclosure, and to the following attached drawings (hereinafter referred to as "Figure" and "FIG."). [Brief explanation of the drawing]

[0016] [Figure 1]

[0017] Figure 1 shows a vector construct for expressing a complement C3 inhibitor (C3i). [Figure 2A]

[0018] Figures 2A and 2B show vector constructs for expressing C3i operably linked to a natriuretic polypeptide. [Figure 2B]

[0018] Figures 2A and 2B show vector constructs for expressing C3i operably linked to a natriuretic polypeptide. [Figure 3]

[0019] Figure 3 shows vector constructs for expressing C3i, CD59, and natriuretic polypeptide. [Figure 4]

[0020] Figure 4 shows an exemplary vector for expressing the angiogenesis inhibitors described herein. [Figure 5A]

[0021] Figures 5A to 5C show the AAV structures fabricated and tested in Example 3. [Figure 5B] Figures 5A to 5C show the AAV structures fabricated and tested in Example 3. [Figure 5C] Figures 5A to 5C show the AAV structures fabricated and tested in Example 3. [Figure 6]

[0022] Figure 6 shows that fusion proteins expressed by cells in a 6-well plate were detected by SDS-PAGE and Western blotting using HRP-conjugated goat anti-human IgG1 Fc antibody. Lane contents: 1. Vector GAM on day 3; 2. Vector EKQ on day 3; 3. Vector CME on day 3; 4. Untransfected on day 3; 5. Vector GAM on day 6; 6. Vector EKQ on day 6; 7. Vector CME on day 6; 8. Untransfected on day 6. [Figure 7]

[0023] Figure 7 shows that the fusion protein expressed by cells in a T125 flask was detected by SDS-PAGE and Western blotting using HRP-conjugated goat anti-human IgG1 Fc antibody. Lane contents: 1. Vector GAM on day 3; 2. Vector EKQ on day 3; 3. Vector CME on day 3; 4. Untransfected on day 3; 5. Vector GAM on day 6; 6. Vector EKQ on day 6; 7. Vector CME on day 6; 8. Untransfected on day 6. [Figure 8]

[0024] Figure 8 shows that fusion proteins expressed by cells in a 6-well plate were detected by SDS-PAGE and Western blotting using rat anti-human CNP antibody. Lane contents: 1. Vector GAM on day 3; 2. Vector EKQ on day 3; 3. Vector CME on day 3; 4. Untransfected on day 3; 5. Vector GAM on day 6; 6. Vector EKQ on day 6; 7. Vector CME on day 6; 8. Untransfected on day 6. [Figure 9]

[0025] Figure 9 shows that the fusion protein expressed by cells in a T125 flask was detected by Western blotting using rat anti-human CNP antibody based on SDS-PAGE and Western blotting analysis. Lane contents: 1. Vector GAM on day 3; 2. Vector EKQ on day 3; 3. Vector CME on day 3; 4. Untransfected on day 3; 5. Vector GAM on day 6; 6. Vector EKQ on day 6; 7. Vector CME on day 6; 8. Untransfected on day 6. [Figure 10]

[0026] Figure 10 shows that membrane-bound CD59 was expressed 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. [Figure 11]

[0027] Figure 11 shows that soluble CD59 (sCD59) protein was detected in the cell culture supernatant on day 3 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. [Figure 12]

[0028] Figure 12 shows that soluble CD59 (sCD59) protein was detected in the cell culture supernatant on day 6 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. [Figure 13]

[0029] Figure 13 shows a schematic diagram of the AAV vector design for Example 5. [Figure 14]

[0030] Figure 14 shows that the large-scale production of soluble CD59 (sCD59) protein in the cell culture supernatant on day 6 was confirmed to be pure using SDS-PAGE gel analysis. [Figure 15A]

[0031] Figures 15A and 15B show graphs illustrating the results of the cell lysis inhibition assay. Figure 15A shows the dose-response curve of % cytotoxicity with increasing concentrations of normal human serum (NHS). [Figure 15B] Figure 15B shows the dose-response curves of normalized % maximum cytotoxicity compared to BSA control, compared to the cytoprotective effects of vectors GEM and CP40. [Figure 16]

[0032] Figure 16 shows a schematic diagram of the AAV vector design for Example 6. [Figure 17]

[0033] Figure 17 shows fusion proteins expressed by cells and detected by Western blotting using SDS-PAGE and HRP-conjugated mouse anti-human Fc antibody. Lane contents: M. Pre-staining protein marker; 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 18]

[0034] Figure 18 shows the fusion protein expressed by cells and detected by SDS-PAGE and Western blotting using biotin-labeled goat anti-human IgG antibody and HRP-streptavidin conjugate. Lane contents: M. Pre-staining protein marker; 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. [Figure 19]

[0035] Figure 19 shows that the vector GGG protein expressed by cells HEK293LTV in a 6-well plate was shown to be pure using SDS-PAGE gel analysis. Lane contents: M. Protein marker; 1. Cell culture medium; 2. Flow-through; 3. Wash 1; 4. Wash 2; 5. Eluted sample; 6. Protein in PBS buffer. [Figure 20]

[0036] Figure 20 shows that the vector GGE protein expressed by cells HEK293LTV in a 6-well plate was shown to be pure using SDS-PAGE gel analysis. Lane contents: M. Protein marker; 1. Cell culture medium; 2. Flow-through; 3. Wash 1; 4. Wash 2; 5. Eluted sample; 6. Protein in PBS buffer. [Figure 21A]

[0037] Figures 21A–21C show mutant fusion proteins expressed by cells and detected by SDS-PAGE and hemolysis inhibition assays. Figure 21A shows the design of the mutant protein derived from the vector GGG. [Figure 21B] Figure 21B shows proteins expressed by Expi293F cells and shown to be of high purity using SDS-PAGE gel analysis. Lane contents: M: Protein marker; 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] Figure 21C shows a graph illustrating the results of hemolysis inhibition assays for all mutant proteins. [Figure 22A]

[0038] Figures 22A and 22B show the fusion proteins expressed by cells and detected by SDS-PAGE, as well as the results from a hemolysis inhibition assay. Figure 22A shows the expressed proteins, which were shown to be pure using SDS-PAGE gel analysis. Lane contents: M. protein marker; 1, vector GGE; 2, vector GGG; 3, vector KAG; 4, vector KKT; 5, vector KKA; 6, vector KPT; 7, vector KPC. [Figure 22B] Figure 22B shows a graph illustrating the results of hemolysis inhibition assays for all mutant proteins. [Figure 23A]

[0039] Figures 23A–23C show graphs illustrating the binding affinity assay results of fusion proteins to C3b, C3c, and C3. Figure 23A shows the estimated IC50 and KD values ​​of vectors KPT, CP40, KKT, KPC, KKA, GGE, KAG, and GGG compared to C3b. [Figure 23B] Figure 23B shows the estimated IC50 and KD values ​​compared to the C3c of vectors KPT, CP40, KKT, KPC, KKA, GGE, KAG, and GGG. [Figure 23C]Figure 23C shows the estimated IC50 and KD values ​​compared to C3 for vectors KPT, CP40, KKT, KPC, KKA, GGE, KAG, and GGG. [Figure 24]

[0040] Figure 24 shows schematic diagrams of exemplary AAV vector designs for vector KMR, vector KKT, and vector KPP. [Figure 25A]

[0041] Figures 25A–25D show protein expression in ARPE-19 cells secreted into the cell culture medium or remaining within the cells. Figure 25A shows the expression of CD59 from vectors KMR, KKT, and KPP. [Figure 25B] Figure 25B shows the expression of C3i from vectors KMR, KKT, and KPP. [Figure 25C] Figure 25C shows the expression of Fc-CNP from the vector KKT. [Figure 25D] Figure 25D shows endostatin expression from the KPP vector. [Figure 26]

[0042] Figure 26 shows a schematic diagram of the AAV vector design for vectors KGQ, KGR, KKT, and KKA. [Figure 27A]

[0043] Figures 27A–27G show the in vivo expression and statistical analysis results of AAV in mouse eyes. Figure 27A shows graphs of CD59 expression results from vectors KGQ, KGR, KKT, and KKA. [Figure 27B] Figure 27B shows a graph illustrating the expression results of Fc-CNP from vector KKT. Figure 27C shows graphs illustrating the expression results of hFc from vectors KGQ, KGR, KKT, and KKA. [Figure 27C] Figure 27C shows graphs illustrating the results of hFc expression from vectors KGQ, KGR, KKT, and KKA. [Figure 27D]Figure 27D shows a graph illustrating the results of endostatin expression from the KKA vector. [Figure 27E] Figure 27E shows graphs illustrating the expression results of CD59 and C3i from vectors KGQ and KGR. [Figure 27F] Figure 27F shows graphs illustrating the expression results of CD59, C3i, and CNP from the vector KKT. [Figure 27G] Figure 27G shows a graph illustrating the expression results of CD59, C3i, and endostatin from the KKA vector. [Figure 28A]

[0044] Figures 28A–28C show the results of ligand binding assays for the C3i fusion protein. Figure 28A shows the results of ELISA assays for the binding of C3b, as well as vectors KMR, KKT, and KPP, and the estimated Kd values. [Figure 28B] Figure 28B shows the results of ELISA assays for C3c and the binding of vectors KMR, KKT, and KPP proteins, as well as the estimated Kd values. [Figure 28C] Figure 28C shows the results of ELISA assays for the binding of C3, as well as vector KMR, vector KKT, and vector KPP proteins, and the estimated Kd values. [Figure 29]

[0045] Figure 29 shows graphs illustrating the results of ligand binding assays for NPR-b, as well as the vector KKT proteins, Fc1-CNP36, and Fc4-CNP36. [Figure 30]

[0046] Figure 30 shows a graph illustrating the results of measuring cGMP production in NIG-ETE cells induced by vector CME protein (Fc4-CNP36), vector KKT protein (C3i-Fc4-CNP36), CNP-22, or Cp40. [Figure 31]

[0047] Figure 31 shows a graph illustrating the results of an endostatin ligand receptor assay for the vector KPP protein and the estimated Kd value. [Figure 32]

[0048] Figure 32 shows a schematic diagram of the AAV vector design for Example 12. [Figure 33]

[0049] Figure 33 shows the results of ligand binding assays for wild-type and variant sCD59 proteins. [Modes for carrying out the invention]

[0017]

[0050] Novel features of this disclosure are described in particular 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 which describes exemplary embodiments. overview

[0051] In this specification, in some embodiments, engineered polynucleotides comprising one or more expression cassettes encoding one or more angiogenesis inhibitors are described. In some embodiments, one or more angiogenesis inhibitors are operably linked (e.g., linked by covalent bonds). In some embodiments, one or more angiogenesis inhibitors include complement inhibitors, natriuretic peptides, membrane invasion complex (MAC) inhibitors, VEGF inhibitors, or a combination thereof. In some embodiments, the complement inhibitors include complement 3 inhibitors (C3i) or complement 3 (C3) degradation fragments. In some embodiments, the C3 degradation fragments include C3a, C3b, iC3b, C3f, C3c, C3d, C3g, or a combination thereof. In some embodiments, the engineered polynucleotides include vectors such as AAV vectors. In some embodiments, the engineered polynucleotides encode a first angiogenesis inhibitor and a second angiogenesis inhibitor. In some embodiments, the engineered polynucleotides encode an angiogenesis inhibitor comprising a complement 3 inhibitor (C3i). For example, Figure 1 shows vector constructs encoding complement 3 inhibitors (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, an engineered polynucleotide encodes a complement 3 inhibitor and a natriuretic peptide (e.g., type C natriuretic peptide or CNP), e.g., vector GGE. In some embodiments, the complement 3 inhibitor and the natriuretic peptide are linked by a covalent bond. For example, Figures 2A and 2B (vector GAM and vector GGE) and Figure 3 (upper panel: vector GKR sCD59-EVQL C3i-Fc4-CNP36; and lower panel: vector KAR sCD59-DK C3i-Fc4-CNP36) show vector constructs encoding complement 3 inhibitors and CNP. In some embodiments, the complement 3 inhibitor includes at least one modification compared to an equivalent wild-type complement 3 inhibitor.In some embodiments, the complement 3 inhibitor comprises an amino acid sequence that is at least 80% identical to one of sequence numbers 1 to 15.

[0018]

[0052] In some embodiments, the engineered polynucleotide encodes one or more angiogenesis inhibitors, one of which includes a CNP. In some embodiments, the CNP is covalently linked to an antibody or a fragment thereof (e.g., a fragment crystallizable region). In some embodiments, the natriuretic peptide includes an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 61-72. In some embodiments, the engineered polynucleotide encodes one or more angiogenesis inhibitors, one of which includes an inhibitor of a membrane invasion complex (MAC). In some embodiments, the MAC inhibitor includes CD59. For example, Figure 3 shows a vector construct encoding CD59. In some embodiments, CD59 includes an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 41-45. In some embodiments, the engineered polynucleotide encodes one or more angiogenesis inhibitors, one of which includes collagen or a fragment thereof (e.g., endostatin or a fragment thereof). In some embodiments, endostatin includes an amino acid sequence that is at least 80% identical to SEQ ID NOs. 51. In some embodiments, the engineered polynucleotide encodes one or more angiogenesis inhibitors, one of which is a VEGF inhibitor. In some embodiments, the VEGF inhibitor comprises an amino acid sequence that is at least 80% identical to any one of sequence numbers 81-92.

[0019]

[0053] In some embodiments, the engineered polynucleotide includes 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., those shown in Tables 13 and 14). In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered AAV capsid includes one amino acid sequence from SEQ ID NOs. 161-182 and SEQ ID NOs. 191-210. In some embodiments, the engineered AAV capsid includes the amino acid sequence from SEQ ID NOs. 169. In some embodiments, the engineered AAV capsid includes one amino acid sequence from SEQ ID NOs. 277-303 and SEQ ID NOs. 312-319 and 325-329.

[0020]

[0054] In some embodiments, the engineered polynucleotide encodes a first angiogenesis inhibitor comprising a complement 3 inhibitor and a second angiogenesis inhibitor comprising 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 Fc-CNP36. In some embodiments, the engineered polynucleotide encodes a third angiogenesis inhibitor. In some embodiments, the third angiogenesis inhibitor comprises an inhibitor of membrane invasion complex (MAC).

[0021]

[0055] In some embodiments, the engineered polynucleotide encodes a first angiogenesis inhibitor comprising a CD59 inhibitor and a second angiogenesis inhibitor comprising 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.

[0022]

[0056] In some embodiments, the engineered polynucleotide encodes a first angiogenesis inhibitor comprising a complement 3 inhibitor, and a second angiogenesis inhibitor comprising an endostatin. In some embodiments, the engineered polynucleotide encodes an Fc region adjacent to 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, and 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, and 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, and 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, and 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, and 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, and 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, engineered polynucleotides, when administered to a subject, inhibit angiogenesis in that subject.In some embodiments, when administered to a subject, the first or second angiogenesis inhibitor exhibits a reduction in the inhibition of angiogenesis in the subject compared to the inhibition of angiogenesis caused by the VEGF inhibitor.

[0023]

[0057] In this specification, in some embodiments, engineered polypeptides comprising a first angiogenesis inhibitor and a second angiogenesis inhibitor are described. In some embodiments, the first angiogenesis inhibitor and the second angiogenesis inhibitor are covalently linked by 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 that is at least 80% identical to any one of SEQ ID NOs: 1 to 15. In some embodiments, the first angiogenesis inhibitor comprises a membrane invasion complex (MAC) inhibitor. In some embodiments, the MAC inhibitor comprises CD59 comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 41 to 45. In some embodiments, the MAC inhibitor comprises CD59 comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 312 to 319. In some embodiments, the MAC inhibitor comprises CD59 comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 325 to 329. In some embodiments, the second angiogenesis inhibitor comprises a natriuretic peptide. In some embodiments, the natriuretic peptide is covalently linked to the antibody or a fragment thereof. In some embodiments, the antibody or a fragment thereof includes a fragment crystallizable (Fc) region. In some embodiments, the natriuretic peptide includes an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 61-72. In some embodiments, the second angiogenesis inhibitor includes collagen or a fragment thereof (e.g., endostatin or a fragment thereof). In some embodiments, the second angiogenesis inhibitor includes an amino acid sequence that is at least 80% identical to SEQ ID NOs. 51. In some embodiments, the second angiogenesis inhibitor includes a VEGF inhibitor. In some embodiments, the VEGF inhibitor includes an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 81-92.

[0024]

[0058] In this specification, in some embodiments, a method is described for treating a disease or condition in a subject by administering an engineered polynucleotide or engineered polypeptide to the subject. In some embodiments, the method comprises a single administration step to cure the disease or condition. In some embodiments, the method does not involve daily administration. In some embodiments, the disease or condition includes eye diseases. In some embodiments, eye diseases include ischemic syndrome of the eye, proliferative retinopathy, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, color blindness, age-related macular degeneration (nAMD), geographic atrophy (GA), atrophic age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), Valdevie-Wiedl syndrome, Best's disease, choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-vertebral dystrophy, vertebral-rod dystrophy, Oguchi disease, autosomal dominant radial drusen (Malattia). This includes leventinese (familial autosomal dominant drusen), blue vertebral monochromatic color blindness, or a combination thereof. Engineered polynucleotides

[0059] In this specification, in some embodiments, an engineered polynucleotide comprising one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor is described. In some embodiments, the engineered polynucleotide comprises one or more expression cassettes for expressing one or more angiogenesis inhibitors. In some embodiments, one or more expression cassettes encode a serial polypeptide. In some embodiments, the serial 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 include serine endoprotease, aspartate endoprotease, cysteine ​​thiol endoprotease, metalloendoprotease, or glutamate and threonine endoproteases. In some embodiments, the protease peptide sequence is cleavable by serine endoprotease. In some embodiments, the protease peptide sequence is cleavable by furin. In some embodiments, the serial polypeptide comprises a protease-cleavable sequence. In some embodiments, the protease-cleavable sequence may be cleaved by any one of the proteases described herein. In some embodiments, the protease-cleavable sequence may be cleavable by furin. In some embodiments, the continuous polypeptide includes a self-cleaving polypeptide sequence. In some embodiments, the self-cleaving polypeptide sequence includes a 2A self-cleaving peptide sequence. Non-limiting examples of 2A self-cleaving peptide sequences include T2A, P2A, E2A, F2A, or combinations thereof. In some embodiments, the self-cleaving polypeptide sequence includes an F2A peptide sequence. In some embodiments, the continuous polypeptide includes a protease-cleavable sequence and a self-cleaving polypeptide sequence. For example, the continuous polypeptide described herein may include a furin-F2A fusion polypeptide sequence. In some embodiments, the engineered polynucleotide includes a viral vector such as an AAV vector.

[0025]

[0060] In some embodiments, the engineered polynucleotide comprises one or more promoters or intra-sequence ribosome entry sites (IRESs). In some embodiments, the expression cassette comprises one or more promoters or IRESs. In some embodiments, the expression cassette is under the expression control of a promoter. In some embodiments, the expression cassette is under the expression control of a promoter. In some embodiments, the expression cassette can exert further expression control via at least one IRES.

[0026]

[0061] 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 are each encoded from one of the one or more expression cassettes.

[0027]

[0062] 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 that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to any one of SEQ ID NOs: 1-15 (Table 10). In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 1. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 2. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 2. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 3. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 3. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 4.In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 5. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 5. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 6. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 6. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 7. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 7. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 8. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 8. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 9. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 9. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 10. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 10.In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 11. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 11. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 12. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 12. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 13. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 13. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 14. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 14. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 15. In some embodiments, the complement 3 inhibitor (C3i) includes the amino acid sequence of SEQ ID NO: 15.

[0028]

[0063] In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of eight consecutive amino acids, at least ten consecutive amino acids, or at least twelve consecutive amino acids from any one of sequence numbers 1 to 15. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of eight consecutive amino acids, at least ten consecutive amino acids, or at least twelve consecutive amino acids from sequence number 1. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of eight consecutive amino acids, at least ten consecutive amino acids, or at least twelve consecutive amino acids from sequence number 2. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of eight consecutive amino acids, at least ten consecutive amino acids, or at least twelve consecutive amino acids from sequence number 3. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of eight consecutive amino acids, at least ten consecutive amino acids, or at least twelve consecutive amino acids from sequence number 4. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of eight consecutive amino acids, at least ten consecutive amino acids, or at least twelve consecutive amino acids from sequence number 5. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as of SEQ ID NO: 6. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as of SEQ ID NO: 7. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as of SEQ ID NO: 8. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as of SEQ ID NO: 9. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as of SEQ ID NO: 10. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as of SEQ ID NO: 11.In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as of SEQ ID NO: 12. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as of SEQ ID NO: 13. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as of SEQ ID NO: 14. In some embodiments, the complement 3 inhibitor (C3i) includes an amino acid sequence of 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids as of SEQ ID NO: 15.

[0029] [Table 1]

[0030]

[0064] In some embodiments, the engineered polynucleotide encoding the complement 3 inhibitor comprises a nucleic acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to any one of SEQ ID NOs. 20–33 (Table 11). In some embodiments, the complement 3 inhibitor (C3i) is encoded by a nucleic acid sequence that is at least 50 consecutive nucleotide bases, at least 60 consecutive nucleotide bases, at least 70 consecutive nucleotide bases, or at least 50 consecutive nucleotide bases of any one of SEQ ID NOs. 20–33.

[0031] [Table 2]

[0032]

[0065] In some embodiments, the engineered polynucleotide encodes a natriuretic peptide or 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 by a linker. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to any one of SEQ ID NOs. 61-72 (Table 12). In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NOs. 61. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 62. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 63. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 64.In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 65. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 66. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 67. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 68. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 69. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 70. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 71.In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 72. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence that is any one of SEQ ID NOs. 61 to 72.

[0033] [Table 3-1]

[0034] [Table 3-2]

[0035]

[0066] In some embodiments, the engineered polynucleotide encodes an inhibitor of the membrane invasion complex (MAC). In some embodiments, the MAC inhibitor includes CD59. In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to any one of SEQ ID NOs. 41-45 (Table 13). In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NOs. 42 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NOs. In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 43. In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 44. In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 45. In some embodiments, CD59 includes an amino acid sequence that is any one of SEQ ID NOs: 41 to 45.

[0036] [Table 4]

[0037]

[0067] In some embodiments, the engineered polynucleotide encodes an inhibitor of the membrane invasion complex (MAC). In some embodiments, the MAC inhibitor includes CD59. In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to any one of SEQ ID NOs. 312-319 (Table 40). In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NOs. 313 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NOs. In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 314. In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 315. In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 316. In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 317. In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 318.In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 319. In some embodiments, CD59 includes an amino acid sequence that is any one of SEQ ID NOs: 312 to 319.

[0038]

[0068] In some embodiments, the engineered polynucleotide encodes an inhibitor of the membrane invasion complex (MAC). In some embodiments, the MAC inhibitor includes CD59. In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to any one of SEQ ID NOs. 325-329 (Table 50). In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NOs. 326 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NOs. In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 327. In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 328. In some embodiments, CD59 includes an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 329. In some embodiments, CD59 includes an amino acid sequence that is any one of SEQ ID NOs. 325 to 329.

[0039] [Table 5-1]

[0040] [Table 5-2]

[0041]

[0069] In some embodiments, the engineered polynucleotide encodes collagen or a fragment thereof. In some embodiments, the collagen or a fragment thereof includes endostatin or a fragment thereof. In some embodiments, the endostatin or a fragment thereof is SEQ ID NO: 51: The amino acid sequence comprises MHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASK which is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical. In some embodiments, endostatin or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 51.

[0042]

[0070] In some embodiments, an engineered polynucleotide encodes a VEGF inhibitor. In some embodiments, the VEGF inhibitor comprises inhibitory RNA for targeting and degrading VEGF transcripts. In some embodiments, the VEGF inhibitor comprises an antibody or a fragment thereof. In some embodiments, the VEGF antibody binds to VEGF and reduces angiogenic signaling, including the VEGF signaling pathway. In some embodiments, the VEGF antibody binds to VEGF-A, VEGF-B, VEGF-C, VEGF-D, or a combination thereof. In some embodiments, the VEGF antibody binds to one or more isoforms of VEGF-A, including VEGF121, VEGF145, VEGF148, VEGF162, VEGF165, VEGF165b, VEGF183, VEGF189, or VEGF206. In some embodiments, the antibody includes monovalent Fab', bivalent Fab2, F(ab)'3 fragment, single-strand variable fragment (scFv), bis-scFv, (scFv)2, diabody, minibody, nanobody, triabody, tetrabody, disulfide-stabilized Fv protein ("dsFv"), single-domain antibody (sdAb), Ig NAR, camel antibody, or a combination thereof, its binding fragment, or a chemically modified derivative thereof. Non-limiting examples of VEGF antibodies include ranibizumab or bevacizumab. In some embodiments, the VEGF antibody includes a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to any one of SEQ ID NOs. 81-87 (Table 14) or a combination thereof or a fragment thereof.

[0043] [Table 6]

[0044]

[0071] In some embodiments, the VEGF inhibitor is not an antibody. For example, the VEGF inhibitors described herein may include VEGF receptors, combinations of VEGF receptors, or fragments thereof for binding to VEGF in order 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 include soluble VEGFR1, soluble VEGFR2, soluble VEGFR3, soluble fragments thereof, or combinations thereof. In some embodiments, the non-antibody VEGF inhibitor includes at least one of FLT1, KDR / FLK1, FLT4, fragments thereof, or combinations thereof. In some embodiments, the non-antibody VEGF inhibitor includes 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 that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to any one of SEQ ID NOs. 88-92 (Table 15).

