Combination therapy for treatment of ocular disease

Abstract

Described herein are compositions for modulating transgene expression of one or more therapeutics. Also described herein are methods for using the compositions described herein for modulating transgene expression and for treating a disease or condition.

Description

WSGR Docket No. 59561-719.601COMBINATION THERAPY FOR TREATMENT OF OCULAR DISEASE CROSS-REFERNCE

[0001] This application claims the benefit of U.S. Provisional Patent Application Number 63 / 721,210 filed on November 15, 