[0045] [Table 7]

[0046]

[0072] In some embodiments, the engineered polynucleotide includes a viral vector, such as an AAV vector, which includes one or more expression cassettes for one or more angiogenesis inhibitors. In some embodiments, the engineered polynucleotide includes a vector. In some embodiments, the vector is a viral vector. In some embodiments, the engineered polynucleotide includes an AAV vector. In some embodiments, the engineered polynucleotide includes an AAV vector encoding an engineered AAV capsid. In some embodiments, the AAV vector includes one or more expression cassettes for encoding an engineered polypeptide, which includes a peptide, or a fusion protein comprising an antibody or fragment thereof operably linked to a peptide.

[0047]

[0073] 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 including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the engineered polynucleotide is an AAV vector containing the AAV2 serotype. In some embodiments, the AAV vector encodes a modified AAV capsid. In some embodiments, the engineered polynucleotide contains a viral vector. In some embodiments, the viral vector contains an AAV vector. In some embodiments, the AAV vector contains an AAV serotype including 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 contains one of the amino acid sequences of SEQ ID NOs. 161-182 and SEQ ID NOs. 191-210. In some embodiments, the engineered AAV capsid contains the amino acid sequence of SEQ ID NO. 169.

[0048]

[0074] In some embodiments, the engineered polynucleotide includes a nucleic acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to any one of sequence numbers 101-103 and 121-129. In some embodiments, the engineered polynucleotide includes a nucleic acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical. In some embodiments, the engineered polynucleotide includes a nucleic acid sequence that is at least 85% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 126. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 127. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 128. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 129. In some embodiments, the engineered polynucleotide includes one of the nucleic acid sequences of SEQ ID NOs: 101-103 and 121-129.

[0049]

[0075] In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to any one of SEQ ID NOs. 111-113 and 43-45.

[0050]

[0076] 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 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 a membrane invasion complex (MAC) inhibitor. In some embodiments, the MAC inhibitor comprises CD59. In some embodiments, the engineered polynucleotide encodes a protease site adjacent to the second and third angiogenesis inhibitors. In some embodiments, the first angiogenesis inhibitor comprises a CD59 inhibitor, and the second angiogenesis inhibitor comprises 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 an endostatin. In some embodiments, an engineered polynucleotide encodes an Fc region adjacent to 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, and the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.In some embodiments, the engineered polynucleotide further encodes a protease site adjacent to the second and third angiogenesis inhibitors. In some embodiments, the protease site includes a furin protease site. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises Fc-CNP36, and 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, and 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, and 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, and the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor.

[0051]

[0077] In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises an endostatin, and an 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, the first and second angiogenesis inhibitors, when administered to a subject, inhibit angiogenesis in the subject. In some embodiments, the first or second angiogenesis inhibitor, when administered to a subject, exhibits a reduction in the inhibition of angiogenesis in the subject compared to the inhibition of angiogenesis caused by a VEGF inhibitor.

[0052]

[0078] 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--sCD59, sCD59-furin2A-C3i-Fc4-CNP36. In some embodiments, the engineered polynucleotide encodes sCD59-furin2A-endostatin-linker-C3i. In some embodiments, the engineered polynucleotide encodes sCD59-furin2A-aflibercept-linker-C3i. In some embodiments, the engineered polynucleotide encodes C3i-Fc4-CNP36-furin-mCD59. In some embodiments, the engineered polynucleotide encodes C3i-Fc4-endostatin-furin-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(+2Y, T14A, -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.

[0053]

[0079] In some embodiments, the engineered polynucleotide includes a nucleic acid sequence that is at least 70% identical to any one of sequence numbers 250-276, 304-311, and 320-324, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical. In some embodiments, the engineered polynucleotide includes a nucleic acid sequence that is at least 75% identical to any one of sequence numbers 250-276, 304-311, and 320-324. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of sequence number 250. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of sequence number 251. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of sequence number 252. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 253. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 254. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 255. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 256. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 257. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 258. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 259. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 260. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 261. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 262. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 263. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 264.In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 265. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 266. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 267. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 268. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 269. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 270. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 271. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 272. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 273. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 274. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 275. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 276. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 304. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 305. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 306. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 307. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 308. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 309. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 310. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 311. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 320. In some embodiments, the engineered polynucleotide includes the nucleic acid sequence of SEQ ID NO: 321. In some embodiments, the engineered polynucleotide includes 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.

[0054]

[0080] In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence that is at least 70% identical to one of SEQ ID NOs. 277-303, 312-319, and 325-329, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical. In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence that is one of SEQ ID NOs. 277-303, 312-319, and 325-329.

[0055]

[0081] In some embodiments, the engineered polynucleotide encodes the sequence shown in Figure 1. In some embodiments, the engineered polynucleotide encodes the sequence shown in Figures 2A-B. In some embodiments, the engineered polynucleotide encodes the sequence shown in Figure 3. In some embodiments, the engineered polynucleotide encodes the sequence shown in Figure 4. In some embodiments, the engineered polynucleotide encodes the sequence shown in Figures 5A-C. In some embodiments, the engineered polynucleotide encodes the sequence shown in Figure 16. In some embodiments, the engineered polynucleotide encodes the sequence shown in Figure 24. In some embodiments, the engineered polynucleotide encodes the sequence shown in Figure 26. In some embodiments, the engineered polynucleotide encodes the sequence shown in Figure 32. In some examples, the engineered polynucleotide includes additional features. Additional features may include sequences such as tags, signal peptides, intron sequences, promoters, stuffer sequences, and the like. In some examples, the engineered polynucleotide encodes a signal peptide. Signal peptides, sometimes also called signal sequences, targeted signals, localization signals, localization sequences, transit peptides, leader sequences, or leader peptides, are short peptides located at the N-terminus of most novel synthetic proteins destined for secretory pathways. These proteins include those located inside certain organelles (endoplasmic reticulum, Golgi apparatus, or endosomes), those secreted from cells, or those inserted into most cell membranes. In some examples, the nucleic acids provided herein may contain signal peptides. Signal peptides can be peptides of any length, but are typically 15–30 amino acid long. Signal peptides can be approximately 10–15, 10–20, 10–30, 15–20, 15–25, 15–30, 20–30, or 25–30 amino acid long. A variety of signal peptides can be utilized, including, but are not limited to, human antibody heavy chains (Vh), human antibody light chains (Vl), and aflibercepts.

[0056]

[0082] In some examples, engineered polynucleotides contain intron sequences. An intron is any nucleotide sequence within a sequence that can be removed by RNA splicing during the maturation of the final RNA product. In other words, an intron is a non-coding region of an RNA transcript, or the DNA that encodes it, that is removed by splicing before translation. Introns do not code for protein products, but they play a role in regulating gene expression. Some introns themselves code for functional RNA through further processing after splicing, generating non-coding RNA molecules. Alternative splicing is widely used to generate multiple proteins from a single gene. Furthermore, some introns play an essential role in a wide range of gene expression regulatory functions, such as nonsense mutation-dependent degradation and mRNA nuclear export. In one embodiment, the intron sequence is included in the nucleic acid of this disclosure and may be selected from hCMV intron A, adenovirus tripartite reader sequence intron, SV40 intron, hamster EF-1 alpha gene intron 1, intercalator sequence intron, human growth hormone intron, and / or human betaglobin intron. Any number of intron sequences are intended. In one embodiment, the intron sequence is SV40. In some examples, the nucleic acid may contain 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.

[0057]

[0083] In one embodiment, the engineered polynucleotide includes additional features, including a promoter. The promoter is a DNA sequence to which a protein binds, initiating the transcription of a single RNA from the DNA downstream of it. This RNA may encode a protein or may function as a tRNA, mRNA, or rRNA, either by itself or alone. The promoter is located upstream of the DNA (towards the 5' region of the sense strand), near the transcription start site of the gene. The promoter may be approximately 100–1000 base pairs long. Various promoters are conceived and may be used in the engineered polynucleotides of this disclosure. In one embodiment, the promoter is the cytomegalovirus (CMV) promoter, the elongation factor 1 alpha (EF1α) promoter, the monkey vacuolar virus (SV40) promoter, the phosphoglycerate kinase (PGK1) promoter, the ubiquitin C (Ubc) promoter, the human beta-actin promoter, the CAG promoter, the tetracycline response element (TRE) promoter, the UAS promoter, the actin 5c (Ac5) promoter, the polyhedron promoter, the Ca2+ / calmodulin-dependent protein kinase II (CaMKIIa) promoter, the GAL1 promoter, the GAL10 promoter, the TEF1 promoter, the glyceraldehyde 3-phosphate dehydrogenase (GDS) promoter, the ADH1 promoter, the CaMV35S promoter, the Ubi promoter, the human polymerase III RNA(H1) promoter, the U6 promoter, its polyadenylated constructs, and any combination thereof. In some examples, the promoter is the CMV promoter.

[0058]

[0084] Any of the provided engineered polynucleotides may contain a viral vector sequence. The viral vector may be a lentivirus, retrovirus, or adeno-associated virus, but is not limited to these. The viral vector may be an adeno-associated virus (AAV) vector. In some examples, the viral vector is an adeno-associated virus vector. Many serotypes of AAV vectors are intended, and are not limited to these, but may include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and / or AAV12. Based on these first serotypes, the AAV capsids of each serotype can be engineered to better suit them for a biological function, tissue, or cell selection. In some embodiments, the AAV vector is AAV2 as well as variants AAV2.N53 and AAV2.N54. Chimeric AAV vectors that may contain at least two AAV serotypes are also intended. 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 with the chimeric AAV vector. In some cases, only a small portion of the AAV is chimeric. For example, a preferred portion may include the capsid, VP1, VP2, or VP3 domain and / or Rep. In some cases, at least one of VP1, VP2, and VP3 has at least one amino acid substitution compared to the wild-type AAV capsid protein, which is otherwise equivalent. 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 1 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3, for example, about 1 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, or about 20 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3. In some examples, VPs can be removed. For example, in some embodiments, the mutant AAV does not contain at least one of VP1, VP2, or VP3.

[0059]

[0085] In some cases, AAV vectors can be modified. For example, modifications to an AAV vector may include insertions, deletions, chemical changes, or synthetic modifications. In some cases, a single nucleotide is inserted into the AAV vector. In other cases, multiple nucleotides are inserted into the vector. Codon optimization

[0086] In one embodiment, the engineered polynucleotides described herein include modifications that confer enhanced expression of one or more angiogenesis inhibitors described herein. For example, one or more angiogenesis inhibitors contain unmodified sequences derived from native gene sequences that are not optimized for introduction and expression in target cells. In one embodiment, the isolated engineered polynucleotide is codon-optimized. Codon optimization may be specific to cell-type-specific codon use. Different organisms and cell types exhibit a bias toward the use of certain codons with respect to the same amino acid. Some species are known to almost completely avoid certain codons. Similarly, certain cell types have a bias toward the use of certain codons with respect to the same amino acid. In one embodiment, a method for optimizing the codons of an engineered polynucleotide may include the step of reassigning codon use based on the frequency of each codon use in target cells. In some examples, the target cells may be cells of a particular tissue or organ. In some examples, the modification is made to increase the guanine and / or cytosine content.

[0060]

[0087] In one embodiment, an engineered nucleic acid sequence may be modified to replace at least one codon with another codon encoding the same amino acid. In some examples, the codon is modified within the coding region of the sequence. In some examples, the codon is modified within the non-coding region of the sequence. In some examples, 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 fit index.

[0061]

[0088] Various modifications are intended herein. In some examples, codons can be swapped. For example, a sequence can be modified to swap AGA with AGG. In other examples, CCC is swapped with CCT. In other examples, AGC is swapped with TCC. In other examples, CCC is swapped with CCG. Any of the non-limiting swaps provided in Table 16 can be applied to modify nucleic acids. Any number of codons in a nucleic acid can be swapped. In some examples, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 42, at least 44, at least 46, at least 48, or up to 50 codons can be replaced. In one embodiment, the engineered polynucleotide contains 3 codon modifications. In one embodiment, the engineered polynucleotide contains 16 codon modifications. In one embodiment, the engineered polynucleotide includes 3-5, 5-10, 5-15, 10-15, 10-20, 15-20, 1-20, 12-20, 12-25, 15-30, or 15-25 codon modifications. In one embodiment, the engineered polynucleotide includes two codon modifications: one from AGA to AGG, and one from CCT to CCC, AGC to TCC, or CCC to CCG. In one embodiment, the engineered polynucleotide includes three codon modifications: one from AGA to AGG, and one from CCT to CCC, AGC to TCC, or CCC to CCG. In one embodiment, the engineered polynucleotide includes four codon modifications: one from AGA to AGG, one from CCT to CCC, AGC to TCC, and CCC to CCG.Additional modifications may include any of the codon modifications provided in Table 16, in combination with any of the above codons and / or any of the additional modifications from Table 16. In one embodiment, the nucleic acid is modified so that AGA is replaced with AGG and CCT is replaced with CCC. In one embodiment, the nucleic acid is modified so that AGA is replaced with AGG and AGC is replaced with TCC. In one embodiment, the nucleic acid is modified so that AGA is replaced with AGG and CCC is replaced with CCG.

[0062] [Table 8]

[0063]

[0089] In some embodiments, the engineered nucleic acid 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 a sequence of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV vector sequence may be a sequence of the AAV2 serotype. In some embodiments, the viral vector sequence may include sequences of at least two AAV serotypes. In some embodiments, at least two serotypes may be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV11, and AAV12.

[0064]

[0090] In some cases, modifications may also include chemical modifications. Modified nucleic acids may involve modifications to their backbone, sugars, or nucleic acid bases, and even to novel bases or base pairs. Modified nucleic acids can have improved chemical and / or biological stability. Modifications with diverse chemical substituents (e.g., hydrophobic groups) can also result in improved properties and functionality, such as novel structural motifs and enhanced target binding.

[0065]

[0091] Examples of chemical modifications, though not limited to these, include 2'F, 2'-fluoro; 2'OMe, 2'-O-methyl; LNA, locked nucleic acid; FANA, 2'-fluoroarabinose nucleic acid; HNA, hexitol nucleic acid; 2'MOE, 2'-O-methoxyethyl; Librona, (1'-3')-β-L-librona, TNA, α-L-threose nucleic acid; tPhoNA, 3'-2'phosphonomethyl-threosyl nucleic acid; dXNA, 2'-deoxyxyl nucleic acid; PS, phosphorothioate; phNA, alkylphosphonate nucleic acid; PNA, and peptide nucleic acid. Modified Capsid

[0092] This specification provides compositions containing modified adeno-associated virus (AAV) capsids and methods for using them. Modified AAV capsids may contain exogenous sequences compared to otherwise equivalent unmodified AAV capsids. The exogenous sequences may refer to exogenous polypeptide sequences. AAV capsids may be modified to impart improved functionality to them, and to any compositions and / or methods in which they are used, thereby resulting in better therapeutic agents, particularly for ophthalmic use.

[0066]

[0093] The AAV wild-type (WT) genome contains at least three genes: rep, cap, and X. The X gene is located at the 3' end of the genome (nucleotides 3929–4393 in AAV2) and appears to encode a protein with an auxiliary function in genome replication. Significantly more information is available regarding rep and cap. The rep gene is located in the first half of the AAV WT genome and encodes a family of non-structural proteins (Rep proteins) necessary for viral transcriptional regulation and replication, as well as packaging the viral genome into a newly produced, pre-assembled capsid. The second half of the AAV genome contains the cap gene, which encodes viral proteins (VPs) VP1, VP2, and VP3, as well as assembly activation protein (AAP). The transcription of all VPs, which are capsid monomers, is controlled by a single promoter (p40 in the case of AAV2) and results in a single mRNA. Splicing (VP1) and an abnormal translation start codon (VP2) are responsible for the presence of VP1 and VP2 being approximately one-tenth or less compared to VP3. When encoded by a single gene, AAV VPs share most of their amino acids. Specifically, the entire VP3 sequence is also contained within VP2 and VP1 ("common VP3 region"), and similarly, VP2 and VP1 also share approximately 65 amino acids ("common VP1 / VP2 region"). Only VP1 contains a unique sequence at its N-terminus (approximately 138 amino acids, VP1 unique). AAP was identified in 2010 as a 23kDa protein encoded in selective cap ORFs. It is used to stabilize newly produced VP proteins and transport them from the cytoplasm to the cell nucleus. AAV serotypes 1-3, 6-9, and rh10 were unable to produce capsids in the absence of AAP, while low but detectable capsid production was reported in AAV4 and AAV5.

[0067]

[0094] In one embodiment, AAV may include modifications. The modifications may be modifications of the rep, cap, and / or X-code polypeptide sequences of AAV. In some examples, the modifications may be modifications of the cap polypeptide. The cap polypeptide may be modified in any one of the VP domains, e.g., VP1, VP2, and / or VP3. In some examples, VP1 is modified. In some examples, VP2 is modified. In some examples, VP3 is modified. In some embodiments, two or all of the VP domains may be modified. In some examples, VP1 and VP2 are modified. In some examples, VP1 and VP3 are modified. In addition, VP2 and VP3 may be modified, or VP1, VP2, and VP3 may be modified. Other combinations, e.g., Rep and Cap, Cap and X, Rep and X, and / or Rep, Cap, and X modifications are contemplated. Any combination of domains may be modified, for example, together with Rep and / or X modifications, as in any one of the VP modifications described above. In some examples, Rep and VP1 and / or VP2 are modified. In some embodiments, the Rep of the subject is modified. The rep modification may include modifications provided herein and may be present in at least one of Rep 78, Rep 68, Rep 52, or Rep 40. In some examples, the Rep is a Rep of a different AAV serotype than the capsid of the subject.

[0068]

[0095] In some cases, the modification is a modification of the AAV capsid. The AAV serotype capsid is assembled from 60 VP monomers, each containing approximately 50 copies of VP3, 5 copies of VP2, and 5 copies of VP1. Topologically prominent capsid surface structures are pores or "channel-like structures" at each 5-fold symmetry axis, depressions at each 2-fold symmetry axis, and three ridges around each 3-fold symmetry axis. The pores allow for exchange between the inside and outside of the capsid. The depressions, more precisely the floors at each 2-fold symmetry axis, are the thinnest parts of the viral capsid. The ridges around the 3-fold symmetry axes contain 5 of 9 so-called variable regions (VRs). Specifically, VR-IV, -V, and -VIII form loops (loops 1-4) at the top of the ridges, while VR-VI and -VII are found at their bases. The VRs differ among serotypes and are responsible for serotype-specific variations in antibody and receptor binding. Due to its exposed location and function in receptor binding, the VR, which forms a loop of ridges, is an ideal location for capsid modifications aimed at redirecting or increasing the directivity (cell surface targeting) of AAV. Redirecting directivity (vector retargeting) involves, for example, removing innate receptor binding by site-directed mutagenesis, combined with the insertion of ligands that mediate transduction through novel non-innate AAV receptors, while the AAV vector with expanded directivity acquires the ability to transduce cells through additional receptors while maintaining its innate receptor binding ability.

[0069]

[0096] In some embodiments, modification of the AAV capsid may refer to the insertion of an exogenous polypeptide sequence. In other embodiments, modification may refer to a deletion in the polypeptide sequence. Modification may also refer to the modification of at least one standard or non-standard amino acid residue in the polypeptide sequence.

[0070]

[0097] The insertion may involve inserting at least one exogenous amino acid residue into the sequence encoding the AAV capsid. The amino acid may be a standard amino acid or a non-standard amino acid. Any number of amino acid residues can be inserted. In some examples, the insertion site may be located in the GH loop or loop IV of the AAV capsid protein, for example, in the solvent-exposed portion of the GH loop or loop IV of the AAV capsid protein.

[0071]

[0098] In some examples, the modification involves the insertion of an exogenous polypeptide sequence containing the sequence of Formula 1: X0-X1-X2-X1-X3-X1-X1-X4. In some examples, X0 is valine (V), isoleucine (I), leucine (L), phenylalanine (F), tryptophan (W), tyrosine (Y), or methionine (M). In some examples, X1 is alanine (A), asparagine (N), glutamine (Q), serine (S), threonine (T), glutamic acid (E), aspartic acid (D), lysine (K), arginine (R), or histidine (H). In some examples, X2 is V, I, L, or M, and X3 is E, S, or Q. In some examples, X4 is K, R, E, or A. In some examples, Formula 1 further includes X5. X5 can be proline (P) or R.

[0072]

[0099] In some examples, formula 1 includes LALG-X3-X1-X1-X4 (sequence number 232), LKLG-X3-X1-X1-X4 (sequence number 233), or VKLG-X3-X1-X1-X4 (sequence number 234). In some examples, formula 1 includes VKLG-X3-X1-X1-X4 (sequence number 235). In some examples, the exogenous polypeptide includes VKLG-X3-X1-T-X4 (sequence number 236) and / or VKLG-X3-X1-X1-K (sequence number 237). In some examples, the exogenous polypeptide includes LALG-X3-X1-X1-X4 (sequence number 238). In some examples, the exogenous polypeptide includes LALG-X3-X1-T-X4 (sequence number 239) and / or LALG-X3-X1-S-X4 (sequence number 240). In some examples, the exogenous polypeptide includes 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 examples, the exogenous polypeptide includes LALG-X3-X1-SK (SEQ ID NO: 246). In some examples, the exogenous polypeptide includes LKLG-X3-X1-X1-X4 (SEQ ID NO: 247). In some examples, the exogenous polypeptide includes LKLG-X3-X1-T-X4 (SEQ ID NO: 248). In some examples, the exogenous polypeptide includes LKLG-X3-X1-TK (SEQ ID NO: 249).

[0073]

[0100] In some cases, the exogenous polypeptide contains the sequence of formula 1. In some cases, the sequence of formula 1 contains polypeptide sequences that are at least 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or up to approximately 100% identical to the sequences in Table 17. In some cases, the exogenous polypeptide is the polypeptide in Table 17 with 0 to 2 modifications per residue.

[0074]

[0101] In some examples, at least two exogenous polypeptides, for example, polypeptides described in Formula 1, are inserted into the AAV capsid sequence provided herein. The at least two exogenous polypeptides may be inserted at the same or different positions. In one embodiment, a modified AAV capsid can be produced by inserting one of the exogenous polypeptide sequences provided in Table 17 into an unmodified AAV capsid sequence, for example, the wild-type sequence provided in Table 18.

[0075] [Table 9-1]

[0076] [Table 9-2]

[0077] [Table 9-3]

[0078] [Table 9-4]

[0079] [Table 9-5]

[0080] [Table 9-6]

[0081] [Table 9-7]

[0082] [Table 9-8]

[0083] [Table 9-9]

[0084] Table 9-10

[0085] Table 9-11

[0086] Table 9-12

[0087] Table 9-13

[0088] Table 10-1

[0089] Table 10-2

[0090]

[0102] Similarly, a deletion may involve the deletion of at least one amino acid residue in the sequence encoding the AAV capsid. Any number of amino acids may be deleted. In some examples, 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 approximately 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 and / or deleted in the polypeptide sequence encoding the AAV capsid. In some cases, approximately or up to approximately 5 to approximately 11 amino acids are inserted at the insertion site in the GH loop or loop IV of the capsid protein compared to the corresponding unmodified AAV capsid protein. For example, the insertion site may be between amino acids 587 and 588 of AAV2, or at the corresponding position in the capsid subunit of another AAV serotype. It should be noted that the insertion site 587–588 is based on the AAV2 capsid protein. Approximately 5 to approximately 11 amino acids may be inserted at the corresponding position in AAV serotypes other than AAV2 (e.g., AAV5, AAV6, AAV8, AAV9, etc.).

[0091]

[0103] In some embodiments, the insertion site is a single insertion site between two adjacent amino acids located between amino acid positions 570-614 of VP1 of any AAV serotype, for example, the insertion site is between two adjacent amino acids located between amino acids 570-610, 570-600, 570-575, 575-580, 580-585, 585-590, 590-600, or 600-614 of VP1 of 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 may be between amino acids 575 and 576, 576 and 577, 577 and 578, 578 and 579, or 579 and 580. The insertion site may 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.

[0092]

[0104] In some embodiments, the insertion site may be between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 588 and 589 of AAV10.

[0093]

[0105] As another example, the insertion site could be between amino acids 450 and 460 of the AAV capsid protein shown in Table 18. For example, the insertion site could be amino acid 453 of AAV2 (e.g., immediately at the N-terminus), 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.

[0094]

[0106] In some embodiments, the capsid protein in question includes a GH loop 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 listed in Table 18. Those skilled in the art will know, based on comparisons of the amino acid sequences of various AAV serotype capsid proteins, where the insertion site corresponding to amino acids 587-588 of AAV2 is located in any given AAV serotype capsid protein.

[0095]

[0107] In some cases, the exogenous polypeptide may have 0 to 4 spacer amino acids (Y1-Y4) at one of the exemplary polypeptides in Table 17 or at the amino and / or carboxyl termini of Formula 1. Preferred spacer amino acids include, but are not limited to, leucine, alanine, glycine, and / or serine.

[0096]

[0108] Modifications to the AAV capsid may involve modifications of at least one amino acid residue in the polypeptide sequence. In some examples, modifications may be made at any AAV capsid position described herein and may involve any number of modifications. In some examples, modifications may involve mutations. Mutations may include point mutations, missense mutations, nonsense mutations, deletions, duplications, frameshifts, and / or repeat extensions.

[0097]

[0109] In one embodiment, the amino acid may be a nonpolar aliphatic residue, such as glycine, alanine, valine, leucine, methionine, isoleucine, or proline. In one embodiment, the amino acid residue may be aromatic, such as phenylalanine, tyrosine, or tryptophan. In one embodiment, the amino acid residue may be polar and uncharged, such as serine, threonine, cysteine, asparagine, or glutamine. In one embodiment, the amino acid may be positively charged, such as lysine, arginine, or histidine. In one embodiment, the amino acid may be negatively charged, such as aspartic acid or glutamic acid.

[0098]

[0110] 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 has a positive charge. In some cases, the charged amino acid has a negative charge.

[0099]

[0111] Point mutations can be conservative mutations. Non-limiting examples of conservative mutations include changes from nonpolar aliphatic amino acids to nonpolar aliphatic amino acids, from polar amino acids to polar amino acids, from positively charged amino acids to positively charged amino acids, from negatively charged amino acids to negatively charged amino acids, and from aromatic amino acids to aromatic amino acids. For example, 20 naturally occurring amino acids may share similar characteristics. Aliphatic amino acids may be glycine, alanine, valine, leucine, or isoleucine. Hydroxyl or sulfur / selenium-containing amino acids may be serine, cysteine, selenocysteine, threonine, or methionine. Cyclic amino acids may be proline. Aromatic amino acids may be phenylalanine, tyrosine, or tryptophan. Basic amino acids may be histidine, lysine, and arginine. Acidic amino acids may be aspartic acid, glutamic acid, asparagine, or glutamine. Conservative mutations can occur from serine to glycine, serine to alanine, serine to serine, serine to threonine, and serine to proline. Conservative mutations can occur from arginine to asparagine, arginine to lysine, arginine to glutamine, arginine to arginine, and arginine to histidine. Conservative mutations can occur from leucine to phenylalanine, leucine to isoleucine, leucine to valine, leucine to leucine, and leucine to methionine. Conservative mutations can occur from proline to glycine, proline to alanine, proline to serine, proline to threonine, and proline to proline. Conservative mutations can occur from threonine to glycine, threonine to alanine, threonine to serine, threonine to threonine, and threonine to proline. Conservative mutations can occur from alanine to glycine, alanine to threonine, alanine to proline, alanine to alanine, and alanine to serine. Conservative mutations can occur from valine to methionine, valine to phenylalanine, valine to isoleucine, valine to leucine, or valine to valine.Conservative mutations can occur from glycine to alanine, glycine to threonine, glycine to proline, glycine to serine, and glycine to glycine. Conservative mutations can occur from isoleucine to phenylalanine, isoleucine to isoleucine, isoleucine to valine, isoleucine to leucine, and isoleucine to methionine. Conservative mutations can occur from phenylalanine to tryptophan, phenylalanine to phenylalanine, and phenylalanine to tyrosine. Conservative mutations can occur from tyrosine to tryptophan, tyrosine to phenylalanine, and tyrosine to tyrosine. Conservative mutations can occur from cysteine ​​to serine, cysteine ​​to threonine, and cysteine ​​to cysteine. Conservative mutations can occur from histidine to asparagine, histidine to lysine, histidine to glutamine, histidine to arginine, and histidine to histidine. Conservative mutations can occur from glutamine to glutamic acid, glutamine to asparagine, glutamine to aspartic acid, and glutamine to glutamine. Conservative mutations can occur from asparagine to glutamic acid, asparagine to asparagine, asparagine to aspartic acid, and asparagine to glutamine. Conservative mutations can occur from lysine to asparagine, lysine to lysine, lysine to glutamine, lysine to arginine, and lysine to histidine. Conservative mutations can occur from aspartic acid to glutamic acid, aspartic acid to asparagine, aspartic acid to aspartic acid, and aspartic acid to glutamine. Conservative mutations can occur from glutamine to glutamine, glutamine to asparagine, glutamine to aspartic acid, and glutamine to glutamine. Conservative mutations can occur from methionine to phenylalanine, methionine to isoleucine, methionine to valine, methionine to leucine, or methionine to methionine. Conservative mutations can also occur from tryptophan to tryptophan, tryptophan to phenylalanine, or tryptophan to tyrosine.

[0100]

[0112] Non-limiting examples of additional amino acid mutations include A to R, A to N, A to D, A to C, A to Q, A to E, A to G, A to H, A to I, A to L, A to K, A to M, A to F, A to P, A to S, A to T, A to W, A to Y, A to V, R to N, R to D, R to C, R to Q, R to E, R to G, R to H, R to I, R to L, R to K, R to M, R to F, R to P, R to S, R to T, R to W, R to Y, R to V, N to D, N to C, N to Q, N to E, N to G, N to H, N to I, N to L, N to K N to M, N to F, N to P, N to S, N to T, N to W, N to Y, N to V, D to C, D to Q, D to E, D to G, D to H, D to I, D to L, D to K, D to M, D to F, D to P, D to S, D to T, D to W, D to Y, D to V, C to Q, C to E, C to G, C to H, C to I, C to L, C to K, C to M, C to F, C to P, C to S, C to T, C to W, C to Y, C to V, Q to E, Q to G, Q to H, Q to I, Q to L, Q to K, Q to M, Q to F, Q to P, Q to S, Q to T, Q to W, Q to Y, Q to V, E to G, E to H, E to I, E to L, E to K, E to M, E to F, E to P, E to S, E to T, E to W, E to Y, E to V, G to H, G to I, G to L, G to K, G to M, G to F, G to P, G to S, G to T, G to W, G to Y, G to V, H to I, H to L, H to K, H to M, H to F, H to P, H to S, H to T, H to W, H to Y, H to V, I to L, I to K, I to M, I to F, I to P, I to S, I to T, I to W, I to Y, I to V, L The mutations may be K, L to M, L to F, L to P, L to S, L to T, L to W, L to Y, L to V, K to M, K to F, K to P, K to S, K to T, K to W, K to Y, K to V, M to F, M to P, M to S, M to T, M to W, M to Y, M to V, F to P, F to S, F to T, F to W, F to Y, F to V, P to S, P to T, P to W, P to Y, P to V, S to T, S to W, S to Y, S to V, T to W, T to Y, T to V, W to Y, W to V, Y to V, and the reverse of any of the mutations already described.

[0101]

[0113] Any one of the aforementioned modifications, insertions, deletions, and / or mutations can occur at any residue in the AAV sequence. The sequence may be a capsid sequence. In other examples, the sequence may be a Rep and / or X sequence, rather than a capsid sequence. The sequence may be located in VP1, VP2, and / or VP3, as previously described. In some examples, the sequence modification is a modification of a loop in the capsid sequence, such as loop 3 and / or loop 4. In some examples, the modification is a modification of a residue in the sequence shown in Table 18.

[0102]

[0114] In some cases, modifications, such as insertions, deletions, and / or mutations, are modifications of residues in the capsid polypeptide sequence shown in Table 18. In some cases, modifications are at positions 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, or combinations thereof. In some cases, modifications are located at residues at positions 200-300, 300-400, 400-500, 500-600, or combinations thereof. In some cases, modifications are located at residues at positions 300-500 or combinations thereof. In one embodiment, the insertion site is located in the GH loop or loop IV of the AAV capsid protein, for example, in the solvent-exposed portion of the GH loop or loop IV of the AAV capsid protein. For example, the insertion site is located within amino acids 570-611 of AAV2, 571-612 of AAV1, 560-601 of AAV5, 571-612 of AAV6, 572-613 of AAV7, 573-614 of AAV8, 571-612 of AAV9, or 573-614 of AAV10.

[0103]

[0115] For example, the insertion site could be between amino acids 587 and 588 in AAV2, between amino acids 590 and 591 in AAV1, between amino acids 575 and 576 in AAV5, between amino acids 590 and 591 in AAV6, between amino acids 589 and 590 in AAV7, between amino acids 590 and 591 in AAV8, between amino acids 588 and 589 in AAV9, or between amino acids 589 and 590 in AAV10. In some examples, the modification is at positions 452, 453, 466, 467, 468, 471, 585, 586, 587, and / or 588 in AAV2. In some examples, the modification is at position 452 or 453 in AAV2. In some examples, the modification is at position 587 or 588 in AAV2. In some cases, the modification is an insertion at position 452, 453, 466, 467, 468, 471, 585, 586, 587, and / or 588 in any of sequence numbers 221-226. In some cases, the modification is an insertion at position 452, 453, 466, 467, 468, 471, 585, 586, 587, and / or 588 in sequence number 221. In some cases, the modification is a mutation, and the mutation is R585A or R588A in any of sequence numbers 221-226. In some cases, the modification is a mutation, and the mutation is R585A or R588A in sequence number 221.

[0104]

[0116] In some embodiments, the modified AAV capsid in question does not contain any other amino acid modifications, mutations, substitutions, insertions, or deletions other than the insertion of approximately 5 to 11 amino acids in the loops (loops 3 and / or 4) compared to the corresponding unmodified AAV capsid protein. In other embodiments, the variant AAV capsid in question contains, in addition to the insertion of approximately 5 to 11 amino acids in loops 3 and / or 4 compared to the unmodified AAV capsid protein, 1 to 25 amino acid insertions, deletions, or substitutions compared to the unmodified AAV capsid protein. In one embodiment, the AAV virion capsid in question does not contain any other amino acid substitutions, insertions, or deletions other than the insertion of approximately 7 to 10 amino acids in the GH loop or loop IV compared to the corresponding parent AAV capsid protein. In other embodiments, the AAV virion capsid in question includes approximately 7 to 10 amino acid insertions in the GH loop or loop IV compared to the corresponding parental AAV capsid protein, in addition to 1 to approximately 25 amino acid insertions, deletions, or substitutions compared to the parental AAV capsid protein. For example, in some embodiments, the AAV virion capsid in question includes approximately 7 to 10 amino acid insertions in the GH loop or loop IV compared to the corresponding parental AAV capsid protein, in addition to 1 to approximately 5, approximately 5 to approximately 10, approximately 10 to approximately 15, approximately 15 to approximately 20, or approximately 20 to approximately 25 amino acid insertions, deletions, or substitutions compared to the parental AAV capsid protein.

[0105]

[0117] In some examples, chimeric AAV capsids are provided herein. A chimeric capsid comprises polypeptide sequences from at least two AAV serotypes. A chimeric capsid may comprise a mixture of sequences selected from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and / or AAV12. In some examples, the chimeric serotype differs between VP1, VP2, and / or VP3. In some examples, the chimeric capsid comprises sequences from at least two serotypes selected from AAV4 and AAV6, AAV5 and AAV6, AAV11 and AAV6, AAV12 and AAV6, and any combination thereof. In some examples, the first AAV serotype 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 may be AAV12 and AAV6. In some cases, the chimeric capsid contains sequences from AAV2 and AAV5 or AAV2 and AAV6. In some cases, the chimeric capsid contains sequences from AAV2 and AAV5, AAV2 and AAV6, AAV2 and AAV8, AAV2 and AAV9, AAV2 and AAV1, and AAV2 and AAV12.

[0106]

[0118] Modifications to AAV provided herein can confer enhanced activity to modified AAV compared to otherwise unmodified or wild-type AAV. Modifications provided herein can improve cell transduction, directivity, and / or reduce capsid-related immunogenicity.

[0107]

[0119] In some cases, the modifications provided herein enhance cellular transduction. Cellular transduction may refer to the ability of AAV to infect cells (in vivo or in vitro) and / or deliver transgenes to cells.

[0108]

[0120] In some examples, the modifications provided herein enhance directivity. Enhanced directivity refers to the acquisition of the ability to transduce cells through additional receptors compared to otherwise unmodified AAV. In some embodiments, enhanced directivity can improve the infectivity of ocular cells by utilizing modified AAV, thereby improving gene therapy. In some examples, the modifications provided herein can improve directivity to ocular cells selected from bipolar cells, retinal ganglion cells, horizontal cells, amacrine cells, epithelial cells, retinal pigment cells, photoreceptor cells, or any combination thereof. In some examples, the modifications improve directivity to retinal cells.

[0121] Similarly, AAV vectors are provided herein. AAV vectors contain terminal inverted repeat sequences (ITRs), Rep, Cap, AAP, and X sequences. Typically, the AAV viral genome is flanked by ITRs that act as packaging signals and replication start sites. Rep genes encode a family of multifunctional proteins (Rep proteins) involved in the regulation of viral transcription, replication, packaging, and integration in AAV2. With respect to AAV2, four Rep proteins are described. The expression of Rep78 and Rep68 is regulated by the AAV2-specific p5 promoter, while p19 regulates 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 and viral capsid proteins VP1 (90 kDa), VP2 (72 kDa), and VP3 (60 kDa), all encoded in the cap gene, is regulated by the p40 promoter. The X gene is located at the 3' end of the genome within a region shared with the cap gene and possesses its own promoter (p81). While the X protein appears to enhance viral replication, AAP is essential for capsid assembly. The three distinct VPs contribute to the polyhedral AAV2 capsid in a ratio of 1 (VP1):1 (VP2):10 (VP3).

[0109]

[0122] The modified capsid proteins disclosed herein can be isolated, for example, purified. In some embodiments, the modified capsids disclosed herein are contained in AAV vectors or AAV virions (e.g., recombinant AAV virions, rAAV, or AAV virus particles). In other embodiments, such modified AAV vectors and / or AAV variant virions are used in in vivo or ex vivo methods to treat eye diseases in the retina of primates, for example, the human retina.

[0110]

[0123] Vectors containing modified AAV capsids are also provided herein. Any one of the previously described modifications may be incorporated into the vectors provided herein. In some examples, the AAV vector contains a modified capsid that includes an exogenous sequence in at least two loops of the VP domain, compared to an otherwise equivalent AAV capsid sequence that lacks the exogenous sequence. In some embodiments, the vectors provided herein may further include a transgene sequence. Engineered polypeptides

[0124] In this specification, in certain embodiments, engineered polypeptides are described. In some embodiments, the engineered polypeptide is encoded by an engineered polynucleotide as described herein. In some embodiments, the engineered polypeptide comprises a first angiogenic inhibitor and a second angiogenic inhibitor as described herein. In some embodiments, the engineered polypeptide comprises a third angiogenic inhibitor. In some embodiments, the engineered polypeptide comprises two or more angiogenic inhibitors covalently linked by an antibody (e.g., an Fc region as described herein) or a linker.

[0111]

[0125] In some embodiments, the engineered polypeptide 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 that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to any one of SEQ ID NOs: 1-15. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at least 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 12 consecutive amino acids of any one of SEQ ID NOs: 1-15.

[0112]

[0126] 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 by a linker. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to any one of SEQ ID NOs. 61-72. In some embodiments, the natriuretic peptide or natriuretic peptide fusion protein comprises an amino acid sequence that is any one of SEQ ID NOs. 61-72.

[0113]

[0127] In some embodiments, the engineered polypeptide comprises a membrane invasion complex (MAC) inhibitor and at least one additional angiogenesis inhibitor. In some embodiments, the MAC inhibitor comprises CD59. In some embodiments, CD59 comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to any one of SEQ ID NOs. 41-45. In some embodiments, CD59 comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to any one of SEQ ID NOs. 312-319. In some embodiments, CD59 comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to any one of SEQ ID NOs. 325-329. In some embodiments, CD59 comprises an amino acid sequence that is at least 95% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to any one of SEQ ID NOs. 41-45. In some embodiments, CD59 includes an amino acid sequence that is one of sequence numbers 312-319. In some embodiments, CD59 includes an amino acid sequence that is one of sequence numbers 325-329.

[0114]

[0128] 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, the endostatin or a fragment thereof comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 51. In some embodiments, the endostatin or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 51.

[0115]

[0129] In some embodiments, the engineered polypeptide comprises a VEGF inhibitor and at least one additional angiogenesis inhibitor. In some embodiments, the VEGF antibody comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to one of SEQ ID NOs: 81-87 or SEQ ID NOs: 88-92, or a combination thereof, or a fragment thereof. In some embodiments, the VEGF antibody comprises a polypeptide sequence that is one of SEQ ID NOs: 81-87 or SEQ ID NOs: 88-92, or a combination thereof, or a fragment thereof.

[0116]

[0130] In some embodiments, the engineered polypeptide comprises a complement 3 inhibitor, a fragment crystallizable (Fc) region, and a natriuretic peptide. In some embodiments, the engineered polypeptide 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 comprises a fragment crystallizable (Fc) region. In some embodiments, the engineered polypeptide further comprises CD59, an endostatin, a VEGF inhibitor, or a combination thereof. In some embodiments, the engineered polypeptide further comprises CD59, an endostatin, and a VEGF inhibitor. In some embodiments, the engineered polypeptide further comprises CD59 and an endostatin. In some embodiments, the engineered polypeptide further comprises an endostatin and a VEGF inhibitor. In some embodiments, the engineered polypeptide further comprises an endostatin and a VEGF inhibitor. In some embodiments, the engineered polypeptide further comprises CD59 and a VEGF inhibitor. In some embodiments, the engineered polypeptide further comprises CD59 and endostatin. In some embodiments, the engineered polypeptide comprises a complement 3 inhibitor and a membrane invasion complex (MAC) inhibitor. In some embodiments, the MAC inhibitor comprises CD59. In some embodiments, the engineered polypeptide comprises a natriuretic peptide, endostatin, a VEGF inhibitor, or a combination thereof. In some embodiments, the engineered polypeptide comprises a natriuretic peptide, endostatin, and a VEGF inhibitor. In some embodiments, the engineered polypeptide 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 polypeptide further comprises a natriuretic peptide, CD59, a VEGF inhibitor, or a combination thereof.In some embodiments, the engineered polypeptide further comprises a natriuretic peptide. In some embodiments, the engineered polypeptide further comprises CD59. In some embodiments, the engineered polypeptide further comprises a VEGF inhibitor. In some embodiments, the engineered polypeptide comprises a complement 3 inhibitor and a VEGF inhibitor.

[0117]

[0131] In some embodiments, the engineered polypeptide 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 fragment crystallizable (Fc) region. In some embodiments, the engineered polypeptide comprises a complement 3 inhibitor, an endostatin, a VEGF inhibitor, or a combination thereof. In some embodiments, the engineered polypeptide comprises a complement 3 inhibitor, an endostatin, and a VEGF inhibitor.

[0118]

[0132] In some embodiments, the engineered polypeptide 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 polypeptide comprises a natriuretic peptide, a complement 3 inhibitor, a VEGF inhibitor, or a combination thereof. In some embodiments, the engineered polypeptide comprises a natriuretic peptide, a complement 3 inhibitor, and a VEGF inhibitor. In some embodiments, the engineered polypeptide comprises a natriuretic peptide and a complement 3 inhibitor.

[0119]

[0133] In some embodiments, the engineered polypeptide contains CD59 and VEGF inhibitors.

[0134] In some embodiments, the engineered polypeptide comprises a natriuretic peptide, a complement 3 inhibitor, an endostatin, or a combination thereof.

[0120]

[0135] In some embodiments, the engineered polypeptide comprises sCD59-Fc4-CNP36. In some embodiments, the engineered polypeptide comprises sCD59-C3i-Fc4-CNP36. In some embodiments, the engineered polynucleotide encodes C3i-Fc4-endostatin. In some embodiments, the engineered polypeptide comprises aflibercept (SEQ ID NO: 71)-linker-C3i. In some embodiments, the engineered polypeptide comprises C3i-Fc4-CNP36-furin--sCD59. In some embodiments, the engineered polypeptide comprises sCD59-furin2A-C3i-Fc4-CNP36. In some embodiments, the engineered polypeptide comprises sCD59-furin2A-endostatin-linker-C3i. In some embodiments, the engineered polypeptide comprises sCD59-furin2A-aflibercept-linker-C3i. In some embodiments, the engineered polypeptide contains C3i-Fc4-CNP36-furin-mCD59. In some embodiments, the engineered polypeptide contains C3i-Fc4-endostatin-furin-sCD59. In some embodiments, the engineered polypeptide contains the sequence shown in Figure 1. In some embodiments, the engineered polypeptide contains the sequence shown in Figures 2A-B. In some embodiments, the engineered polypeptide contains the sequence shown in Figure 3. In some embodiments, the engineered polypeptide contains the sequence shown in Figure 4. In some embodiments, the engineered polypeptide contains the sequence shown in Figures 5A-C. In some embodiments, the engineered polynucleotide encodes the sequence shown in Figure 16. In some embodiments, the engineered polynucleotide encodes the sequence shown in Figure 24. In some embodiments, the engineered polynucleotide encodes the sequence shown in Figure 26. In some embodiments, the engineered polynucleotide encodes the sequence shown in Figure 32.

[0121]

[0136] In some embodiments, the engineered polypeptide can be administered to a subject to treat a disease or condition. In some embodiments, the engineered polypeptide can be formulated into a pharmaceutical composition administered to a subject to treat a disease or condition. In some embodiments, the engineered polypeptide can increase activity or signal cascades related to the complement pathway. In some embodiments, the engineered polypeptide can increase activity or signal cascades related to natriuretic peptide receptors (NPRs). In some embodiments, the engineered polypeptide can increase activity or signal cascades related to the cyclic GMP (cGMP) signaling pathway. In some embodiments, the engineered polypeptide can increase activity or signal cascades related to CD59. In some embodiments, the engineered polypeptide can increase activity or signal cascades related to endostatins. In some embodiments, the engineered polypeptide can decrease activity or signal cascades related to VEGFs.

[0122]

[0137] In some embodiments, engineered polypeptides can be administered to subjects to treat a disease or condition by increasing activity or signaling cascades related to the complement pathway. In some embodiments, engineered polypeptides can be administered to subjects to treat a disease or condition by increasing activity or signaling cascades related to natriuretic peptide receptors (NPRs). In some embodiments, engineered polypeptides can be administered to subjects to treat a disease or condition by increasing activity or signaling cascades related to the cGMP signaling pathway. In some embodiments, engineered polypeptides can be administered to subjects to treat a disease or condition by increasing activity or signaling cascades related to CD59. In some embodiments, engineered polypeptides can be administered to subjects to treat a disease or condition by increasing activity or signaling cascades related to endostatins. In some embodiments, engineered polypeptides can be administered to subjects to treat a disease or condition by decreasing activity or signaling cascades related to VEGFs. Pharmaceutical composition

[0138] This specification describes pharmaceutical compositions comprising engineered polynucleotides, AAV vectors containing engineered polynucleotides, engineered polypeptides, cells transduced to AAV vectors containing engineered polynucleotides, viral particles containing engineered polynucleotides, or combinations thereof. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition comprises two or more activators disclosed herein. In some embodiments, the pharmaceutical composition comprising an engineered polynucleotide, an AAV vector containing an engineered polynucleotide, or an AAV vector containing an engineered polynucleotide treats a disease or condition described herein. In some embodiments, the disease or condition includes eye diseases. In some embodiments, the disease or condition includes ischemic syndrome of the eye, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color blindness, age-related macular degeneration (nAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, traumatic glaucoma, Valdevie-Wiedl syndrome, Best's disease, choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-vertebral dystrophy, vertebral-rod dystrophy, Oguchi disease, autosomal dominant radial drusen (familial autosomal dominant drusen), blue vertebral monochromatic color blindness, or a combination thereof.

[0123]

[0139] For in vivo delivery, engineered polynucleotides, AAV vectors containing engineered polynucleotides, engineered polypeptides, cells transduced to AAV vectors containing engineered polynucleotides, or combinations thereof can be formulated into pharmaceutical compositions and generally administered intravitreally or parenterally (e.g., via routes of administration such as intramuscular, subcutaneous, intratumoral, transdermal, intrathecal, etc.). In some embodiments, the pharmaceutical compositions are formulated for administration to subjects requiring the administration of the pharmaceutical composition via intrathecal, intraocular, intravitreous, retinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, subretinal, superchoroidal, intratumoral, lung, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray, intraluminal-GI route, or combinations thereof.

[0124]

[0140] In some embodiments, the pharmaceutical composition can be used to treat subjects such as humans or mammals that require the pharmaceutical composition. In some examples, the subject may be diagnosed with a disease, such as an eye disease. In some embodiments, the pharmaceutical composition of the subject is administered in combination with a second-line treatment. The second-line treatment may include any treatment for ophthalmic use. In some examples, the second-line treatment includes nutritional therapy, vitamins, laser treatments such as laser photocoagulation, photodynamic therapy, Visudyne, anti-VEGF therapy, eyewear, eye drops, numbing agents, treatments for binocular vision impairment, behavioral / visual cognitive therapy, and similar. In some embodiments, any of the previously described biological products may be considered a second-line treatment.

[0125]

[0141] In some embodiments, an effective amount of the pharmaceutical composition results in a reduction of the rate of loss of retinal function, anatomical integrity, or retinal health, and therefore the rate of disease progression, for example, to half, one-third, one-quarter, or one-fifth, or less, for example, to one-tenth, or less, of the rate of loss of disease, and therefore the rate of disease progression.

[0126]

[0142] In some embodiments, an effective amount of the pharmaceutical composition reduces angiogenic 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 angiogenic signaling in cells not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition reduces angiogenesis in subjects requiring treatment with the pharmaceutical composition by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more, compared to angiogenesis in subjects not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition reduces vascular leakage in a subject requiring treatment with the pharmaceutical composition by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more, compared to vascular leakage in a subject not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition reduces inflammation in a subject requiring treatment with the pharmaceutical composition by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more, compared to inflammation in a subject not treated with the pharmaceutical composition.

[0127]

[0143] In some embodiments, an effective amount of the subject rAAV virions results in an increase in visual function, retinal function, improvement in retinal anatomy or health, and / or improvement in eye movement, and / or improvement in nerve function, such as a 2-fold, 3-fold, 4-fold, or 5-fold or greater improvement in retinal function, retinal anatomy or health, and / or eye movement, such as a 10-fold or greater improvement in retinal function, retinal anatomy or health, and / or eye movement. As will be readily appreciated by those skilled in the art, the dosage required to achieve the desired treatment effect is typically 1×10 8 ~ about 1×10 15 recombinant virions, and is typically 1×10 8 ~ about 1×10 15 is referred to as the "vector genome."

[0128]

[0144] In some aspects, the compositions provided herein, such as pharmaceutical compositions, are administered to a subject in need thereof. In some examples, the administration involves delivering a dosage of about 0.5×10 9 vg, 1.0×109vg, 1.0×10 10 , 1.0×10 11 vg, 3.0×10 11 vg, 6×10 11 vg, 8.0×10 11 vg, 1.0×10 12 vg, 1.0×10 13 vg, 1.0×10 14 vg, 1.0×10 15 vg, 1.5×10 15 vg. For example, in the case of in vivo injection, such as direct injection into the eye, the therapeutically effective dosage is about 10 6 ~ about 10 15 times, such as about 10 8 ~ 10 12 times the number of engineered AAV virions. In the case of in vitro transduction, the effective amount of engineered AAV virions delivered to the cells is about 10 8 ~ about 10 13This is the order of the dose. Other effective doses can be readily established by those skilled in the art through the usual trials for establishing dose-response curves.

[0129]

[0145] The administration can be repeated for any period of time. In some embodiments, administration may be performed twice daily, every other day, twice weekly, every two months (bimonthly), every three months, once a month, every other month (every other month), every six months, once a year, or once every two years.

[0130]

[0146] Dosage administration can be a single-dose schedule or a multi-dose schedule. Furthermore, the subject may be administered as many doses as necessary. Those skilled in the art can easily determine an appropriate number of doses. In some embodiments, the pharmaceutical composition is administered via intravitreous injection, subretinal injection, microinjection, or choroidal injection.

[0131]

[0147] In the practice of the treatment or method of use provided herein, the therapeutically effective dose of the pharmaceutical composition described herein is administered to a mammal having the disease, disorder, or condition being treated, such as cancer. In some embodiments, the mammal is a human. The therapeutically effective dose may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used, and other factors. The therapeutic agents, and in some examples the compositions described herein, may be used alone or in combination with one or more therapeutic agents as components of a mixture.

[0132]

[0148] The pharmaceutical compositions described herein, but are not limited to those described herein, may be administered to a target by an appropriate route of administration, including intravenous, intra-arterial, oral, parenteral, oral cavity, topical surface, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration routes. The compositions described herein, but are not limited to those described herein, may include aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, sustained-release formulations, fast-melt formulations, tablets, capsules, pills, delayed-release formulations, sustained-release formulations, pulsed-release formulations, multi-particle formulations, as well as mixed immediate-release formulations and controlled-release formulations.

[0133]

[0149] Pharmaceutical compositions may be manufactured by conventional methods, for example, by conventional mixing, dissolution, granulation, grinding, emulsification, encapsulation, capture, or compression processes.

[0150] In certain embodiments, the pharmaceutical compositions provided herein include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances, such as merfen and thimerosal; stabilized chlorine dioxide; and quaternary ammonium compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0134]

[0151] In some embodiments, the pharmaceutical compositions described herein are formulated into any suitable dosage form, including, but are not limited to, aqueous oral dispersants, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled-release formulations, fast-melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, sugar-coated tablets, capsules, delayed-release formulations, sustained-release formulations, pulsed-release formulations, multiparticle formulations, and mixed immediate-release formulations and controlled-release formulations. In one embodiment, the therapeutic agents disclosed herein, for example, the therapeutic agents are formulated into pharmaceutical compositions suitable for intramuscular, subcutaneous, or intravenous injection. In one embodiment, the formulation suitable for intramuscular, subcutaneous, or intravenous injection comprises a physiologically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension, or emulsion, and a sterile powder for rehydration in a sterile injection solution or dispersion. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, cremophor, and the like), suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters, such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersant, and by using a surfactant. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial growth can be ensured by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some examples, it is desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Sustained absorption of the injectable pharmaceutical form can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin.

[0135]

[0152] In another embodiment, the dosage form includes 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, disintegration accelerators, defoamers, antioxidants, flavorings, and carrier materials, such as binders, suspenders, disintegrants, fillers, surfactants, solvents, stabilizers, lubricants, wetting agents, and diluents.

[0136]

[0153] The dosage form of a liquid formulation for oral administration is an aqueous suspension selected as necessary from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersants, emulsions, solutions, elixirs, gels, and syrups. In addition to the therapeutic agent, the liquid dosage form may optionally include additives, such as (a) disintegrants; (b) dispersants; (c) wetting agents; (d) at least one preservative; (e) thickeners; (f) at least one sweetener; and (g) at least one flavoring agent. In some embodiments, the aqueous dispersant further includes a crystal formation inhibitor.

[0137]

[0154] In some embodiments, the pharmaceutical compositions described herein are self-emulsifying drug delivery systems (SEDDS). Emulsions are typically dispersants in the form of droplets in one immiscible phase and the other immiscible phase. Generally, emulsions are produced by vigorous mechanical dispersion. In contrast to emulsions or microemulsions, SEDDS spontaneously form emulsions when an excess amount of water is added, without any external mechanical dispersion or agitation. The advantage of SEDDS is that only very light mixing is required to distribute the droplets throughout the solution. In addition, water or the aqueous phase is added as needed immediately before administration, thereby ensuring the stability of unstable or hydrophobic active ingredients. Thus, SEDDS provide an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provide improved bioavailability of hydrophobic active ingredients.

[0138]

[0155] Furthermore, the pharmaceutical composition may optionally include 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 trishydroxymethylaminomethane; 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.

[0139]

[0156] In addition, the pharmaceutical composition may optionally contain one or more salts in amounts necessary to bring the osmotic pressure of the composition into an acceptable range. Such salts include salts having sodium, potassium, or ammonium cations, and salts having chloride, citric acid, ascorbic acid, boric acid, phosphoric acid, bicarbonate, sulfuric acid, thiosulfate, or bisulfite anions; preferred salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate. kit

[0157] In this specification, in some embodiments, kits for use are disclosed, comprising engineered polynucleotides, AAVs containing engineered polynucleotides, engineered polypeptides, cells transduced to an AAV vector containing an engineered polynucleotide, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof. In some embodiments, the kits disclosed herein may be used to treat a disease or condition in a subject. In some embodiments, the kits comprise a collection of materials or components, apart from comprising engineered polynucleotides, AAVs containing engineered polynucleotides, engineered polypeptides, cells transduced to an AAV vector containing an engineered polynucleotide, or pharmaceutical compositions.

[0140]

[0158] In some embodiments, the kits described herein include components for selecting a homogeneous population of AAVs containing the engineered polynucleotides described herein. In some embodiments, the kits include components for assaying the number of units of a synthesized and / or released biomolecule (e.g., AAV) or expressed on the surface of a host cell. In some embodiments, the kits include components for performing an assay such as an enzyme-linked immunosorbent assay (ELISA). The exact nature of the components comprising the kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating a disease or condition disclosed herein (e.g., cancer) in a subject. In some embodiments, the kits are configured for the purpose of treating a mammalian subject in particular. In some embodiments, the kits are configured for the purpose of treating a human subject in particular.

[0141]

[0159] Instructions for use may be included in the kit. In some embodiments, the kit includes instructions for administering an engineered polynucleotide, an AAV vector containing an engineered polynucleotide, an engineered polypeptide, an AAV containing an engineered polynucleotide, cells transduced to the AAV vector, a pharmaceutical composition, or a combination thereof to a subject requiring administration of the kit. In some embodiments, the kit includes instructions for further engineering cells to express a biomolecule (e.g., an engineered polynucleotide, an AAV vector containing an engineered polynucleotide, an engineered polypeptide, an AAV containing an engineered polynucleotide, or cells transduced to the AAV vector). In some embodiments, the kit includes instructions for thawing or otherwise restoring the bioactivity of an engineered polynucleotide, an AAV vector containing an engineered polynucleotide, or an AAV containing an engineered polynucleotide, which may be cryopreserved or lyophilized during storage or transport. In some embodiments, the kit includes a restored engineered polynucleotide, an AAV vector containing the engineered polynucleotide, and instructions for use to measure the viability of the AAV containing the engineered polynucleotide, in order to ensure effectiveness for its intended purpose (e.g., therapeutic effectiveness when used to treat a subject).

[0142]

[0160] If necessary, the kit may also contain other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandage materials, or other useful instruments. The materials or components assembled in the kit may be stored in any convenient and suitable manner that preserves their operability and usefulness and provided to the physician. For example, components may be in soluble, dehydrated, or lyophilized form; they may be provided at room temperature, refrigerated, or freezing temperature. Components are typically contained in suitable packaging material(s). Delivery method

[0161] Engineered polynucleotides can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method available 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 to cells by physical methods, such as calcium phosphate precipitation, lipofection, particle impact, microinjection, gene guns, electroporation, and the like.

[0143]

[0162] Physical methods for introducing engineered polynucleotides into cells may include calcium phosphate precipitation, lipofection, particle impact, microinjection, gene guns, electroporation, and similar methods. One method for introducing engineered polynucleotides into host cells is calcium phosphate transfection.

[0144]

[0163] Chemical means for introducing engineered polynucleotides encoding non-natural molecules into cells may include colloidal dispersion systems, e.g., polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, globular nucleic acids (SNAs), liposomes, or lipid nanoparticles. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). Other nucleic acid-targeted delivery methods of the latest technology are available, e.g., delivery of engineered polynucleotides or vectors encoding engineered polynucleotides by targeted nanoparticles.

[0145]

[0164] When nonviral delivery systems are utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is intended to introduce engineered polynucleotides or vectors encoding engineered polynucleotides into cells (in vitro, ex vivo, or in vivo). In other embodiments, the vector can associate with lipids. A lipid-associated vector can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, bound to a liposome via a linking molecule that associates with both the liposome and the engineered polynucleotide, captured by a liposome and forming a complex with a liposome, dispersed in a lipid-containing solution, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in micelles or forming a complex 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 a bilayer structure as micelles or as a "broken" structure. Alternatively, they are simply dispersed in solution and may form aggregates that are not uniform in size or shape. Lipids are fatty substances that, in some embodiments, are naturally occurring or synthetic lipids. For example, lipids include naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0146]

[0165] Lipids suitable for use are obtained from suppliers, and storage solutions of lipids in chloroform or chloroform / methanol are often 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 single-lamellar and multi-lamellar lipid vehicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes are often characterized as having a vesicular structure with a phospholipid bilayer and an internal aqueous medium. Multi-lamellar liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess amount of aqueous solution, they form spontaneously. After the lipid components undergo self-rearrangement, they form a closed structure that traps water and dissolved solutes between the lipid bilayers. However, compositions that have structures in solution different from normal vesicular structures are also included. For example, lipids, in some embodiments, take on micelle-like structures or simply exist as heterogeneous aggregates of lipid molecules. Similarly, lipofectamine-nucleic acid complexes are also considered.

[0147]

[0166] In some examples, nonviral delivery methods include lipofection, nucleofection, microinjection, bioristics, virosomes, liposomes, immunoliposomes, exosomes, polycations or lipids: cargo conjugates (or aggregates), naked polypeptides (e.g., recombinant polypeptides), naked DNA, artificial virions, and activator-enhanced uptake of polypeptides or DNA. In some embodiments, the delivery method includes conjugating or encapsulating the compositions or engineered polynucleotides described herein with or with at least one polymer, such as a natural polymer or synthetic material. The polymer may be biocompatible or biodegradable. Non-limiting examples of suitable biocompatible, biodegradable synthetic polymers include aliphatic polyesters, poly(amino acids), copoli(ether-esters), polyalkylene oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamide esters, polyoxaesters containing amine groups, and poly(acid anhydrides). Such synthetic polymers may be two or more homopolymers or copolymers (e.g., random, block, segmented, graft) of multiple different monomers, such as lactic acid, lactide, glycolic acid, glycolide, epsilon-caprolactone, trimethylene carbonate, p-dioxanone, etc. In one example, the scaffold may consist of polymers containing glycolic acid and lactic acid, for example, 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, glycosaminoglycans (GAGs) and fragments(s) derived from these components, elastin, laminin, dechlorin, fibrinogen / fibrin, fibronectin, osteopontin, tenacin, hyaluronic acid, collagen, chondroitin sulfate, heparin, heparan sulfate, ORC, carboxymethylcellulose, and chitin.

[0148]

[0167] In some examples, the engineered polynucleotides described herein may be packaged via extracellular vesicles and delivered to cells. Extracellular vesicles may be any membrane-bound proteins. In some embodiments, extracellular vesicles may be any membrane-bound particles secreted by at least one cell. In some examples, extracellular vesicles may be any membrane-bound particles synthesized in vitro. In some examples, extracellular vesicles may be any membrane-bound particles synthesized without cells. In some examples, extracellular vesicles may be exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apotosomes, oncosomes, exophers, enveloped viruses, exomers, or other very large extracellular vesicles.

[0149]

[0168] In some embodiments, engineered polynucleotides may be delivered to cells via biological methods such as the use of DNA and RNA vectors. Viral vectors, and retroviral vectors in particular, have become the most widely used method for inserting genes into mammalian cells, e.g., human cells. Other viral vectors, in some embodiments, are derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses, and similar species. Exemplary viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors (AAV vectors), pox vectors, parvovirus vectors, baculovirus vectors, measles virus vectors, or herpes simplex virus vectors (HSV). In some examples, retroviral vectors include gamma-retroviral vectors, e.g., Moloney's mouse leukemia (Keukemia) virus (MoMLV, MMLV, MuLV, or MLV), or vectors derived from the mouse stem cell virus (MSCV) genome. In some examples, retroviral vectors also include lentiviral vectors, e.g., lentiviral vectors derived from the human immunodeficiency virus (HIV) genome. In some examples, the AAV includes serotypes comprising AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or combinations thereof. Based on these initial serotypes, the AAV capsid of each serotype may be engineered to be more favorable for biological function, tissue, or cell selection. In some embodiments, the AAV is AAV2, as well as variants AAV2.N53 and AAV2.N54, which are used in the examples of this disclosure. Chimeric AAVs, which may contain at least two AAV serotypes, are also contemplated. In some examples, 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. In some examples, only a small portion of the AAV is chimeric. For example, a preferred portion may comprise the capsid, VP1, VP2, or VP3 domain and / or Rep.In some examples, at least one of VP1, VP2, and VP3 has at least one amino acid substitution compared to the wild-type AAV capsid protein, which is otherwise equivalent. In some examples, 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 1 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3, for example, about 1 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, or about 20 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3. In some examples, VPs can be removed. For example, in some embodiments, the mutant AAV does not contain at least one of VP1, VP2, or VP3. Methods for modifying cells

[0169] In one embodiment, a method for modifying cells to generate engineered cells is also provided herein. Cells may refer to primary cultured cells, recombinant cells, or cell lines. In some examples, cells are packaging cells. Packaging cells may be, for example, HEK293 cells, HeLa cells, and Vero cells. Engineered cells may be primary cultured cells. In some examples, engineered cells may be eye cells. Preferred eye cells include, but are not limited to, photoreceptors, ganglion cells, RPE cells, amacrine cells, horizontal cells, Müller cells, and the like.

[0150]

[0170] 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, e.g., CaPO4 transfection or electroporation, and / or infection of cell lines with hybrid adenovirus / AAV vectors, e.g., human embryonic kidney cell line HEK293 (a human kidney cell line containing a functional adenovirus E1 gene that provides a transactivated E1 protein), are used. Suitable transfection methods include calcium phosphate coprecipitation, direct microinjection, electroporation, liposome-mediated gene transfer, and nucleic acid delivery using fast microlaunchers, all of which are known in the art.

[0151]

[0171] To engineer cells, multiple cells may be brought into contact with isolated, engineered polynucleotides. The contact step may include any duration, ranging from about 5 minutes to about 5 days. The contact step may last for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some examples, the contact step may last for 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, or up to about 5 days.

[0152]

[0172] In some examples, the supernatant of the packaging cell line is treated with PEG precipitation to concentrate the virus. In other examples, the virus can be concentrated using a centrifugation step. For example, the virus can be concentrated during centrifugation using a column. In some embodiments, precipitation is carried out at a temperature of about 4°C or lower (e.g., about 3°C, about 2°C, about 1°C, or about 1°C) for at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours, or at least about 24 hours. In some embodiments, recombinant AAV is isolated from the PEG precipitate supernatant by CsCl gradient separation after slow centrifugation. Slow centrifugation may be at about 4000 RPM, about 4500 RPM, about 5000 RPM, or about 6000 RPM for about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes. In some cases, recombinant AAV is isolated from the PEG precipitate supernatant by performing CsCl gradient separation after centrifugation at approximately 5000 RPM for approximately 30 minutes. In some cases, CsCl purification can be replaced with IDX gradient ultracentrifugation. The supernatant can be collected at approximately 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, or any time between these two points in time after transfection. The supernatant can also be purified, concentrated, or combined. For example, the concentration or viral titer can be determined by qPCR or silver staining.

[0153]

[0173] In one embodiment, a plurality of AAV particles (containing the engineered polynucleotides described herein) isolated from engineered cells are also provided. The viral titer is approximately 10 2 vp / mL, approx. 10 3 vp / mL, approx. 10 4 vp / mL, approx. 10 5 vp / mL, approx. 10 6 vp / mL, approx. 10 7 vp / mL, approx. 10 8 vp / mL, or up to approximately 10 9 It may be vp / mL. The viral titer is approximately 10 2GC / mL, approx. 10 3 GC / mL, approx. 10 4 GC / mL, approx. 10 5 GC / mL, approx. 10 6 GC / mL, approx. 10 7 GC / mL, approx. 10 8 GC / mL, or up to approximately 10 9 It can be GC / mL. In some cases, the viral titer is about 10 2 TU / mL, approximately 10 3 TU / mL, approximately 10 4 TU / mL, approximately 10 5 TU / mL, approximately 10 6 TU / mL, approximately 10 7 TU / mL, approximately 10 8 TU / mL, or up to approximately 10 9 It may be TU / mL. The optimal viral titer may vary depending on the type of cell being transduced. The viral range may be approximately 1000 MOI to 2000 MOI, approximately 1500 MOI to 2500 MOI, approximately 2000 MOI to 3000 MOI, approximately 3000 MOI to 4000 MOI, approximately 4000 MOI to 5000 MOI, approximately 5000 MOI to 6000 MOI, approximately 6000 MOI to 7000 MOI, approximately 7000 MOI to 8000 MOI, approximately 8000 MOI to 9000 MOI, and approximately 9000 MOI to 10,000 MOI. For example, to infect 1 million cells using MOI 10,000, you would need 10,000 × 1,000,000 = 10 10 Garbage collection is required.

[0154]

[0174] In some cases, multiple AAV particles can be formulated into a single-dose dosage form. Various formulations are intended for delivery to adults or children, but are not limited to 0.5 × 10⁻⁶. 9 vg, 1.0 × 10 9 vg, 1.0 × 10 10 , 1.0 × 10 11 vg, 3.0×10 11 vg, 6×10 11 vg, 8.0×10 11 vg, 1.0 × 10 12 vg, 1.0 × 10 13 vg, 1.0 × 1014 vg, 1.0 × 10 15 vg, or up to 1.5 × 10 15 Contains VG. The virus particle composition can be stored frozen or in a suitable container.

[0155]

[0175] The compositions and methods provided herein may be sufficient to enhance the delivery and / or expression of the biological product of interest 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 equivalent unmodified nucleic acids. In some examples, otherwise equivalent unmodified nucleic acids are nucleic acids encoding VEGF-Trap. In some cases, the modification was at least approximately 1x, 6x, 11x, 16x, 21x, 26x, 31x, 36x, 41x, 46x, 51x, 56x, 61x, 66x, 71x, 76x, 81x, 86x, 91x, 96x, 101x, 106x, 111x, 116x, 121x, 126x, 131x, 136x, 141x, 146x, 151x, 156x, 161x, 166x, 171x, 176x, 181x, 186x, This may be sufficient to enhance the delivery and / or expression of the target biologic by approximately 191 times, 196 times, 201 times, 206 times, 211 times, 216 times, 221 times, 226 times, 231 times, 236 times, 241 times, 246 times, 251 times, 256 times, 261 times, 266 times, 271 times, 276 times, 281 times, 286 times, 291 times, 296 times, 301 times, 306 times, 311 times, 316 times, 321 times, 326 times, 331 times, 336 times, 341 times, 346 times, or approximately 350 times more. In one embodiment, the increase in 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 blotting, Luminex, microscopy, imaging, and / or flow cytometry.

[0156]

[0176] The AAV virions in question may exhibit an increased infectivity of retinal cells (photoreceptors, ganglion cells, RPE cells, amacrine cells, horizontal cells, Müller cells, and similar) by at least 1-fold, at least 6-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or greater than 50-fold compared to the infectivity of retinal cells (photoreceptors, ganglion cells, RPE cells, amacrine cells, horizontal cells, Müller cells, and similar) by AAV virions otherwise containing equivalent WT AAV capsid proteins. Treatment method

[0177] This specification provides methods for treating diseases or conditions described herein. In some embodiments, the methods provide protection against the disease or condition. A method of treatment may include the step of introducing an engineered polynucleotide, an engineered polypeptide, an AAV vector containing an engineered polynucleotide, an AAV containing an engineered polynucleotide, a cell transduced to an AAV vector, a viral particle containing an engineered polynucleotide, a pharmaceutical composition, or a combination thereof, into a subject requiring treatment. Similarly, methods for treating a disease or condition are also provided, including the step of administering a pharmaceutical composition to a subject requiring treatment of the disease or condition. A pharmaceutical composition may include a sequence encoding a biologic comprising an engineered polynucleotide, an AAV vector containing an engineered polynucleotide, an AAV vector containing an engineered polynucleotide, a viral particle containing an engineered polynucleotide, or a combination thereof. In some embodiments, administration is by any preferred method of administration, including systemic administration (e.g., intravenous, intravitreal, subretinal, etc.). In some embodiments, the subject is human.

[0157]

[0178] In some embodiments, the method includes the step of treating a disease or condition in a subject requiring treatment of the disease or condition by administering to the subject a therapeutically effective amount of an engineered polynucleotide, engineered polypeptide, cell transduced with an engineered polynucleotide, or pharmaceutical composition described herein. In some embodiments, the method treats the disease or condition, and a single administration of the engineered polynucleotide, engineered polypeptide, cell transduced with an engineered polynucleotide, or pharmaceutical composition described herein cures the disease or condition. In some embodiments, the step of treating the disease or condition and administering the engineered polynucleotide, engineered polypeptide, cell transduced with an engineered polynucleotide, or pharmaceutical composition described herein does not involve daily administration. In some embodiments, the disease or condition includes eye diseases. Non-specific examples of eye diseases include ischemic syndrome of the eye, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color blindness, age-related macular degeneration (nAMD), geographic atrophy (GA), atrophic age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, traumatic glaucoma, Valdevie's syndrome, Best's disease, This may include choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-vertebral dystrophy, vertebral-rod dystrophy, Oguchi disease, autosomal dominant radial drusen (familial autosomal dominant drusen), blue vertebral monochromatic vision, or a combination thereof. In some embodiments, the disease or condition is neovascular glaucoma (NG). In some embodiments, the disease or condition is glaucoma. In some embodiments, the disease or condition is traumatic glaucoma.

[0158]

[0179] In another embodiment, the Specified Provision provides a pharmaceutical composition comprising an engineered polynucleotide, an engineered polypeptide, a vector, a viral particle, a cell, or a composition disclosed herein. In some embodiments, the pharmaceutical composition is formulated for administration to a subject requiring administration of the pharmaceutical composition via intrathecal, intraocular, intravitreous, retinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, subretinal, superchoroidal, intratumoral, lung, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray, intraluminal-GI route, or a combination 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 intravitreous, subretinal, or superchoroidal administration.

[0159]

[0180] In another embodiment, a method is provided herein that includes the step of contacting cells obtained from a subject with an engineered polynucleotide, an engineered polypeptide, a vector, a viral particle, a cell, a composition, or a pharmaceutical composition disclosed herein.

[0160]

[0181] In another embodiment, the Specified Provision provides a method for treating a disease or condition in a subject, comprising the step of administering to the subject an engineered polynucleotide, an engineered polypeptide, a vector, a viral particle, a cell, a composition, or a pharmaceutical composition as disclosed herein. In some embodiments, the administration step cures the disease or condition. In some embodiments, the administration step does not involve daily administration. In some embodiments, the administration step involves weekly administration, bi-weekly administration, monthly administration, bi-monthly administration, semi-annual administration, or annual administration, or a combination thereof. In some embodiments, the disease or condition includes eye diseases. In some embodiments, eye diseases include ischemic syndrome of the eye, proliferative retinopathy, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, color blindness, age-related macular degeneration (nAMD), geographic atrophy (GA), atrophic age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), Valdevie's syndrome, and Beth This includes ocular diseases such as ophthalmos, choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-vertebral dystrophy, vertebral-rod dystrophy, Oguchi disease, autosomal dominant radial drusen (familial autosomal dominant drusen), blue vertebral monochromatic vision, or combinations thereof. In some embodiments, ocular diseases include GA or dAMD.

[0161]

[0182] In another embodiment, this specification provides a method for treating a disease or condition in a subject, comprising the step of administering to the subject an 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 one or more expression cassettes. In some embodiments, the first angiogenesis inhibitor and the second angiogenesis inhibitor are operably linked. In some embodiments, the first angiogenesis inhibitor and the second angiogenesis inhibitor are covalently linked by a linker. In some embodiments, the first angiogenesis inhibitor includes a complement inhibitor, such as a complement 3 inhibitor. In some embodiments, the complement 3 inhibitor includes an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1 to 15. In some embodiments, the complement 3 inhibitor is encoded by a nucleic acid sequence that is at least 80% identical to any one of SEQ ID NOs: 20 to 33. In some embodiments, the first angiogenesis inhibitor includes an inhibitor of a membrane invasion complex (MAC), such as CD59. In some embodiments, CD59 contains an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 41-45. In some embodiments, the second angiogenesis inhibitor contains a natriuretic peptide. In some embodiments, the natriuretic peptide contains an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 61-72. In some embodiments, the second angiogenesis inhibitor contains collagen or a fragment thereof. In some embodiments, the second angiogenesis inhibitor contains endostatin or a fragment thereof. In some embodiments, the second angiogenesis inhibitor contains an amino acid sequence that is at least 80% identical to SEQ ID NOs. 51. In some embodiments, the second angiogenesis inhibitor contains a VEGF inhibitor. In some embodiments, the VEGF inhibitor contains an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 81-92.

[0162]

[0183] 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 comprising 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 comprising one of the amino acid sequences of SEQ ID NOs: 161-182 and SEQ ID NOs: 191-210. In some embodiments, the engineered AAV capsid comprises the amino acid sequence of SEQ ID NO: 169.

[0163]

[0184] In some embodiments, the method includes a single administration step to cure the disease or condition. In some embodiments, the administration step does not include daily administration. In some embodiments, the administration step includes weekly administration, bi-weekly administration, monthly administration, bi-monthly administration, semi-annual administration, or annual administration, or a combination thereof. In some embodiments, the disease or condition includes eye diseases. In some embodiments, eye diseases include ischemic syndrome of the eye, proliferative retinopathy, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, color blindness, age-related macular degeneration (nAMD), geographic atrophy (GA), atrophic age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), Valdevie's syndrome, Beth This includes ocular diseases such as ophthalmos, choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinolysis (XLRS), rod-vertebral dystrophy, vertebral-rod dystrophy, Oguchi disease, autosomal dominant radial drusen (familial autosomal dominant drusen), blue vertebral monochromatic vision, or combinations thereof. In some embodiments, the eye disease includes GA or dAMD. In some embodiments, the first and second angiogenesis inhibitors, when administered to a subject, inhibit angiogenesis in the subject.

[0164]

[0185] In some embodiments, the first or second angiogenesis inhibitor, upon administration to a subject, exhibits a reduction in angiogenesis inhibition in the subject compared to the inhibition of angiogenesis caused by a VEGF inhibitor. In such examples, the reduction in angiogenesis inhibition induced by the first or second angiogenesis inhibitor may be more therapeutically effective in treating a disease or condition. For example, the reduction in angiogenesis inhibition allows for the presence of blood vessels to transport and deliver the first or second angiogenesis inhibitor to the site associated with the disease or condition.

[0165]

[0186] In some embodiments, the step of administering a therapeutically effective amount of an engineered polynucleotide, engineered polypeptide, transduced cell with an engineered polynucleotide, or pharmaceutical composition described herein to a subject protects the subject from disease or condition. For example, the step of administering a therapeutically effective amount of an engineered polynucleotide, engineered polypeptide, transduced cell with an engineered polynucleotide, or pharmaceutical composition can protect the subject from developing a disease or condition resulting from injury. In some embodiments, the step of administering a therapeutically effective amount of an engineered polynucleotide, engineered polypeptide, transduced cell with an engineered polynucleotide, or pharmaceutical composition protects or promotes cell survival in the subject. In some embodiments, the step of administering a therapeutically effective amount of an engineered polynucleotide, engineered polypeptide, transduced cell with an engineered polynucleotide, or pharmaceutical composition protects or promotes cell survival in the eye in the subject. In some embodiments, the step of administering a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, cells transduced with an engineered polynucleotide, or a pharmaceutical composition protects or promotes the survival of retinal ganglion cells in a subject. In some embodiments, the step of administering a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, cells transduced with an engineered polynucleotide, or a pharmaceutical composition reduces intraocular pressure in a subject.

[0166]

[0187] In some embodiments, an engineered polynucleotide, an engineered polypeptide, an AAV vector containing an engineered polynucleotide, an AAV containing an engineered polynucleotide, a cell transduced from an AAV vector, or a pharmaceutical composition is administered at least once over a period of time (e.g., once every two days, twice a week, once a week, weekly, three times a month, twice a month, once a month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven 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, 100 times). In some embodiments, the administration described herein includes a single dose. In some embodiments, the administration described herein does not include daily administration.

[0167]

[0188] In some embodiments, the method includes the step of administering a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, an AAV vector containing an engineered polynucleotide, an AAV containing an engineered polynucleotide, cells transduced to an AAV vector, or a pharmaceutical composition in various dosage forms and routes, such as oral or topical administration. In some embodiments, the composition may be administered intravitreously, subretinally, suprachoroidally, parenterally, intravenously, subcutaneously, intramuscularly, intradermally, intraperitoneally, intracerebrally, subarachnoidally, intraocularly, intracisternally, ocularly, endothelially, topically, intranasally, intrapulmonaryly, rectally, intraarterially, intrathecally, by inhalation, intralesionally, intradermally, intradurally, intradermally, intracapsularly, intracardiacly, transtracheally, subepidermally, or intraspinally, for example, by injection or infusion. In some embodiments, the composition may be administered by absorption through the epithelium or mucocutaneous lining (e.g., oral mucosal, rectal, and intestinal mucosal administration). In some embodiments, the composition is delivered via multiple routes of administration.

[0168]

[0189] In some embodiments, the method includes administering an engineered polynucleotide, an engineered polypeptide, an AAV vector containing an engineered polynucleotide, an AAV containing an engineered polynucleotide, a cell transduced from an AAV vector, a viral particle containing an engineered polynucleotide, a pharmaceutical composition, or a combination thereof by intravenous infusion. In some embodiments, the engineered polynucleotide, an AAV vector containing an engineered polynucleotide, an AAV containing an engineered polynucleotide, a cell transduced from an AAV vector, a viral particle containing an engineered polynucleotide, a pharmaceutical composition, or a combination thereof is administered by slow, sustained infusion over a long period, for example, longer than 24 hours. In some embodiments, the engineered polynucleotide, an AAV vector containing an engineered polynucleotide, an AAV containing an engineered polynucleotide, a cell transduced from an AAV vector, a viral particle containing an engineered polynucleotide, a pharmaceutical composition, or a combination thereof is administered by intravenous injection or short-duration infusion. In some embodiments, engineered polynucleotides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced to the AAV vector, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof are administered via the vitreous pathway. In some embodiments, engineered polynucleotides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced to the AAV vector, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof may be administered topically, for example by direct injection of the agent into an organ, as a depot if necessary, or as a sustained-release formulation or implant.

[0169]

[0190] In some embodiments, engineered polynucleotides, engineered polypeptides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced from AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof may be administered in conjunction with other treatments, 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 from AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof may be administered before, during, or after the onset of a disease or condition, and the timing of administration of compositions containing therapeutic agents may vary. In some examples, compositions may be used as prophylactic agents and may be administered continuously to subjects susceptible to coronavirus or prone to developing coronavirus-related conditions or diseases (e.g., subjects for immunization or treatment). Prophylactic administration may reduce the probability of developing an infection, disease, or condition, or reduce the severity of an infection, disease, or condition.

[0170]

[0191] Engineered polynucleotides, engineered polypeptides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced from AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof may be administered to a subject before the onset of symptoms. In some embodiments, engineered polynucleotides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced from AAV vectors, viral particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof may be administered to a subject (e.g., a subject for immunization or treatment) after (e.g., as soon as possible) test results, such as test results providing a diagnosis, a test indicating the presence of coronavirus in the subject (e.g., a subject for immunization or treatment), or a test indicating disease progression, such as a decrease in blood oxygen levels. Therapeutic agents may be administered after the onset of a disease or condition is detected or suspected (e.g., as soon as practically possible). Therapeutic agents may be administered after potential exposure to the coronavirus (e.g., as soon as feasible), for example, after a subject (e.g., a subject for immunization or treatment) has come into contact with an infected subject, or after it is known that the subject has come into contact with an infectious subject.

[0171]

[0192] The actual dose levels of the activators of this disclosure (e.g., engineered polynucleotides or pharmaceutical compositions) may vary to obtain an amount of the activator that achieves the desired therapeutic response with respect to a particular subject, composition, and mode of administration without causing toxicity to the subject (e.g., a subject for immunization or a subject for treatment). The selected dose level may depend on a variety of pharmacokinetic factors, including the activity of the particular composition of this disclosure used, the route of administration, the time of administration, the elimination rate, the duration of treatment, other drugs, compounds, and / or materials used with the particular composition used, the age, sex, weight, condition, overall health, and prior medical history of the patient being treated, and similar factors known in the medical technology.

[0172]

[0193] Dosage regimens can be adjusted to provide the optimal desired response (e.g., therapeutic and / or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased depending on the urgency of the treatment situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions into unit dosing forms. As used herein, unit dosing forms refer to physically distinct units suitable as unit doses of a subject (e.g., a subject for immunization or a subject for treatment); each unit contains a predetermined amount of the activator calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications of the unit dosing forms in this disclosure may depend on (a) the unique characteristics of the activator and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art to synthesize such activators for the treatment of susceptibility in an individual. Dosages may be determined by reference to plasma or topical concentrations of cyclic polyribonucleotides or antibodies or their antigen-binding fragments. The dose may be determined by reference to the plasma or local concentration of the linear polyribonucleotide or antibody or its antigen-binding fragment.

[0173]

[0194] The engineered polynucleotides, engineered polypeptides, AAV vectors containing engineered polynucleotides, AAVs containing engineered polynucleotides, cells transduced to AAV vectors, virus particles containing engineered polynucleotides, pharmaceutical compositions, or combinations thereof described herein may be suitable unit-dose formulations for a single dose of a precise amount. In unit-dose formulations, the formulation may be divided into unit doses containing an appropriate amount of the composition. In unit-dose formulations, the formulation may be divided into unit doses containing an appropriate amount of one or more linear polyribonucleotides, antibodies, or antigen-binding fragments thereof, and / or therapeutic agents. The unit doses may be in the form of packaging containing separate amounts of the formulation. Non-limiting examples include packaged injectables, vials, and ampoules. The aqueous suspension compositions disclosed herein may be packaged in single-dose, non-reusable containers. Multiple-dose, reusable containers may be used, for example, with or without preservatives. The injectable formulations disclosed herein may be in unit-dose formulations, such as ampoules, or in multi-dose containers containing preservatives.

[0174]

[0195] In some cases, an increase in the level of biologics in the subject results in an increase of at least 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, or 500-fold, as determined by a diagnostic assay.

[0175]

[0196] Suitable diagnostic assays may include ocular diagnostic assays. Ocular diagnostic assays may include ophthalmic examinations, such as refraction tests, eye scans, optical coherence tomography, Farnsworth-Munsell 100 Hue tests, computer-aided imaging of the optic nerve head, and nerve fiber layer analysis (GDX, HRT, OCT), corneal topography, electroretinography (ERG), electrooculography (EOG), visual evoked potentials (VEP), visual evoked responses (VER), fluorescein angiography, optical coherence tomography (OCT), retinal photography, fundus photography, specular microscopy, Goldmann perimeter, Humphrey perimeter, FDT, Octopus perimeter, biometry / IOL calculations, A-scan, B-scan, and combinations thereof.

[0176]

[0197] In some cases, retinal examinations can be utilized. Non-limited methods for assessing retinal function and its changes include assessment of visual acuity (e.g., best corrected visual acuity [BCVA], gait, navigation, object detection, and discrimination), assessment of visual fields (e.g., static and dynamic perimetry), performance of clinical examinations (e.g., anterior and posterior segment slit-lamp examination), and assessment of electrophysiological responses to all wavelengths of light and dark (e.g., all forms of electroretinography (ERG) [whole field, multifocal, and pattern], 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-assisted scanning laser ophthalmoscope (AO-SLO), fluorescence and / or autofluorescence; measurement of eye movements and ocular motion (e.g., nystagmus, fixation preference, and stability); measurement of reported outcomes (patient-reported changes in visually and non-visually induced behaviors and activities, patient-reported outcomes [PRO]), questionnaire-based quality of life assessments, and measurement of daily activities and neurological function (e.g., magnetic resonance imaging (MRI)).

[0177]

[0198] In some embodiments, an engineered polynucleotide, an AAV vector containing an engineered polynucleotide, an AAV containing an engineered polynucleotide, a cell transduced to an AAV vector, a viral particle containing an engineered polynucleotide, a pharmaceutical composition, or a combination thereof is 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more, compared to an equivalent cell that has not been in contact with the engineered polynucleotide, the AAV vector containing an engineered polynucleotide, the AAV containing an engineered polynucleotide, or the pharmaceutical composition.

[0178]

[0199] In some embodiments, the treatment methods described herein can treat eye diseases. Relevant eye diseases and conditions may include, but are not limited to, blindness, color blindness, age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma, Valdevee-Wiedl syndrome, Best's disease, choroideremia, Leber congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-vertebral dystrophy, vertebral-rod dystrophy, Oguchi disease, autosomal dominant radial drusen (familial autosomal dominant drusen), and blue vertebral monochromatic color vision. In one embodiment, the eye disease or condition is AMD. AMD may be exudative AMD or atrophic AMD.

[0179]

[0200] In some cases, administration of a pharmaceutical composition is sufficient to reduce at least one symptom of a disease or condition, treat the disease or condition, and / or eliminate the disease or condition. In some cases, improvement in the disease or condition can be confirmed by one of the provided diagnostic assays. In other words, improvement can be obtained through a medical history taken of the treated subject. For example, the subject may tell their physician that their vision has improved compared to before administration of the drug in question. In other cases, in vivo animal models may be used to confirm the reduction of the disease or condition after treatment. Suitable animal models include mouse models, primate models, rat models, dog models, and similar models.

[0180]

[0201] The use of absolute or sequential terms, such as “will,” “will not,” “shall,” “shall not,” “must,” “must not,” “first,” “first,” “next,” “then,” “before,” “after,” “finally,” and “ultimately,” is illustrative and not intended to limit the scope of the embodiments disclosed herein.

[0181]

[0202] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Further, the terms "including", "includes", "having", "has", "together with", or variations thereof are used in any of the detailed description and / or claims to the extent that such terms are intended to be construed in a manner similar to the term "comprising".

[0182]

[0203] As used herein, the phrases "at least one", "one or more", and "and / or" are unrestricted expressions whose function is both conjunctive and disjunctive. For example, 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" each mean A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0183]

[0204] As used herein, "or" can refer to "and", "or", "and / or" and can be used both exclusively and inclusively. For example, the term "A or B" can refer to "A or B", "A but not B", "B but not A", and "A and B". In some instances, the context may indicate a particular meaning.

[0184]

[0205] Any system, method, software, and platform described herein is modular. Accordingly, terms such as "first" and "second" do not necessarily imply a priority, importance, or order of implementation.

[0185]

[0206] The term "about," when referring to a number or numerical range, means that the recited number or numerical range is an approximation within experimental variations (or within the statistical error range of experiments), and that the number or numerical range can vary, for example, from 1% to 15% of the recited number or numerical range. In the example, the term "about" refers to ±10% of the recited number or value.

[0186]

[0207] The terms "increased," "increasing," or "increase" are used herein to generally mean an increase in a statistically significant amount. In some embodiments, the terms "increased" or "increase" mean an increase of at least 10%, for example, at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase including up to and including 100%, or any increase between 10 and 100% compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or greater compared to a reference level.

[0187]

[0208] The terms “decreased,” “decreasing,” or “decreasing” generally mean a statistically significant decrease. In some embodiments, the terms “decreased” or “decreasing” mean a decrease of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% and 100% (e.g., a level that does not exist, or a level that is undetectable compared to a reference level), or any decrease between 10% and 100% compared to a reference level. In the context of markers or symptoms, these terms mean a statistically significant decrease of such a level. The decrease may be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or greater, and preferably decrease to a level that is acceptable as within the normal range for an individual without the disease in question.

[0188]

[0209] "AAV," "AAV construct," "recombinant AAV," or "AAV" refers to any known adeno-associated virus of any serotype, including AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, or scAAV, rh10, chimeric or hybrid AAV, or any combination, derivative, or variant thereof. AAVs are small, non-enveloped, single-stranded DNA viruses. They are non-pathogenic parvoviruses and may require helper viruses such as adenoviruses, herpes simplex viruses, vaccinia viruses, and CMV for replication. Wild-type AAVs are common in the general population and are not associated with any known pathogenicity. Hybrid AAVs are AAVs that contain the capsid protein of one AAV serotype and genomic material from another AAV serotype. A chimeric AAV comprises gene sequences and / or protein sequences derived from two or more AAV serotypes, and may include mutations made against the gene sequences of those two or more AAV serotypes. An exemplary chimeric AAV may include a chimeric AAV capsid, for example, a capsid protein having one or more regions of amino acids derived from two or more AAV serotypes. An AAV variant is an AAV that has one or more amino acid mutations in its genome or protein compared to its parent AAV, and one or more amino acid mutations in its capsid protein compared to its parent AAV. As used herein, AAV includes avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV, where primate AAV refers to an AAV that infects non-primates, and non-primate AAV refers to an AAV that infects non-primate animals, for example, avian AAV that infects birds. In some cases, wild-type AAV contains rep and cap genes, the rep gene being necessary for viral replication and the cap gene being necessary for the synthesis of the capsid protein. As used herein, the terms “recombinant AAV” and “rAAV” are interchangeable.

[0189]

[0210] 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 that are deleted in whole or in part, such as rep and / or cap genes, but which contain functional elements necessary for packaging and using the AAV virus for gene therapy. For example, functional terminal inverted repeat sequences or ITR sequences adjacent to open reading frames or cloned exogenous sequences are known to be important for the replication and packaging of AAV virions, but ITR sequences can be modified from the wild-type nucleotide sequence, including nucleotide insertions, deletions, or substitutions, so that the AAV is suitable for use in the embodiments described herein, such as gene therapy or gene delivery systems. In some embodiments, self-complementary vectors (sc), such as self-complementary AAV vectors, can be used, which can avoid the need for viral double-strand DNA synthesis and result in higher expression of transgene proteins. In some embodiments, AAV vectors may be generated to allow for the selection of optimal serotypes, promoters, and transgenes. In some examples, the vector may be a targeted or modified vector that selectively binds to or infects immune cells.

[0190]

[0211] The term “AAV virion” or “AAV virion” refers to a viral particle comprising a capsid containing at least one AAV capsid protein that capsids an AAV vector as described herein, the vector further comprising heterologous nucleic acid sequences or transgenes in some embodiments. The virion may be an engineered virion.

[0191]

[0212] The terms “control,” “host,” “individual,” and “patient” are used interchangeably herein and refer to animals, typically mammals. Any suitable mammal may be administered with the compositions described herein (e.g., engineered guide RNA) or treated by the methods described herein. The subject may be a vertebrate or an invertebrate. The subject may be an experimental animal. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and similar species), domesticated animals (e.g., dogs and cats), livestock (e.g., horses, cattle, goats, sheep, pigs), and experimental animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal may be of any age or any developmental stage (e.g., adult, teenager, child, infant, or fetus). The mammal may be male or female. In some embodiments, the subject is a human. The subject may be a patient. The subject may be suffering from a disease. The subject may exhibit symptoms of the disease. The subject may not exhibit symptoms of the disease, but still possesses the disease. The subject may be receiving medical care from a caregiver (for example, the subject is hospitalized and being treated by a doctor).

[0192]

[0213] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably in their broadest sense to refer to compounds of two or more subunit amino acids, amino acid analogs, or peptide mimetic compounds. The terms also encompass modified amino acid polymers, such as disulfide bond formation, glycosylation, lipid addition, acetylation, phosphorylation, or any other operation, such as conjugation with labeling components. As used herein, the term “amino acid” refers to any natural and / or unnatural or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptide mimetic compounds. Subunits may be linked by peptide bonds. In another embodiment, subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may constitute a protein or peptide sequence. As used herein, the term “amino acid” refers to any natural and / or unnatural or synthetic amino acids, including glycine and its D and L optical isomers, amino acid analogs, and peptide mimetic compounds. As used herein, the term “fusion protein” refers to a protein consisting of one or more naturally occurring or recombinantly produced protein domains, each generally performing a different function. In this context, the term “linker” refers to a protein fragment used to link these domains together, to preserve the three-dimensional structure of the fusion protein domains as needed, and / or to prevent undesirable interactions between the fusion protein domains, which may include their respective functions.

[0193]

[0214] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, meaning that when the two sequences are aligned, they will have the same percentage of bases or amino acids. Sequence similarity can be determined in several different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, which is available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package.

[0194]

[0215] While preferred embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. The present invention is not intended to be limited by the specific embodiments provided herein. While the present invention has been described with reference to the above specification, the descriptions and illustrations of embodiments herein are not intended to be constrained. Numerous variations, alterations, and substitutions will be conceivable to those skilled in the art without departing from the present invention. Furthermore, it is understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions described herein, depending on a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in practice of the present invention. Therefore, the present invention is intended to encompass any such alternatives, alterations, variations, or equivalents. The following claims define the scope of the present invention, and methods and structures within the scope of these claims and their equivalents are intended to be included therein. Embodiment

[0216] Embodiment 1: An engineered polynucleotide comprising one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor.

[0195]

[0217] Embodiment 2: An engineered polynucleotide comprising a complement 3 (C3) inhibitor or C3 degradation fragment; and one or more expression cassettes encoding a natriuretic peptide.

[0196]

[0218] Embodiment 3: An engineered polynucleotide according to Embodiment 2, wherein a complement 3 inhibitor or C3 degradation fragment and a natriuretic peptide are covalently linked by a linker.

[0197]

[0219] Embodiment 4: An engineered polynucleotide according to Embodiment 2 or 3, wherein the complement 3 inhibitor comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1 to 15.

[0198]

[0220] Embodiment 5: An engineered polynucleotide according to any one of Embodiments 2 to 4, wherein the complement 3 inhibitor comprises an amino acid sequence including one of SEQ ID NOs: 1 to 15.

[0199]

[0221] Embodiment 6: An engineered polynucleotide according to any one of Embodiments 2 to 5, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence that is at least 80% identical to any one of SEQ ID NOs. 20 to 33.

[0200]

[0222] Embodiment 7: An engineered polynucleotide according to any one of Embodiments 2 to 6, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence comprising any one of SEQ ID NOs: 20 to 33.

[0201]

[0223] Embodiment 8: An engineered polynucleotide according to any one of Embodiments 2 to 7, wherein the natriuretic peptide comprises a C-type natriuretic peptide (CNP).

[0224] Embodiment 9: An engineered polynucleotide according to any one of Embodiments 2 to 8, wherein a natriuretic peptide is covalently linked to an antibody or a fragment thereof.

[0202]

[0225] Embodiment 10: The engineered polynucleotide according to Embodiment 9, wherein the antibody or fragment thereof contains a fragment crystallizable (Fc) region.

[0226] Embodiment 11: The engineered polynucleotide according to any one of Embodiments 8 to 10, wherein the natriuretic peptide contains an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 61 to 72.

[0203]

[0227] Embodiment 12: The engineered polynucleotide according to any one of Embodiments 8 to 11, wherein the natriuretic peptide contains an amino acid sequence that is any one of SEQ ID NOs: 61 to 72.

[0204]

[0228] Embodiment 13: The engineered polynucleotide according to Embodiment 1, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are each encoded by one of the one or more expression cassettes.

[0205]

[0229] Embodiment 14: The engineered polynucleotide according to Embodiment 1 or 13, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are encoded by one of the one or more expression cassettes.

[0206]

[0230] Embodiment 15: The engineered polynucleotide according to any one of Embodiments 1 or 13 to 14, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are operably linked.

[0207]

[0231] Embodiment 16: The engineered polynucleotide according to any one of Embodiments 1 or 13 to 15, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are covalently connected by a linker.

[0208]

[0232] Embodiment 17: The engineered polynucleotide according to any one of Embodiments 1 or 13 to 16, wherein the first angiogenesis inhibitor contains a complement inhibitor.

[0233] Embodiment 18: An engineered polynucleotide according to Embodiment 1 or any one of Embodiments 13-17, wherein the complement inhibitor comprises a complement 3 inhibitor or a C3 degradation fragment.

[0209]

[0234] Embodiment 19: An engineered polynucleotide according to Embodiment 1 or any one of Embodiments 13-18, wherein the complement 3 inhibitor comprises an amino acid sequence that is at least 80% identical to any one of Sequence IDs 1-15.

[0210]

[0235] Embodiment 20: An engineered polynucleotide according to Embodiment 1 or any one of Embodiments 13-19, wherein the complement 3 inhibitor comprises an amino acid sequence including one of SEQ ID NOs: 1-15.

[0211]

[0236] Embodiment 21: An engineered polynucleotide according to Embodiment 1 or any one of Embodiments 13-20, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence that is at least 80% identical to any one of Sequence IDs 20-33.

[0212]

[0237] Embodiment 22: An engineered polynucleotide according to Embodiment 1 or any one of Embodiments 13-21, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence comprising any one of Sequence IDs 20-33.

[0213]

[0238] Embodiment 23: An engineered polynucleotide according to Embodiment 1 or any one of Embodiments 13-22, wherein the first angiogenesis inhibitor comprises an inhibitor of membrane invasion complex (MAC).

[0214]

[0239] Embodiment 24: An engineered polynucleotide according to Embodiment 1 or any one of Embodiments 13-23, wherein the MAC inhibitor comprises CD59.

[0240] Embodiment 25: An engineered polynucleotide according to Embodiment 1 or any one of Embodiments 13-24, wherein CD59 comprises an amino acid sequence that is at least 80% identical to any one of Sequence IDs 41-45.

[0215]

[0241] Embodiment 26: An engineered polynucleotide according to Embodiment 1 or any one of Embodiments 13-25, wherein CD59 comprises an amino acid sequence which is one of SEQ ID NOs. 41-45.

[0216]

[0242] Embodiment 27: An engineered polynucleotide according to Embodiment 1 or any one of Embodiments 13-26, wherein the second angiogenesis inhibitor comprises an inhibitor of membrane invasion complex (MAC).

[0217]

[0243] Embodiment 28: An engineered polynucleotide according to Embodiment 1 or any one of Embodiments 13-27, wherein the MAC inhibitor comprises CD59.

[0244] Embodiment 29: An engineered polynucleotide according to Embodiment 1 or any one of Embodiments 13-28, wherein CD59 comprises an amino acid sequence that is at least 80% identical to any one of Sequence IDs 41-45.

[0218]

[0245] Embodiment 30: An engineered polynucleotide according to Embodiment 1 or any one of Embodiments 13-29, wherein CD59 comprises an amino acid sequence which is one of SEQ ID NOs. 41-45.

[0219]

[0246] Embodiment 31: An engineered polynucleotide according to Embodiment 1 or any one of Embodiments 13-30, wherein the second angiogenesis inhibitor comprises a natriuretic peptide.

[0247] Embodiment 32: The engineered polynucleotide according to Embodiment 31, wherein the natriuretic peptide comprises a C-type natriuretic peptide (CNP).

[0220]

[0248] Embodiment 33: An engineered polynucleotide according to Embodiment 31 or 32, wherein a natriuretic peptide is covalently linked to an antibody or a fragment thereof.

[0249] Embodiment 34: An engineered polynucleotide according to Embodiment 33, wherein the antibody or its fragment comprises a fragment crystallizable (Fc) region.

[0221]

[0250] Embodiment 35: An engineered polynucleotide according to any one of Embodiments 31 to 34, wherein the natriuretic peptide comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 61 to 72.

[0222]

[0251] Embodiment 36: An engineered polynucleotide according to any one of Embodiments 31 to 35, wherein the natriuretic peptide comprises an amino acid sequence which is one of SEQ ID NOs. 61 to 72.

[0223]

[0252] Embodiment 37: An engineered polynucleotide according to any one of Embodiments 1, 13 to 36, wherein the second angiogenesis inhibitor comprises collagen or a fragment thereof.

[0253] Embodiment 38: An engineered polynucleotide according to any one of Embodiments 1, 13 to 37, wherein the second angiogenesis inhibitor comprises endostatin or a fragment thereof.

[0224]

[0254] Embodiment 39: An engineered polynucleotide according to any one of Embodiments 1, 13 to 38, wherein the second angiogenesis inhibitor comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 51.

[0225]

[0255] Embodiment 40: An engineered polynucleotide according to any one of Embodiments 1, 13 to 39, wherein the second angiogenesis inhibitor comprises the amino acid sequence of Sequence ID No. 51.

[0226]

[0256] Embodiment 41: An engineered polynucleotide according to any one of Embodiments 1, 13 to 40, wherein the second angiogenesis inhibitor comprises a VEGF inhibitor.

[0257] Embodiment 42: The engineered polynucleotide according to Embodiment 41, wherein the VEGF inhibitor comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 81-92.

[0227]

[0258] Embodiment 43: An engineered polynucleotide according to Embodiment 41 or 42, wherein the VEGF inhibitor comprises an amino acid sequence which is one of SEQ ID NOs: 81-92.

[0228]

[0259] Embodiment 44: An engineered polynucleotide according to any one of Embodiments 3 to 43, wherein the linker includes a cleavable linker.

[0260] Embodiment 45: The engineered polynucleotide according to Embodiment 44, wherein the cleavable linker comprises a furin protease linker.

[0229]

[0261] Embodiment 46: An engineered polynucleotide according to any one of Embodiments 1 to 45, further encoding a third angiogenic inhibitor.

[0262] Embodiment 47: An engineered polynucleotide according to any one of Embodiments 2 to 46, wherein the C3 degradation fragment comprises C3a, C3b, iC3b, C3f, C3c, C3d, C3g, or a combination thereof.

[0230]

[0263] Embodiment 48: An engineered polynucleotide according to any one of Embodiments 1 to 47, comprising a viral vector.

[0264] Embodiment 49: The viral vector is an engineered polynucleotide as described in Embodiment 48, comprising an AAV vector.

[0231]

[0265] Embodiment 50: An engineered polynucleotide as in Embodiment 49, wherein the AAV vector comprises an AAV serotype including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof.

[0232]

[0266] Embodiment 51: An engineered polynucleotide according to Embodiment 49 or 50, wherein the AAV vector is an AAV2 vector.

[0267] Embodiment 52: An engineered polynucleotide according to any one of Embodiments 49-51, wherein the AAV vector encodes an engineered AAV capsid.

[0233]

[0268] Embodiment 53: The engineered polynucleotide according to Embodiment 52, wherein the engineered AAV capsid comprises one amino acid sequence from SEQ ID NOs. 161-182 and SEQ ID NOs. 191-210.

[0234]

[0269] Embodiment 54: An engineered polynucleotide according to Embodiment 52 or 53, wherein the engineered AAV capsid comprises the amino acid sequence of SEQ ID NO: 169.

[0270] Embodiment 55: An engineered polynucleotide according to any one of Embodiments 1 to 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 a CNP.

[0235]

[0271] Embodiment 56: An engineered polynucleotide according to any one of Embodiments 1 to 55, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises CNP36.

[0236]

[0272] Embodiment 57: An engineered polynucleotide according to any one of Embodiments 1 to 56, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises Fc-CNP36.

[0237]

[0273] Embodiment 58: An engineered polynucleotide according to any one of Embodiments 1 to 57, further encoding a third angiogenic inhibitor.

[0274] Embodiment 59: The engineered polynucleotide according to Embodiment 58, wherein the third angiogenesis inhibitor comprises an inhibitor of membrane invasion complex (MAC).

[0238]

[0275] Embodiment 60: The engineered polynucleotide according to Embodiment 59, wherein the MAC inhibitor comprises CD59.

[0276] Embodiment 61: An engineered polynucleotide according to any one of Embodiments 58-60, encoding a protease site adjacent to a second angiogenesis inhibitor and a third angiogenesis inhibitor.

[0239]

[0277] Embodiment 62: An engineered polynucleotide according to any one of Embodiments 1 to 61, wherein the first angiogenesis inhibitor comprises a CD59 inhibitor and the second angiogenesis inhibitor comprises CNP.

[0240]

[0278] Embodiment 63: An engineered polynucleotide according to any one of Embodiments 1 to 62, wherein the first angiogenesis inhibitor comprises CD59 and the second angiogenesis inhibitor comprises CNP36.

[0241]

[0279] Embodiment 64: An engineered polynucleotide according to any one of Embodiments 1 to 63, wherein the first angiogenesis inhibitor comprises CD59 and the second angiogenesis inhibitor comprises Fc-CNP36.

[0242]

[0280] Embodiment 65: An engineered polynucleotide according to any one of Embodiments 1 to 64, wherein the first angiogenesis inhibitor comprises CD59 and the second angiogenesis inhibitor comprises a complement 3 inhibitor fused to Fc-CNP36.

[0243]

[0281] Embodiment 66: An engineered polynucleotide according to any one of Embodiments 1 to 65, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor and the second angiogenesis inhibitor comprises an endostatin.

[0244]

[0282] Embodiment 67: An engineered polynucleotide according to any one of Embodiments 1 to 66, encoding an Fc region adjacent to a first angiogenesis inhibitor and a second angiogenesis inhibitor.

[0245]

[0283] Embodiment 68: An engineered polynucleotide according to any one of Embodiments 1 to 67, wherein the first angiogenesis inhibitor comprises a VEGF inhibitor and the second angiogenesis inhibitor comprises a complement 3 inhibitor.

[0246]

[0284] Embodiment 69: An engineered polynucleotide according to any one of Embodiments 1 to 68, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises Fc-CNP36, and the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.

[0247]

[0285] Embodiment 70: An engineered polynucleotide according to any one of Embodiments 1 to 69, further encoding a protease site adjacent to the second and third angiogenesis inhibitors.

[0248]

[0286] Embodiment 71: An engineered polynucleotide according to any one of Embodiments 1 to 70, wherein the protease moiety comprises a furin protease moiety.

[0287] Embodiment 72: The engineered polynucleotide according to any one of Embodiments 1 to 71, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises Fc-CNP36, and the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.

[0249]

[0288] Embodiment 73: An engineered polynucleotide according to any one of Embodiments 1 to 72, wherein the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises a complement 3 inhibitor, and the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising Fc-CNP36.

[0250]

[0289] Embodiment 74: The engineered polynucleotide according to any one of Embodiments 1 to 73, wherein the first angiogenesis inhibitor comprises CD59, and the second angiogenesis inhibitor comprises endostatin, and the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor.

[0251]

[0290] Embodiment 75: The engineered polynucleotide according to any one of Embodiments 1 to 74, wherein the first angiogenesis inhibitor comprises CD59, the second angiogenesis inhibitor comprises a VEGF inhibitor, and the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor.

[0252]

[0291] Embodiment 76: The engineered polynucleotide according to any one of Embodiments 1 to 75, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor, the second angiogenesis inhibitor comprises an endostatin, and the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.

[0253]

[0292] Embodiment 77: An engineered polynucleotide according to any one of Embodiments 1 to 76, wherein the first angiogenesis inhibitor, the second angiogenesis inhibitor, and the third angiogenesis inhibitor are not VEGF inhibitors.

[0254]

[0293] Embodiment 78: An engineered polynucleotide according to any one of Embodiments 1 to 77, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor inhibit angiogenesis in the subject when administered to the subject.

[0255]

[0294] Embodiment 79: An engineered polynucleotide according to any one of Embodiments 1 to 78, wherein when the first or second angiogenesis inhibitor is administered to a subject, it exhibits a reduction in inhibition of angiogenesis in the subject compared to the inhibition of angiogenesis caused by the VEGF inhibitor.

[0256]

[0295] Embodiment 80: An engineered polypeptide comprising a first angiogenesis inhibitor and a second angiogenesis inhibitor.

[0296] Embodiment 81: The engineered polypeptide according to Embodiment 80, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are covalently linked by a linker.

[0257]

[0297] Embodiment 82: An engineered polypeptide according to any one of Embodiments 80 to 81, wherein the first angiogenic inhibitor comprises a complement inhibitor.

[0298] Embodiment 83: An engineered polypeptide according to any one of Embodiments 80 to 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.

[0258]

[0299] Embodiment 84: An engineered polypeptide according to any one of Embodiments 80 to 83, wherein the complement 3 inhibitor comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1 to 15.

[0259]

[0300] Embodiment 85: An engineered polypeptide according to any one of Embodiments 80 to 84, wherein the complement 3 inhibitor comprises an amino acid sequence including one of SEQ ID NOs: 1 to 15.

[0260]

[0301] Embodiment 86: An engineered polypeptide according to any one of Embodiments 80 to 85, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence that is at least 80% identical to any one of SEQ ID NOs. 20 to 33.

[0261]

[0302] Embodiment 87: An engineered polypeptide according to any one of Embodiments 80 to 86, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence comprising any one of SEQ ID NOs. 20 to 33.

[0262]

[0303] Embodiment 88: An engineered polypeptide according to any one of Embodiments 80-87, wherein the first angiogenesis inhibitor comprises an inhibitor of membrane invasion complex (MAC).

[0304] Embodiment 89: An engineered polypeptide according to any one of Embodiments 80 to 88, wherein the MAC inhibitor comprises CD59.

[0263]

[0305] Embodiment 90: An engineered polypeptide according to any one of Embodiments 80 to 89, wherein CD59 comprises an amino acid sequence that is at least 80% identical to any one of Sequence IDs 41 to 45.

[0264]

[0306] Embodiment 91: An engineered polypeptide according to any one of Embodiments 80 to 90, wherein CD59 comprises an amino acid sequence which is one of SEQ ID NOs. 41 to 45.

[0265]

[0307] Embodiment 92: An engineered polypeptide according to any one of Embodiments 80 to 91, wherein the second angiogenesis inhibitor comprises a natriuretic peptide.

[0308] Embodiment 93: An engineered polypeptide according to any one of Embodiments 80 to 92, wherein a natriuretic peptide is covalently linked to an antibody or a fragment thereof.

[0266]

[0309] Embodiment 94: An engineered polypeptide according to any one of Embodiments 80 to 93, wherein the antibody or a fragment thereof comprises a fragment crystallizable (Fc) region.

[0310] Embodiment 95: An engineered polypeptide according to any one of Embodiments 80 to 94, wherein the natriuretic peptide comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 61 to 72.

[0267]

[0311] Embodiment 96: An engineered polypeptide according to any one of Embodiments 80 to 95, wherein the natriuretic peptide comprises an amino acid sequence which is one of SEQ ID NOs. 61 to 72.

[0268]

[0312] Embodiment 97: An engineered polypeptide according to any one of Embodiments 80 to 96, wherein the second angiogenesis inhibitor comprises collagen or a fragment thereof.

[0313] Embodiment 98: An engineered polypeptide according to any one of Embodiments 80 to 97, wherein the second angiogenesis inhibitor comprises endostatin or a fragment thereof.

[0269]

[0314] Embodiment 99: An engineered polypeptide according to any one of Embodiments 80 to 98, wherein the second angiogenesis inhibitor comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 51.

[0270]

[0315] Embodiment 100: An engineered polypeptide according to any one of Embodiments 80 to 99, wherein the second angiogenesis inhibitor comprises the amino acid sequence of SEQ ID NO: 51.

[0316] Embodiment 101: An engineered polypeptide according to any one of Embodiments 80 to 100, wherein the second angiogenesis inhibitor comprises a VEGF inhibitor.

[0271]

[0317] Embodiment 102: An engineered polypeptide according to any one of Embodiments 80 to 101, wherein the VEGF inhibitor comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 81 to 92.

[0272]

[0318] Embodiment 103: An engineered polypeptide according to any one of Embodiments 80 to 102, wherein the VEGF inhibitor comprises an amino acid sequence which is any one of SEQ ID NOs: 81 to 92.

[0273]

[0319] Embodiment 104: An engineered polypeptide according to any one of Embodiments 80 to 103, wherein the linker includes a severable linker.

[0320] Embodiment 105: An engineered polypeptide according to any one of Embodiments 80 to 104, wherein the cleavable linker comprises a furin protease linker.

[0274]

[0321] Embodiment 106: An engineered polypeptide according to any one of the prior embodiments 80-105, further encoding a third angiogenic inhibitor.

[0322] Embodiment 107: The engineered polypeptide according to Embodiment 106, wherein the third angiogenesis inhibitor is different from the first and second angiogenesis inhibitors.

[0275]

[0323] Embodiment 108: A vector comprising an engineered polynucleotide as described in any one of Embodiments 1 to 79.

[0324] Embodiment 109: A vector encoding an engineered polypeptide as described in any one of Embodiments 80 to 107.

[0276]

[0325] Embodiment 110: A vector according to Embodiment 108 or 109 that encodes an AAV capsid.

[0326] Embodiment 111: A vector according to any one of Embodiments 108 to 110, wherein the AAV capsid comprises an engineered AAV capsid.

[0277]

[0327] Embodiment 112: A vector according to any one of Embodiments 108 to 111, wherein the modified AAV capsid comprises an engineered AAV2 capsid.

[0328] Embodiment 113: A viral particle comprising an engineered polynucleotide as described in any one of Embodiments 1 to 79, or a vector as described in any one of Embodiments 108 to 112.

[0278]

[0329] Embodiment 114: The viral particle according to Embodiment 113, comprising an AAV capsid.

[0330] Embodiment 115: A viral particle according to any one of Embodiments 113 to 114, wherein the AAV capsid comprises an engineered AAV capsid.

[0279]

[0331] Embodiment 116: A viral particle according to any one of Embodiments 113 to 115, wherein the modified AAV capsid comprises an engineered AAV2 capsid.

[0332] Embodiment 117: A cell comprising an engineered polynucleotide according to any one of Embodiments 1 to 79, an engineered polypeptide according to any one of Embodiments 80 to 107, a vector according to any one of Embodiments 108 to 112, or a viral particle according to any one of Embodiments 113 to 116.

[0280]

[0333] Embodiment 118: A composition comprising a complement 3 inhibitor, or a C3 degradation fragment containing C3a, C3b, iC3b, C3f, C3c, C3d, C3g, or a combination thereof; and a natriuretic peptide.

[0281]

[0334] Embodiment 119: The composition according to Embodiment 118, wherein the natriuretic peptide comprises C-type natriuretic peptide (CNP).

[0335] Embodiment 120: The composition according to any one of Embodiments 118 to 119, wherein a natriuretic peptide is covalently linked to an antibody or a fragment thereof.

[0282]

[0336] Embodiment 121: The composition according to any one of Embodiments 118 to 120, wherein the antibody or a fragment thereof comprises a fragment crystallizable (Fc) region.

[0337] Embodiment 122: The composition according to any one of Embodiments 118 to 121, wherein the natriuretic peptide comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 61 to 72.

[0283]

[0338] Embodiment 123: The composition according to any one of Embodiments 118 to 122, wherein the natriuretic peptide comprises an amino acid sequence which is one of SEQ ID NOs. 61 to 72.

[0284]

[0339] Embodiment 124: The composition according to any one of Embodiments 118 to 123, further comprising CD59, endostatin, a VEGF inhibitor, or a combination thereof.

[0340] Embodiment 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 membrane invasion complex (MAC).

[0285]

[0341] Embodiment 126: The composition according to any one of Embodiments 118 to 125, wherein the MAC inhibitor comprises CD59.

[0342] Embodiment 127: The composition according to any one of Embodiments 118 to 126, wherein CD59 comprises an amino acid sequence that is at least 80% identical to any one of Sequence IDs 41 to 45.

[0286]

[0343] Embodiment 128: The composition according to any one of Embodiments 118 to 127, wherein CD59 comprises an amino acid sequence which is one of SEQ ID NOs. 41 to 45.

[0344] Embodiment 129: The composition according to any one of Embodiments 118 to 128, further comprising a natriuretic peptide, an endostatin, a VEGF inhibitor, or a combination thereof.

[0287]

[0345] Embodiment 130: A composition comprising a complement 3 inhibitor, or a C3 degradation fragment containing C3a, C3b, iC3b, C3f, C3c, C3d, C3g, or a combination thereof; and collagen or a fragment thereof.

[0288]

[0346] Embodiment 131: A composition according to any one of Embodiments 118 to 130, wherein collagen or a fragment thereof comprises endostatin or a fragment thereof.

[0347] Embodiment 132: The composition according to any one of Embodiments 118 to 131, wherein the endostatin or a fragment thereof comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 51.

[0289]

[0348] Embodiment 133: The composition according to any one of Embodiments 118 to 132, wherein the endostatin or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 51.

[0349] Embodiment 134: The composition according to any one of Embodiments 118 to 133, further comprising a natriuretic peptide, CD59, a VEGF inhibitor, or a combination thereof.

[0290]

[0350] Embodiment 135: A composition comprising a complement 3 inhibitor, or a C3 degradation fragment containing C3a, C3b, iC3b, C3f, C3c, C3d, C3g, or a combination thereof; and a VEGF inhibitor.

[0291]

[0351] Embodiment 136: The composition according to any one of Embodiments 118 to 135, wherein the VEGF inhibitor comprises an amino acid sequence that is at least 80% identical to any one of Sequence IDs 81 to 92.

[0292]

[0352] Embodiment 137: The composition according to any one of Embodiments 118 to 136, wherein the VEGF inhibitor comprises an amino acid sequence which is any one of SEQ ID NOs: 81 to 92.

[0353] Embodiment 138: A composition according to any one of Embodiments 118 to 137, further comprising a natriuretic peptide, CD59, endostatin, or a combination thereof.

[0293]

[0354] Embodiment 139: The composition according to any one of Embodiments 118 to 138, wherein the complement 3 inhibitor comprises an amino acid sequence that is at least 80% identical to any one of Sequence ID No. 1 to 15.

[0294]

[0355] Embodiment 140: The composition according to any one of Embodiments 118 to 139, wherein the complement 3 inhibitor comprises an amino acid sequence including any one of SEQ ID NOs: 1 to 15.

[0356] Embodiment 141: The composition according to any one of Embodiments 118 to 140, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence that is at least 80% identical to any one of Sequence IDs 20 to 33.

[0295]

[0357] Embodiment 142: The composition according to any one of Embodiments 118 to 141, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence comprising any one of Sequence IDs 20 to 33.

[0296]

[0358] Embodiment 143: A composition comprising CD59 and a natriuretic peptide.

[0359] Embodiment 144: The composition according to any one of Embodiments 118 to 143, wherein the natriuretic peptide comprises C-type natriuretic peptide (CNP).

[0297]

[0360] Embodiment 145: The composition according to any one of Embodiments 118 to 144, wherein a natriuretic peptide is covalently linked to an antibody or a fragment thereof.

[0361] Embodiment 146: The composition according to any one of Embodiments 118 to 145, wherein the antibody or a fragment thereof comprises a fragment crystallizable (Fc) region.

[0298]

[0362] Embodiment 147: The composition according to any one of Embodiments 118 to 146, wherein the natriuretic peptide comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 61 to 72.

[0299]

[0363] Embodiment 148: The composition according to any one of Embodiments 118 to 147, wherein the natriuretic peptide comprises an amino acid sequence which is one of SEQ ID NOs. 61 to 72.

[0300]

[0364] Embodiment 149: The composition according to any one of Embodiments 143 to 148, further comprising a complement 3 inhibitor, an endostatin, a VEGF inhibitor, or a combination thereof.

[0365] Embodiment 150: A composition comprising CD59 and collagen or a fragment thereof.

[0301]

[0366] Embodiment 151: A composition according to any one of Embodiments 118 to 150, wherein collagen or a fragment thereof comprises endostatin or a fragment thereof.

[0367] Embodiment 152: The composition according to any one of Embodiments 118 to 151, wherein the endostatin or a fragment thereof comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 51.

[0302]

[0368] Embodiment 153: The composition according to any one of Embodiments 118 to 152, wherein the endostatin or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 51.

[0369] Embodiment 154: A composition according to any one of Embodiments 118 to 153, further comprising a natriuretic peptide, a complement 3 inhibitor, a VEGF inhibitor, or a combination thereof.

[0303]

[0370] Embodiment 155: A composition comprising CD59 and a VEGF inhibitor.

[0371] Embodiment 156: The composition according to any one of Embodiments 118 to 155, wherein the VEGF inhibitor comprises an amino acid sequence that is at least 80% identical to any one of Sequence IDs 81 to 92.

[0304]

[0372] Embodiment 157: The composition according to any one of Embodiments 118 to 156, wherein the VEGF inhibitor comprises an amino acid sequence which is one of SEQ ID NOs: 81 to 92.

[0373] Embodiment 158: A composition according to any one of Embodiments 118 to 157, further comprising a natriuretic peptide, a complement 3 inhibitor, an endostatin, or a combination thereof.

[0305]

[0374] Embodiment 159: The composition according to any one of Embodiments 118 to 158, wherein CD59 comprises an amino acid sequence that is at least 80% identical to any one of Sequence IDs 41 to 45.

[0306]

[0375] Embodiment 160: The composition according to any one of Embodiments 118 to 159, wherein CD59 comprises an amino acid sequence which is one of SEQ ID NOs. 41 to 45.

[0376] Embodiment 161: A pharmaceutical composition comprising an engineered polynucleotide according to any one of Embodiments 1 to 79, an engineered polypeptide according to any one of Embodiments 80 to 107, a vector according to any one of Embodiments 108 to 112, a viral particle according to any one of Embodiments 113 to 116, a cell according to Embodiment 117, or a composition according to any one of Embodiments 118 to 160.

[0307]

[0377] Embodiment 162: The pharmaceutical composition according to Embodiment 161, formulated for administration to a subject requiring administration of the pharmaceutical composition via intrathecal cavity, intraocular, intravitreous, retinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, subretinal, superchoroidal, intratumoral, lung, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray, intraluminal-GI route, or a combination thereof.

[0308]

[0378] Embodiment 163: A pharmaceutical composition according to any one of Embodiments 160 to 162, formulated for intravitreous, subretinal, or choroidal administration.

[0379] Embodiment 164: A method comprising contacting cells obtained from a subject with an engineered polynucleotide described in any one of Embodiments 1 to 79, an engineered polypeptide described in any one of Embodiments 80 to 107, a vector described in any one of Embodiments 108 to 112, a viral particle described in any one of Embodiments 113 to 116, a cell described in Embodiment 117, a composition described in any one of Embodiments 118 to 160, or a pharmaceutical composition described in any one of Embodiments 161 to 163.

[0309]

[0380] Embodiment 165: A method for treating a disease or condition in a subject, comprising the step of administering to the subject an engineered polynucleotide according to any one of Embodiments 1 to 79, an engineered polypeptide according to any one of Embodiments 80 to 107, a vector according to any one of Embodiments 108 to 112, a viral particle according to any one of Embodiments 113 to 116, a cell according to Embodiment 117, a composition according to any one of Embodiments 118 to 160, or a pharmaceutical composition according to any one of Embodiments 161 to 163.

[0310]

[0381] Embodiment 166: The method according to either Embodiment 164 or 165, wherein a single administration step cures a disease or condition.

[0382] Embodiment 167: The method according to any one of Embodiments 164 to 166, wherein the administration step does not include daily administration.

[0311]

[0383] Embodiment 168: The method according to any one of Embodiments 164 to 167, wherein the administration step includes weekly administration, bi-weekly administration, monthly administration, bi-monthly administration, semi-annual administration, or annual administration, or a combination thereof.

[0312]

[0384] Embodiment 169: The method according to any one of Embodiments 164 to 168, wherein the disease or condition includes an eye disease.

[0385] Embodiment 170: Eye diseases include ischemic syndrome of the eye, proliferative retinopathy, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, color blindness, age-related macular degeneration (nAMD), geographic atrophy (GA), atrophic age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), Valdevie's syndrome, Best's disease, colloideremia, and leva The method according to any one of Embodiments 164 to 169, including congenital amaurosis, macular degeneration, polypoid choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-vertebral dystrophy, vertebral-rod dystrophy, Oguchi disease, autosomal dominant radial drusen (familial autosomal dominant drusen), blue vertebral monochromatic color vision, or a combination thereof.

[0313]

[0386] Embodiment 171: The method according to any one of Embodiments 164 to 170, wherein the eye disease includes GA or dAMD.

[0387] Embodiment 172: A method for treating a disease or condition in a subject, comprising the step of administering to the subject an engineered polynucleotide comprising one or more expression cassettes encoding a first angiogenic inhibitor and a second angiogenic inhibitor.

[0314]

[0388] Embodiment 173: The method according to any one of Embodiments 164 to 172, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are each encoded by one expression cassette out of one or more expression cassettes.

[0315]

[0389] Embodiment 174: The method according to any one of Embodiments 164 to 173, wherein a first angiogenic inhibitor and a second angiogenic inhibitor are operably linked.

[0390] Embodiment 175: The method according to any one of Embodiments 164 to 174, wherein the first angiogenesis inhibitor and the second angiogenesis inhibitor are covalently linked by a linker.

[0316]

[0391] Embodiment 176: The method according to any one of Embodiments 164 to 175, wherein the first angiogenesis inhibitor comprises a complement inhibitor.

[0392] Embodiment 177: The method according to any one of Embodiments 164 to 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.

[0317]

[0393] Embodiment 178: The method according to any one of Embodiments 164 to 177, wherein the complement 3 inhibitor comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1 to 15.

[0318]

[0394] Embodiment 179: The method according to any one of Embodiments 164 to 178, wherein the complement 3 inhibitor comprises an amino acid sequence including any one of SEQ ID NOs: 1 to 15.

[0395] Embodiment 180: The method according to any one of Embodiments 164 to 179, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence that is at least 80% identical to any one of Sequence IDs 20 to 33.

[0319]

[0396] Embodiment 181: The method according to any one of Embodiments 164 to 180, wherein the complement 3 inhibitor is encoded by a nucleic acid sequence comprising any one of SEQ ID NOs. 20 to 33.

[0397] Embodiment 182: The method according to any one of Embodiments 164 to 181, wherein the first angiogenesis inhibitor comprises an inhibitor of membrane invasion complex (MAC).

[0320]

[0398] Embodiment 183: The method according to any one of Embodiments 164 to 182, wherein the MAC inhibitor comprises CD59.

[0399] Embodiment 184: The method according to any one of Embodiments 164 to 183, wherein CD59 contains an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 41 to 45.

[0321]

[0400] Embodiment 185: The method according to any one of Embodiments 164 to 184, wherein CD59 comprises an amino acid sequence which is one of SEQ ID NOs. 41 to 45.

[0401] Embodiment 186: The method according to any one of Embodiments 164 to 185, wherein the second angiogenesis inhibitor comprises a natriuretic peptide.

[0322]

[0402] Embodiment 187: The method according to any one of Embodiments 164 to 186, wherein a natriuretic peptide is covalently linked to an antibody or a fragment thereof.

[0403] Embodiment 188: The method according to any one of Embodiments 164 to 187, wherein the antibody or fragment thereof includes a fragment crystallizable (Fc) region.

[0323]

[0404] Embodiment 189: The method according to any one of Embodiments 164 to 188, wherein the natriuretic peptide comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 61 to 72.

[0324]

[0405] Embodiment 190: The method according to any one of Embodiments 164 to 189, wherein the natriuretic peptide comprises an amino acid sequence which is one of SEQ ID NOs. 61 to 72.

[0406] Embodiment 191: The method according to any one of Embodiments 164 to 190, wherein the second angiogenesis inhibitor comprises collagen or a fragment thereof.

[0325]

[0407] Embodiment 192: The method according to any one of Embodiments 164 to 191, wherein the second angiogenesis inhibitor comprises endostatin or a fragment thereof.

[0408] Embodiment 193: The method according to any one of Embodiments 164 to 192, wherein the second angiogenesis inhibitor comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 51.

[0326]

[0409] Embodiment 194: The method according to any one of Embodiments 164 to 193, wherein the second angiogenesis inhibitor comprises the amino acid sequence of SEQ ID NO: 51.

[0410] Embodiment 195: The method according to any one of Embodiments 164 to 194, wherein the second angiogenesis inhibitor comprises a VEGF inhibitor.

[0327]

[0411] Embodiment 196: The method according to any one of Embodiments 164 to 195, wherein the VEGF inhibitor comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 81 to 92.

[0328]

[0412] Embodiment 197: The method according to any one of Embodiments 164 to 196, wherein the VEGF inhibitor comprises an amino acid sequence which is one of SEQ ID NOs: 81 to 92.

[0413] Embodiment 198: The method according to any one of Embodiments 164 to 197, wherein the linker includes a severable linker.

[0329]

[0414] Embodiment 199: The method according to any one of Embodiments 164 to 198, wherein the severable linker includes a furin protease linker.

[0415] Embodiment 200: The method according to any one of Embodiments 164-199, wherein an engineered polynucleotide further encodes a third angiogenic inhibitor.

[0330]

[0416] Embodiment 201: The method according to any one of Embodiments 164 to 200, wherein the third angiogenesis inhibitor is different from the first angiogenesis inhibitor and the second angiogenesis inhibitor.

[0417] Embodiment 202: The method according to any one of Embodiments 164 to 201, wherein the engineered polynucleotide comprises a viral vector.

[0331]

[0418] Embodiment 203: The method according to any one of Embodiments 164 to 202, wherein the viral vector comprises an AAV vector.

[0419] Embodiment 204: The method according to any one of Embodiments 164 to 203, wherein the AAV vector comprises an AAV serotype including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof.

[0332]

[0420] Embodiment 205: The method according to any one of Embodiments 164 to 204, wherein the AAV vector is an AAV2 vector.

[0421] Embodiment 206: The method according to any one of Embodiments 164 to 205, wherein the AAV vector encodes an engineered AAV capsid.

[0333]

[0422] Embodiment 207: The method according to any one of Embodiments 164 to 206, wherein the engineered AAV capsid comprises one amino acid sequence from SEQ ID NOs. 161 to 182 and SEQ ID NOs. 191 to 210.

[0334]

[0423] Embodiment 208: The method according to any one of Embodiments 164 to 207, wherein the engineered AAV capsid comprises the amino acid sequence of SEQ ID NO: 169.

[0424] Embodiment 209: The method according to any one of Embodiments 164 to 208, wherein a single administration step cures a disease or condition.

[0335]

[0425] Embodiment 210: The method according to any one of Embodiments 164 to 209, wherein the administration step does not include daily administration.

[0426] Embodiment 211: The method according to any one of Embodiments 164 to 210, wherein the administration step includes weekly administration, bi-weekly administration, monthly administration, bi-monthly administration, semi-annual administration, or annual administration, or a combination thereof.

[0336]

[0427] Embodiment 212: The method according to any one of Embodiments 164 to 211, wherein the disease or condition includes an eye disease.

[0428] Embodiment 213: Eye diseases include ischemic syndrome of the eye, proliferative retinopathy, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, color blindness, age-related macular degeneration (nAMD), geographic atrophy (GA), atrophic age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), Valdevie's syndrome, Best's disease, colloideremia, and leva The method according to any one of Embodiments 164 to 212, including congenital amaurosis, macular degeneration, polypoid choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-vertebral dystrophy, vertebral-rod dystrophy, Oguchi disease, autosomal dominant radial drusen (familial autosomal dominant drusen), blue vertebral monochromatic vision, or a combination thereof.

[0337]

[0429] Embodiment 214: The method according to any one of Embodiments 164 to 213, wherein the eye disease includes GA or dAMD.

[0430] Embodiment 215: The method according to any one of Embodiments 164 to 214, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises an endostatin, and an engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.

[0338]

[0431] Embodiment 216: The method according to any one of Embodiments 164 to 215, wherein the first angiogenesis inhibitor, the second angiogenesis inhibitor, and the third angiogenesis inhibitor are not VEGF inhibitors.

[0339]

[0432] Embodiment 217: The method according to any one of Embodiments 164 to 216, wherein a first angiogenesis inhibitor and a second angiogenesis inhibitor inhibit angiogenesis in a subject when administered to the subject.

[0340]

[0433] Embodiment 218: The method according to any one of Embodiments 164 to 217, wherein when the first or second angiogenesis inhibitor is administered to a subject, it exhibits a reduction in the inhibition of angiogenesis in the subject compared to the inhibition of angiogenesis caused by the VEGF inhibitor. [Examples]

[0341]

[0434] The following exemplary embodiments are presentations of embodiments of the stimuli, systems, and methods described herein and are not intended to limit them in any way. Example 1. Vector for expressing multiple angiogenesis inhibitors

[0435] Vectors can be designed to express any of the angiogenesis inhibitors described herein. Figure 1 shows a vector map (top) for expressing a complement 3 inhibitor (C3i) operably ligated to a read sequence (LS). The vector map (bottom) of Figure 1 shows a complement 3 inhibitor (C3i) operably ligated to a human IgG Fc region. The complement 3 inhibitor (C3i) can be modified to include at least one amino acid substitution compared to wild-type complement 3 inhibitor (C3i). Table 1 shows exemplary C3i amino acid sequences that can be encoded by the vectors described herein. Table 2 shows exemplary nucleic acid sequences encoding exemplary C3i.

[0342] [Table 11]

[0343] [Table 12]

[0344]

[0436] Figures 2A and 2B show complement 3 inhibitors (C3i) operably linked to a natriuretic polypeptide (e.g., CNP36) by a human IgG Fc region (e.g., vector GAM or vector GGE constructs as described herein). As shown in Figure 2A, a 15-mer-C3i is cloned between the heavy chain leader sequence and Fc4 by ligation with 2×GGGS in vector CPE (SEQ ID NO: 15). Figure 2B shows a similar vector in which C3i further contains an EVQL peptide or a DK peptide at the N-terminus of C3i. Figure 3 shows vectors for expressing CD59, C3i, and the natriuretic polypeptide (CNP36). The C3i shown in Figure 3 is any one of the C3i described herein and may include the C3i shown in Figure 2B. Figure 4 shows additional exemplary vectors for expressing angiogenesis inhibitors as described herein. Table 3 shows exemplary clone identification numbers and construct names for the vectors described herein.

[0345] [Table 13-1]

[0346] [Table 13-2]

[0347] [Table 13-3]

[0348]

[0437] 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. After confirming the expression of multiple angiogenesis inhibitors, the therapeutic efficacy of these inhibitors can be determined in both cell and animal models. For example, the therapeutic efficacy of co-expressing multiple angiogenesis inhibitors from a vector can be evaluated in N-methyl-D-aspartate (NMDA) excitotoxicity mouse models of retinal degeneration; rat partial optic nerve transection (pONT) models; or neuroprotective and treatment efficacy glaucoma models.

[0349] Example 2. rBV amplification and AAV protection Maintenance of cell culture

[0438] SF-RVF cells were cultured at 28°C in Corning bottles containing ESF AF medium (Expression Systems, Davis, CA) with gentle shaking at 160 RPM. The cell density was approximately 1 × 10⁶ 7 Once the cell count reached 100 cells / mL, the cells were divided into a 1:2–1:8 ratio in fresh medium and continuously cultured for maintenance. Bacmid transfection and rBV amplification

[0439] Recombinant baculoviruses expressing capsid proteins (AAV2.N54 or AAV6.N54) and GOP were generated using the Bac-to-Bac baculovirus expression system (Thermo Fischer Scientific, Fremont, CA) and GenJet DNA transfection reagent. The recombinant baculoviruses were amplified by infecting 50-100 mL of 1:200 (v / v) SF-RVF cell culture in 250 mL Corning bottles with shaking at 180 RPM and 28 °C for 3 days. Baculovirus titers were detected by qPCR. AAV production and purification

[0440] AAV, 5x10 6RBV-GOI was produced by co-infecting 200 mL of SF-RVF cell culture with a cell / mL concentration with a mixture of rBV-AAV2.N54 (AAV6.N54) and rBV-GOI at an MOI of 400-800, based on the rBV titer measured by QPCR analysis. The rBV-infected cultures were incubated with shaking at 180 RPM and 28°C for 3 days. Cell pellets were collected and lysed in lysis buffer (1% (w / v) sarcosyl, 1% (v / v) Triton X-100, 10 mM Tris·HCl pH 8.0, 2 M urea, 2 mM MgCl2, and 25 IU / mL Benzonase®) with shaking at 37°C and 300 RPM / min for 1 hour. At the end of incubation, NaCl preservative (5M) was added to the lysate to a final concentration of 0.5–1M, and the lysate was centrifuged at 8000 RPM for 20 minutes. The cleared lysate was purified by two rounds of CsCl gradient ultracentrifugation. The complete AAV band was collected with a needle, and the buffer was replaced with ACI formulation buffer 1a using a PD-10 desalting column. The filtered AAV was quantified by ddPCR analysis using GOI-specific primers / probes.

[0350] Example 3. Cloning and expression of the C3i-Fc4-CNP36 fusion gene

[0441] A C3i-Fc4-CNP36 fusion gene, having a human antibody heavy chain secretion signal peptide coding sequence at its 5' end, was constructed in a single-stranded AAV vector, vector ETP, and named vector GAM (Figure 5A, 5B, or 5C). The designed human antibody heavy chain secretion signal peptide-C3i-3xGGGGS fusion protein was back-translated into a DNA sequence using the Jcat program (Technische Universitat Braunschweig, Lower Saxony, Germany) with human (homo sapiens) codon output. The partial fusion gene was then manually modified to adjust the GC content and sent for DNA synthesis at Integrated DNA Technologies, Inc. (Coralville, Iowa). After the synthetic DNA fragment was successfully cloned in vector ETP, plasmid DNA was identified by restriction enzyme digestion, and the sequence between two ITRs from the plasmid DNA of the correct colony was confirmed by competitive sequencing. Table 4. Transient expression of vectors in mammalian cell culture systems

[0442] Human HEK293LTV cells were used for fusion gene expression in this study. The cells were cultured in DMEM medium (Thermo Fisher Scientific) containing 10% FBS (ATCC, Manassas, VA) in a CO2 incubator at 37°C. For maintenance subculturing, the cells were divided into 1:10 portions twice a week. For transfection, the cells were placed in 6-well plates (Corning, NY) at a ratio of 0.75 × 10⁶. 6Cells were seeded overnight in 2 mL of medium per well. One or two hours before transfection, the culture medium was replaced with DMEM medium containing 2% FBS. For each well, 2 μg of plasmid DNA was diluted in 300 μL of 150 mM NaCl, and then 8 μL of PEImax (Polysciences, Germany) was added. After incubating the mixture at room temperature for 20 minutes, it was added dropwise to the cells and incubated in a 37°C CO2 incubator for 3 days. The medium was then taken out for further experimentation, and 3 mL of fresh medium was added to the cells. The medium was collected again in the same manner after 3 days. In addition, if a relatively large amount of protein was required, a T125 flask was used, and the amount of plasmid DNA and reagents was increased accordingly.

[0351]

[0443] The GAM plasmid was prepared using Xtra Maxi Plus EF (Macherey-Nagel SAS, France) and confirmed by restriction enzyme digestion using four different enzymes. In this study, the EKQ plasmid containing the EGFP gene was used as a transfection control, and the CPE plasmid containing the Fc4-CNP36 gene was used as a protein expression control. HEK293LTV cells were seeded in 6-well plates one day before transient transfection, and each transfection was performed using 2 μg / well DNA with PEImax. To evaluate transfection efficiency, cells transfected with vector EKQ were observed by fluorescence microscopy (data not shown). Cell culture supernatants were collected on days 3 and 6 post-transfection and analyzed for protein expression. Detection and quantification of C3i-Fc4-CNP36 fusion protein

[0444] The C3i-Fc4-CNP36 fusion protein (Table 5) was detected by SDS-PAGE and Western blot analysis. Plasmid DNA was transfected into cells in 6-well plates or T125 flasks, and the HEK293LTV cell medium (supernatant) was collected on days 3 and 6. The total volume of 26 μL of supernatant was mixed with 10 μL of 4× loading buffer and 4 μL of 10× reducing buffer and loaded onto Novex® WedgeWell® Tris-Glycine gel (Thermo Fisher Scientific) for electrophoresis. After electrophoresis at 150 V for approximately 1.5 hours, one gel was stained with SimplyBlue® SafeStain (Thermo Fisher Scientific), destained with water, and imaged using a digital camera (Figures 6, 7, 8, and 9). On the other hand, another gel was transferred to a PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). Immediately after treating the membranes with a PBS solution of casein blocker (Thermo Scientific, Waltham, MA, USA) at room temperature for 1 hour, they were probed using 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 goat-HRP-conjugated goat anti-rat IgG secondary antibody (1:10,000) (31470) (Thermo Fisher Scientific). Proteins were detected using the Supersignal® West Atto Ultimate sensitivity kit (Thermo Fisher Scientific), and images were acquired using Gel Doc™ XR+ with Image Lab® software (Bio-Rad, Hercules, CA) (Figures 6, 7, 8, and 9).SDS-PAGE and Western blot results indicate that the C3i-Fc4-CNP36 fusion protein was detected in the culture supernatant of vector GAM-transfected cells for 3 or 6 days, but not in the culture supernatant of vector EKQ or non-transfected cells. However, the expression level of the C3i-Fc4-CNP36 fusion protein in the vector GAM plasmid was considerably lower than that of the Fc4-CNP36 fusion protein expressed by the plasmid, vector CPE, in a 6-well plate or T125 flask using either HRP-conjugated goat anti-human IgG1 Fc antibody or rat anti-human CNP antibody.

[0352]

[0445] Similarly, 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), incubated overnight at 4°C, and then treated with a casein blocker in PBS solution 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). The color reaction was developed using 1-step® Ultra TMB-ELISA substrate solution (ab171523) (Abcam, Waltham, MA) and stopped by the addition of 2M HCl. Finally, the microplates were read and recorded using SpectaMax iD3 (Molecular Devices, San Jose, CA). ELISA data were plotted and analyzed using GraphPad Prism 9.5.1 (Dotmatics, Boston, Massachusetts). Table 6 demonstrates that the C3i-Fc4-CNP36 fusion protein concentrations in the supernatant on day 3 and day 6 were 4.87 and 8.97 μg / mL, respectively. Similarly, using a T125 flask, the fusion protein concentrations in the supernatant on day 3 and day 6 were 29.02 and 16.09 μg / mL, respectively (Table 7). The fusion protein concentrations measured by ELISA were considerably higher than those obtained by SDS-PAGE and Western blotting.

[0353]

[0446] In 6-well plates, the C3i-Fc4-CNP36 fusion protein was detected in the culture supernatant of cells transfected with vector GAM for 3 or 6 days using HRP-conjugated goat anti-human IgG1 Fc antibody, but not in the culture supernatant of vector EKQ or untransfected cells. However, the expression level of the C3i-Fc4-CNP36 fusion protein from the vector GAM plasmid was considerably lower than that of the Fc4-CNP36 fusion protein expressed by the plasmid and vector CPE. The results of the obtained Western blots are shown in Figure 6. Lane contents: 1. Vector GAM on day 3; 2. Vector EKQ on day 3; 3. Vector CME on day 3; 4. Untransfected on day 3; 5. Vector GAM on day 6; 6. Vector EKQ on day 6; 7. Vector CME on day 6; 8. Untransfected on day 6.

[0354]

[0447] The C3i-Fc4-CNP36 fusion protein was detected in the culture supernatant of cells transfected with vector GAM for 3 or 6 days in a T125 flask using HRP-conjugated goat anti-human IgG1 Fc antibody, but not in the culture supernatant of vector EKQ or untransfected cells. However, the expression level of the C3i-Fc4-CNP36 fusion protein in the vector GAM plasmid was considerably lower than that of the Fc4-CNP36 fusion protein expressed by the plasmid and vector CPE. The results of the obtained Western blots are shown in Figure 7. Lane contents: 1. Vector GAM on day 3; 2. Vector EKQ on day 3; 3. Vector CME on day 3; 4. Untransfected on day 3; 5. Vector GAM on day 6; 6. Vector EKQ on day 6; 7. Vector CME on day 6; 8. Untransfected on day 6.

[0355]

[0448] In 6-well plates, the C3i-Fc4-CNP36 fusion protein was detected in the culture supernatant of cells transfected with vector GAM for 3 or 6 days using rat anti-human CNP antibody, but not in the culture supernatant of vector EKQ or untransfected cells. However, the expression level of the C3i-Fc4-CNP36 fusion protein from the vector GAM plasmid was considerably lower than that of the Fc4-CNP36 fusion protein expressed by the plasmid and vector CPE. The results of the obtained Western blots are shown in Figure 8. Lane contents: 1. Vector GAM on day 3; 2. Vector EKQ on day 3; 3. Vector CME on day 3; 4. Untransfected on day 3; 5. Vector GAM on day 6; 6. Vector EKQ on day 6; 7. Vector CME on day 6; 8. Untransfected on day 6.

[0356]

[0449] The C3i-Fc4-CNP36 fusion protein was detected by rat anti-human CNP antibody in the culture supernatant of cells transfected with vector GAM for 3 or 6 days in a T125 flask, but not in the culture supernatant of vector EKQ or untransfected cells. However, the expression level of the C3i-Fc4-CNP36 fusion protein in the vector GAM plasmid was considerably lower than that of the Fc4-CNP36 fusion protein expressed by the plasmid and vector CPE. The results of the obtained Western blots are shown in Figure 9. Lane contents: 1. Vector GAM on day 3; 2. Vector EKQ on day 3; 3. Vector CME on day 3; 4. Untransfected on day 3; 5. Vector GAM on day 6; 6. Vector EKQ on day 6; 7. Vector CME on day 6; 8.

[0357] [Table 14-1]

[0358] [Table 14-2]

[0359] [Table 14-3]

[0360] Table 14-4

[0361] Table 14-5

[0362] Table 14-6

[0363] Table 14-7

[0364] Table 14-8

[0365] Table 14-9

[0366] Table 14-10

[0367] Table 14-11

[0368] Table 14-12

[0369] Table 15

[0370] Table 16

[0371] [Table 17]

[0372] Example 4. Cloning and expression of membrane-bound and soluble CD59 genes Design and molecular cloning of membrane-bound and soluble CD59 (sCD59) genes

[0450] Membrane-bound CD59 (mCD59) with a native signal peptide coding sequence at its 5' end was constructed in the self-complementary AAV vector, vector CPE. The mCD59 protein was backtranslated to a human codon-output DNA sequence using four different programs from Snapgene, Jcat, Geneart, and Genscript. The full-length mCD59 gene with different codon optimizations was then manually modified to adjust the GC content and sent for DNA synthesis at Twist Bioscience (South San Francisco, CA). After the synthetic DNA fragments were successfully cloned in vector CPE, all plasmid DNA was identified by restriction enzyme digestion. Wild-type mCD59 was named vector GTM, Snapgene-optimized mCD59 vector GTP, Jcat-optimized vector GTQ, Geneart-optimized vector GTR, and Genscript-optimized vector GAT (Figure 10). Sequences from all these plasmids between the two ITRs were confirmed by complete sequencing (Table 8). Additional information regarding sequence information is shown in Table 9.

[0373]

[0451] Wild-type and Snapgene-optimized mCD59 showed high expression levels among these genes, and were therefore selected for soluble CD59 expression. The GPI anchor peptide (GGTSLSEKTVLLLVTPFLAAAWSLHP) coding sequence was excised from the full-length CD59 gene, and wild-type and Snapgene-optimized sCD59, which has a native or human antibody heavy chain secretion signal peptide coding sequence at its 5' end, were subcloned in the single-stranded AAV vector, vector ETP. Four different plasmids were obtained, for example, 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. Similarly, all of these plasmids were identified by restriction enzyme digestion, and the sequences from all of these plasmids between the two ITRs were confirmed by complete sequencing (Table 8). Transient expression of the mCD59 gene in mammalian cell culture systems

[0452] Human EXPI293 suspension cells were used for mCD59 expression. The cells were cultured in BalanCD medium (Fujifilm Irvine Scientific, Santa Ana, CA) containing 5 mM glutaMax® (Thermo Fisher Scientific) at 37°C in an orbital shaker. Immediately before transfection with Mirus transfection reagent (San Francisco, CA), the cells were transfected in a shaking flask (Corning, NY) in 25 mL of medium, resulting in a total volume of 2.0 × 10⁶ cells. 6The solution was diluted to 100⁴ cells / mL. For each shaking flask, 50 μg of plasmid DNA was diluted in 2.5 ml of VirusGEN® SELECT AAV Complex Formation Solution and Enhancer in a sterile tube, and 75 μl of TransIT-VirusGEN® SELECT reagent was added to the diluted DNA. The mixture was incubated at room temperature for 15–30 minutes without further agitation to form a transfection complex, which was then added dropwise to the cells and incubated in an orbital shaker at 37°C for 2 days. The cells were then harvested for further experiments. After washing the cells once with 1×PBS, 1 mL of RIPA buffer (89901) (Thermo Fisher Scientific) containing 1× protease inhibitor cocktail (1183670001) (Millipore Sigma, Burlington, MA) was added in a 10×10⁶ solution. 6 The sample was added to individual cells. The cells were sonicated three times for 10 seconds at 10-second intervals, and then the samples were centrifuged at 12000 × g for 15 minutes. Cell lysates were collected, and the protein concentration was measured using the Pierce® BCA Protein Assay Kit (23225) (Thermo Fisher Scientific).

[0374]

[0453] Plasmid vectors GTM, GTP, GTQ, GTR, and GAT were prepared using Xtra Maxi Plus EF (Macherey-Nagel SAS, France) and identified by restriction enzyme digestion. Vector EKQ, containing the EGFP gene, was used as a transfection control. EXPI293 suspension cells were raised to 2.0 × 10⁶ cells in 25 mL of culture medium. 6 After dilution to 100 cells / mL, each transfection was performed using Mirus transfection reagent with 50 μg of DNA. To evaluate transfection efficiency, cells transfected with vector EKQ were observed under a fluorescence microscope. Cell lysates were prepared, collected, and analyzed for protein expression.

[0375]

[0454] The mCD59 protein in cell lysates was detected by SDS-PAGE and Western blot analysis. EXPI293 cells were collected 2 days after plasmid DNA transfection. The total volume of 26 μL of cell lysates was mixed with 10 μL of 4× loading buffer and 4 μL of 10× reducing buffer and loaded onto NuPAGE® 4-12% Bis-Tris gel (Thermo Fisher Scientific) for electrophoresis. The proteins were run at 150 V for approximately 1 hour, one gel was stained with SimplyBlue® SafeStain (Thermo Fisher Scientific), destained with water, and imaged using a digital camera (Figure 11). The other gel was then transferred to a PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). Immediately after treating the membranes with a casein blocker PBS solution (Thermo Scientific, Waltham, MA, USA) at room temperature for 1 hour, they were probed with HRP-conjugated mouse anti-human CD59 monoclonal antibody (sc-133170) (Santa Cruz Biotechnology, Dallas, TX). The protein was detected using the Supersignal® West Atto Ultimate sensitivity kit (Thermo Fisher Scientific), and images were acquired by Gel Doc™ XR+ using Image Lab® software (Bio-Rad, Hercules, CA). SDS-PAGE and Western blotting results showed that mCD59 was expressed in vector GTM, vector GTP, vector GTQ, vector GTR, and vector GAT transfected cells, but not in vector EKQ or untransfected cells (Figure 11). Furthermore, Snapgene-optimized mCD59 exhibits the highest expression level, followed by wild-type mCD59, Genscript-optimized mCD59, Jcat-optimized mCD59, and Geneart-optimized mCD59, which shows the lowest expression level. Transient expression of the sCD59 gene in mammalian cell culture systems

[0455] Human HEK293LTV cells were used for sCD59 gene expression and cultured in DMEM medium (Thermo Fisher Scientific) containing 10% FBS (ATCC, Manassas, VA) in a CO2 incubator at 37°C. For maintenance subculturing, cells were divided into a 1:10 ratio twice a week. For transfection, cells were placed in 2 mL of medium in a 6-well plate (Corning, NY) at a rate of 0.6 × 10⁶ 6 Cells were seeded overnight in 1 cell / well. One or two hours before transfection, the culture medium was replaced with DMEM medium containing 2% FBS. For each well, 2 μg of plasmid DNA was diluted in 300 μL of 150 mM NaCl, and then 8 μL of PEImax (Polysciences, Germany) was added. The mixture was incubated at room temperature for 20 minutes, then added dropwise to the cells and incubated in a CO2 incubator at 37°C. After 3 days, all the medium from each well was collected for further experimentation, and 3 mL of fresh medium was added to the cells. The medium was collected again in the same manner on day 3.

[0376]

[0456] Plasmids, vectors GCK, GCM, GEM, and GEP were prepared using Xtra Maxi Plus EF (Macherey-Nagel SAS, France) and identified by restriction enzyme digestion. In this study, the plasmid and vector EKQ containing the EGFP gene were used as transfection controls. HEK293LTV cells were seeded in 6-well plates one day before transient transfection, and each transfection was performed using PEImax with 2 μg / well DNA. To evaluate transfection efficiency, cells transfected with vector EKQ were observed under a fluorescence microscope. Cell culture supernatants were collected on days 3 and 6 post-transfection and analyzed for protein expression.

[0377]

[0457] The sCD59 protein was detected by SDS-PAGE and Western blot analysis. Cell culture supernatants were collected on days 3 and 6 after plasmid DNA transfection of cells. The total volume of each supernatant (26 μL) was mixed with 10 μL of 4× loading buffer and 4 μL of 10× reducing buffer and loaded onto NuPAGE® 4-12% Bis-Tris gels (Thermo Fisher Scientific) for electrophoresis. After electrophoresis at 150 V for approximately 1 hour, one gel was stained with SimplyBlue® SafeStain (Thermo Fisher Scientific), destained with water, and imaged using a digital camera (Figures 11 and 12). The other gel was then transferred to a PVDF membrane using a Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). Immediately after treating the membranes with a casein blocker PBS solution (Thermo Scientific, Waltham, MA, USA) at room temperature for 1 hour, the membranes were probed with HRP-conjugated mouse anti-human CD59 monoclonal antibody (sc-133170) (Santa Cruz Biotechnology, Dallas, TX). The protein was detected using the Supersignal® West Atto Ultimate sensitivity kit (Thermo Fisher Scientific), and images were acquired by Gel Doc™ XR+ with Image Lab® software (Bio-Rad, Hercules, CA). Western blotting results showed that sCD59 was expressed in vector GCK, 426, 436, and 437 transfected cells, but not in vector EKQ or untransfected cells (Figures 11 and 12). Furthermore, the sCD59 gene from plasmids, vector GCK, and vector GEM demonstrated significantly higher expression levels than from vector GCM and vector GEP.

[0378]

[0458] Membrane-bound CD59 (mCD59) from cell lysates was detected by HRP-conjugated anti-human CD59 antibody, but not in vector EKQ or untransfected cells. The results of the obtained Western blots are shown in Figure 10. 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.

[0379]

[0459] Soluble CD59 (sCD59) protein in the cell culture supernatant on day 3 was detected using an HRP-conjugated anti-human CD59 antibody. Soluble CD59 (sCD59) from the cell culture supernatant on day 3 was detected by the HRP-conjugated anti-human CD59 antibody, but not in vector EKQ or untransfected cells. The results of the Western blots are shown in Figure 11. Lane contents: 1. Vector GCK; 2. Vector GCM; 3. Vector GEM; 4. Vector GEP; 5. Vector EKQ; 6. Untransfected; 7. Purified CD59 protein.

[0380]

[0460] Soluble CD59 (sCD59) in the cell culture supernatant on day 6 was detected by HRP-conjugated anti-human CD59 antibody, but not in vector EKQ or untransfected cells. The resulting Western blot images are shown in Figure 12. Lane contents: 1. Vector GCK; 2. Vector GCM; 3. Vector GEM; 4. Vector GEP; 5. Vector EKQ; 6. Untransfected; 7. Purified CD59 protein. Purification of sCD59 protein

[0461] Large cell cultures were prepared for the purification of vector GEM proteins. The sCD59 protein was purified in four steps using an AKTA Explorer 100. First, the sample was cleared and centrifugated, followed by removal of cell debris by 0.2 μm filtration (Nalgene, Rochester, NY). The cleared collection was directly transferred for downstream purification. Second, gel filtration column chromatography was performed using a Sephadex G-25 (Cytiva, Marlborugh, MA) to remove the cell culture medium from the cell culture supernatant containing the CD59 protein. The G-25 desalting column was equilibrated with Tris buffer (20 mM Tris pH 7.3, 100 mM NaCl, 0.005% F-68), and then loaded with the cell culture supernatant containing the sCD59 protein. The sCD59 protein was collected when UV A280 increased to 5 mAU and stopped when UV A280 decreased to 5 mAU. Thirdly, strong anion exchange column chromatography using HQ resin (ThermoFisher, Walthan, MA) was performed on the G-25 desalted pool. The G-25 desalted product was diluted with XQ column equilibrium buffer (20 mM Tris pH 7.3, 0.005% F-68) to a conductivity of <3 mS / cm and loaded onto an HQ column equilibrated with equilibrium buffer (20 mM Tris pH 7.3, 10 mM NaCl, 0.005% F-68). After loading and washing with equilibrium buffer, the column was eluted using a 10–500 mM NaCl gradient. Elution peaks were collected when A280 increased to 5 mAU, and collection was terminated when UV A280 decreased to 5 mAU. Fourthly, size exclusion column (SEC) chromatography using Superdex 75 (Cytiva, Marlborugh, MA) was performed to further remove impurities from the HQ purified collectibles. After equilibrating the SEC column with PBS buffer, the HQ column chromatography collectibles were loaded. The CD59 protein elution peak was collected when A280 increased to 5 mAU, and collection was terminated when UV A280 decreased to 5 mAU.

[0381]

[0462] Purified CD59 samples were separated by SDS-PAGE using a 4-12% Bis-Tris gradient gel (Invitrogen® Novex) according to the manufacturer's instructions. Protein bands were visualized by staining with Coomassie blue. In the gel, the protein size was estimated to be approximately 14 kDa, about 5 kDa larger than the theoretical molecular weight (Figure 14). The results indicate that the sCD59 was of high purity. The increase in size may be due to protein glycosylation. Lane contents: 1. Vector GEM. Determination of the N-terminal sequence of sCD59

[0463] Purified sCD59 protein was sent for N-terminal sequencing for protein identification. The sCD59 protein was transferred to a PVDF membrane, stained with SimplyBlue SafeStain (Thermo Fisher Scientific, Cat.#465034), and 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 with 50%, 60%, and 70% methanol sequentially for 15 minutes. 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. The sample, along with a coolant, was then transported to Creative Proteomics (Shirley, NY, USA) for identification of the first five amino acids of the N-terminus. N-terminal sequencing of the protein showed that the first four of the five amino acids were identical to the expected result (Table 42). The results indicate that the sCD59 protein was accurately translated and processed for secretion.

[0382] [Table 18]

[0383] sCD59 functional assay

[0464] To test the biological function of sCD59, a cell lysis inhibition assay was performed using Hep1c1c7 cells. After determining the serum dose that would induce approximately 70% cell lysis for novel normal human serum (NHS), purified sCD59 protein was used for the cell lysis inhibition assay. Three hours before the experiment, 1 × 10⁶ cells were used. 4 Cells were seeded in 96-well plates at doses of 100 cells / well. Various doses of NHS were added to GVB2++ buffer (Ca 2+ and Mg 2+ The cells were mixed with (Complement Technology, Cat.B100). 100 μL of the mixture was added to each well. Background lysis was measured using GVB2++ buffer alone, and maximum lysis was measured by lysing all cells with Maximum Lysis Buffer (GVB2++ plus 1% SDS). After incubating the cells with the mixture for 1 hour, 10 μL of WST-8 was added to each well of the plate. The cells were incubated again with WST-8 for 1–4 hours. Absorbance at 450 nm was measured using a microplate reader. To calculate the percentage of viable cells, the absorbance was subtracted from the maximum lysis, divided by the difference between background lysis and maximum lysis, and multiplied by 100; this was equal to the percentage of viable cells. The percentage of dead cells was calculated by subtracting the viable cells from 100. In GraphPad Prism, the percentage of cell death was plotted against the dose of NHS to determine the NHS doses that caused 50% and 70% hemolysis, respectively (Figure 15A).

[0384]

[0465] Purified sCD59 protein was used for the cell lysis inhibition assay using an NHS dose that induces 70% cell lysis. The sCD59 protein was serially diluted 2-fold and mixed with GVB2++ buffer in the presence of NHS, yielding final concentrations of 30.11, 15.06, 7.53, 3.76, 1.88, and 0.94 μM. A reference CP40 peptide was also used for this test. The mixture of each sample was added to four wells. The four-well readings were subtracted from the maximum lysis, averaged, divided by 70% of the difference between cell lysis and maximum lysis, and multiplied by 100. The percentage of cell lysis was considered to be the percentage of cell lysis inhibition. Using GraphPad Prism software, the percentage of cell lysis inhibition was plotted against the molecular concentration of sCD59 protein, and based on the data plot, the IC50 value was estimated to be approximately 20 μM (Figure 15B). The results demonstrated that purified sCD59 could dramatically inhibit cell lysis at gradually increasing concentrations, and CP40 also showed slight inhibition, while the BSA control had no inhibitory effect at all.

[0385] [Table 19-1]

[0386] [Table 19-2]

[0387] [Table 19-3]

[0388] [Table 19-4]

[0389] [Table 19-5]

[0390] [Table 19-6]

[0391] Table 19-7

[0392] Table 19-8

[0393] Table 19-9

[0394] Table 19-10

[0395] Table 19-11

[0396] Table 19-12

[0397] Table 19-13

[0398] Table 19-14

[0399] Table 19-15

[0400] Table 19-16

[0401] Table 19-17

[0402] Table 19-18

[0403] Table 19-19

[0404] Table 19-20

[0405] Table 19-21

[0406] Table 19-22

[0407] Table 19-23

[0408] Table 19-24

[0409] Table 19-25

[0410] Table 19-26

[0411] Table 19-27

[0412] [Table 19-28]

[0413] [Table 19-29]

[0414] [Table 19-30]

[0415] [Table 20]

[0416] Example 5. Development of a macular-selective adeno-associated vector (AAV) containing multiple target genes to target atrophic age-related macular degeneration.

[0466] Age-related macular degeneration (AMD) is the leading cause of blindness in people over 60, affecting 19.8 million people in the United States and 198 million worldwide. Currently, there is no effective treatment to halt geographic atrophy (GA) and improve vision. Adeno-associated virus (AAV) vectors may be a method of gene delivery. AAV capsids can be engineered to improve tissue targeting. In this example, a capsid with improved targeting to the macula (AAV2.N54) was identified through multi-species screening in mice, pigs, rabbits, and monkeys. Across the four species, AAV2.N54 showed improved targeting compared to wild-type AAV2 and AAV2.7m8, effectively delivering to the macula without targeting retinal ganglion cells (RGCs). AAV2.N54, possessing multiple target genes (GOIs), encodes complement 3 inhibitory protein (C3IP), an engineered C3 / C3b inhibitory peptide (C3ip) fused to a human IgG Fc fragment, C3IP fused at its terminus to C3ip (C3IP-C3ip), C3IP fused at its C-terminus to either a C-type natriuretic peptide (CNP36) (C3IP-CNP36) or a 36-amino acid peptide containing endostatin (C3IP-Endo), and soluble CD59 (sCD59). The resulting AAV vectors selected for systematic analysis are shown in Figure 13: AAV2.N54-C3IP-C3ip, sCD59, AAV2.N54-C3IP-CNP36, sCD59, and AAV2.N54-C3IP-endo, sCD59. C3IP demonstrated similar affinity (KD) to C3b as determined by Biacore and ELISA, which was equivalent to the positive control peptide CP40. C3IP-C3ip showed half the IC50 of CP40 in a hemolysis assay using human red blood cells (RBCs). 50The results showed that both C3IP-CNP36 and C3IP-Endo demonstrated hemolysis and proliferation inhibition in VEGF-A165-stimulated HUVEC cells. C3IP-CNP36 also induced cyclic guanidine monophosphate (cGMP) formation and showed binding to natriuretic peptide receptor B (NPR-B). Fc-CNP induced dose-dependent RGC protection in a rat partial optic nerve transection (pONT) model and a mouse N-methyl-D-aspartate (NMDA) excitotoxicity model after a single intravitreal (IVT) injection of AAV2.N54. AAV2.N54-C3IP, sCD59, AAV2.N54-C3IP-CNP36, sCD59, and AAV2.N54-C3IP-Endo, sCD59 demonstrated good expression in RPE-19 and HEK293 cells in mice after IVT injection. These AAV vectors are currently being evaluated in a sodium iodate-damaged retinal model using cynomolgus monkeys.

[0417] Example 6. Optimization of fusion protein expression

[0467] The C3i-Fc4-CNP36 fusion gene in vector GAM expression was further optimized by applying five different strategies shown in Table 5: 1) the promoter was changed from CBA to CAG (vector GGQ); 2) the recognition site of the EVQL leader sequence was replaced with DK (vector GGE); 3) the C3i position was switched from the C-terminus to the N-terminus (vector GGG); 4) the Fc fragment of IgG4 was replaced with the Fc fragment of IgG1 (vector GKK); and 5) the GOI at the 3' end was changed from CNP36 to endostatin (vector GKA). The ORF sequences of the GOI and the protein sequences they encode are shown in Tables 19 and 20, respectively. In addition, the structures of 1. vector GGE, 2. vector GGG, 3. vector GGQ, 4. vector GKA, and 5. vector GKK are shown in Figure 16.

[0418]

[0468] Plasmids were prepared using Xtra Maxi Plus EF (Macherey-Nagel SAS, France) and confirmed by restriction enzyme digestion and partial plasmid sequencing. They were then used for transfection into HEK293LTV cells according to the protocol described in Example 3. In this study, the EKQ vector containing the EGFP gene was used as a transfection control, and the CPE vector containing the Fc4-CNP36 gene was used as a protein expression control. To evaluate transfection efficiency, cells transfected with vector EKQ were observed using a fluorescence microscope (data not shown). Cell culture supernatants were collected 5 days after transfection and analyzed for protein expression. Fusion proteins were detected using the method described in Example 3 with a mouse-anti-human Fc antibody conjugated with HRP (Cat.#A01854-200, GenScript, Nanjing, China). Lane contents: M. Pre-staining 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).

[0419]

[0469] Western blotting results showed that the C3i fusion protein was highly expressed by plasmids, vectors GGE and GGG, but weakly expressed by plasmids, vectors GGQ, GKA, and GKK (Figure 17). The results indicated that changes in the promoter, Fc fragment, or GOI did not contribute to the improvement of the C3i fusion protein. However, changes in the recognition site of the Vh reader sequence (EVQL--->DK) or the placement of Fc4 at the 5' end of the C3i fusion gene dramatically improved gene expression. Notably, the Fc4 fragment in constructor vector GGG uses the same recognition site (DK) as vector GGE. Thus, in this study, the recognition site of the Vh reader sequence played a crucial role in optimizing C3i fusion gene expression. (DK)C3i-Fc-endostatin protein expression

[0470] The C3i-Fc-endostatin (vector GKA) fusion gene exhibited low expression. Since altering the recognition site of the Vh reader sequence enhanced the expression of the C3i-Fc4-CNP36 fusion protein, vector GKA was also converted to DK in the novel plasmid and vector GKC. Table 19 shows the C3i-related GOI sequences in the plasmid and vector GKC, and Table 20 shows the encoded protein sequences.

[0420]

[0471] Protein expression using vector GKC was not tested. Instead, bacmids were prepared, AAVs were packaged, and used directly for protein expression testing. Figure 18 shows that vector GKC AAV had similar C3i fusion protein expression levels to vector GGE and vector GGG AAV, suggesting that the recognition site modification of the Vh reader sequence also improved C3i-Fc4-endostatin fusion gene expression. 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. In addition, when the Vh reader sequence was used 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, overall, optimizing the recognition site of the leader sequence was shown to be an important consideration for improving the gene expression of secreted proteins.

[0421] [Table 21-1]

[0422] [Table 21-2]

[0423] [Table 21-3]

[0424] [Table 21-4]

[0425] [Table 21-5]

[0426] [Table 21-6]

[0427] [Table 21-7]

[0428] [Table 21-8]

[0429] [Table 22-1]

[0430] [Table 22-2]

[0431] [Table 22-3]

[0432] Example 7. Purification of C3i fusion protein and determination of its N-terminal sequence

[0472] In the final experiment, vectors GGE and GGG were shown to highly express the C3i fusion gene. In this experiment, adherent HEK293LTV cells were seeded in 6-well plates, and numerous plasmids were prepared and transfected into suspension Expi293F cells for large-scale protein purification. Expi293F cells were cultured in BalanCD HEP293 medium (Cat.#91165, Fujifim, Tokyo, Japan) containing 25 mM L-glutamine (Cat.#35050-61, ThermoFisher Scientific, Waltham, MA, USA). 1–2 hours before transfection, cells were placed in a transfection culture vessel containing 150 mL of culture medium at a rate of 1 × 10⁶ 6 The plasmid DNA was diluted to a density of 100 cells / ml. 600 μg of plasmid DNA and 15 mL of diluent were combined in individual sterile tubes and vortexed briefly. 1200 μL of PEImax was added to the diluted DNA, the mixture was vortexed for 5 minutes, and then incubated at room temperature for 20 minutes without further stirring to form the transfection complex. After gently mixing the solution by moving the pipette up and down, the entire solution was added to 150 mL of suspension cell culture. The flasks were shaken in an incubator for 2-3 hours, and then 150 mL of fresh medium was added to each flask. After incubation of the cell cultures for 5 days, they were harvested.

[0433]

[0473] C3i fusion proteins were detected on SDS-PAGE gels. All cell culture supernatants were collected for protein purification using Protein A resin (Cat.#L00210, GenScript USA, Piscataway, NJ, USA). After completely resuspending the resin, 2 ml of resin was transferred to a new tube. The beads were centrifuged and the supernatant was discarded. 12 ml of binding / wash buffer (150 mM NaCl, 20 mM Na2HPO4, pH 7.0) was added to the beads and vortexed for 20 seconds. The beads were centrifuged and the supernatant was discarded again. The beads were diluted in binding / wash 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 was loaded onto the column along with the remaining supernatant, and the flow-through sample was collected for analysis. 90 mL of washing buffer was added to the resin in the column, and the washed sample was collected for analysis. The washing process was repeated. Finally, the protein was eluted three times with 3 mL of elution buffer (0.1 M glycine, pH 3.0). The eluate was neutralized to pH 7.4 using 300 μL of neutralizing buffer (1 M Tris·Cl, pH 8.5). Finally, the sample was concentrated using a 3 kDa cutoff Amicon® Ultra Centrifugal Filter column (Cat.#UFC900308, Millipore-Sigma, Burlington, MA, USA), and buffer exchange was performed.

[0434]

[0474] After collecting the flow-through, washing solution, and eluted samples of vectors GGE and GGG as described above, they were analyzed by SDS-PAGE gel. The majority of the Fc-fusion proteins were able to bind to the protein A resin and eluted with low pH glycine buffer. Both purified vector GGE (Figure 19) and vector GGG (Figure 20) proteins were of high purity, and dimer bands were observed for each. Lane contents of both gels: M. Protein marker; 1. Cell culture medium; 2. Flow-through; 3. Wash 1; 4. Wash 2; 5. Eluted sample; 6. Protein in PBS buffer. The results indicated that vectors GGE and GGG were purified very well, and that some dimers were formed even in the presence of a reducing loading buffer.

[0435]

[0475] After replacing the elution buffer with PBS buffer, the purified protein was quantified using the BCA protein assay kit (Cat.#23225, Thermo Scientific, Waltham, MA, USA) and stored at ≤-60°C until testing. N-terminal sequencing of C3i fusion protein

[0476] To further identify the purified vector GGE and vector GGG proteins, the purified proteins were sent for N-terminal sequencing. The proteins were blotted onto a PVDF membrane and stained with SimplyBlue SafeStain (Cat.#465034, Thermo Fisher Scientific). Bands from each sample containing approximately 8 μg of protein were individually excised and m...

Claims

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

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

3. The engineered polynucleotide according to claim 1 or 2, wherein the first angiogenesis inhibitor comprises a complement inhibitor.

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

5. The engineered polynucleotide according to claim 4, wherein the complement 3 inhibitor comprises an amino acid sequence that is at least 80% identical to any one of sequence numbers 1 to 15.

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

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

8. The engineered polynucleotide according to claim 7, wherein CD59 comprises an amino acid sequence that is at least 80% identical to one of sequence numbers 41-45, 312-319, or 325-329.

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

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

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

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

13. An engineered polynucleotide according to any one of claims 9 to 12, wherein the natriuretic peptide comprises an amino acid sequence that is at least 80% identical to any one of sequence numbers 61 to 72.

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

15. An engineered polynucleotide according to any one of claims 1 to 14, further encoding a third angiogenesis inhibitor.

16. An engineered polynucleotide according to any one of claims 1 to 15, comprising a viral vector.

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

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

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

20. The engineered polynucleotide according to claim 19, wherein the engineered AAV capsid comprises one amino acid sequence from SEQ ID NOs. 161-182 and SEQ ID NOs. 191-210.

21. An engineered polynucleotide according to any one of claims 1 to 20, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises CNP36.

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

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

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

25. An engineered polynucleotide according to any one of claims 1 to 24, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises an endostatin.

26. The engineered polynucleotide according to any one of claims 1 to 25, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor, the second angiogenesis inhibitor comprises Fc-CNP36, and the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.

27. The engineered polynucleotide according to any one of claims 1 to 26, wherein the first angiogenesis inhibitor comprises CD59, the second angiogenesis inhibitor comprises a complement 3 inhibitor, and the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising Fc-CNP36.

28. The engineered polynucleotide according to any one of claims 1 to 27, wherein the first angiogenesis inhibitor comprises CD59, the second angiogenesis inhibitor comprises endostatin, and the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor.

29. The engineered polynucleotide according to any one of claims 1 to 28, wherein the first angiogenesis inhibitor comprises a complement 3 inhibitor, the second angiogenesis inhibitor comprises an endostatin, and the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising CD59.

30. Engineered polypeptides comprising a first angiogenic inhibitor and a second angiogenic inhibitor.

31. The engineered polypeptide according to claim 30, wherein a first angiogenesis inhibitor and a second angiogenesis inhibitor are covalently linked by a linker.

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

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

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

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

36. An engineered polypeptide according to any one of claims 1 to 35, further encoding a third angiogenic inhibitor.

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

38. The vector according to claim 37, wherein the vector encodes an AAV capsid, and the AAV capsid comprises an engineered AAV capsid.

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

40. The virus particle according to claim 39, wherein the virus particle comprises an AAV capsid, and the AAV capsid comprises an engineered AAV capsid.

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

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

43. The composition according to 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 containing C3a, C3b, iC3b, C3f, C3c, C3d, C3g, or a combination thereof; and an inhibitor of the membrane invasion complex (MAC).

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

46. A composition comprising CD59 and natriuretic peptide.

47. The composition according to 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 to 29, an engineered polypeptide according to any one of claims 30 to 36, a vector according to any one of claims 37 to 38, a viral particle according to any one of claims 39 to 40, a cell according to claim 41, or a composition according to any one of claims 42 to 47.

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

50. A method for treating a disease or condition in a subject, comprising the step of administering to the subject an engineered polynucleotide according to any one of claims 1 to 29, an engineered polypeptide according to any one of claims 30 to 36, a vector according to any one of claims 37 to 38, a viral particle according to any one of claims 39 to 40, a cell according to claim 41, or a composition according to any one of claims 42 to 47, or a pharmaceutical composition according to claim 48.

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

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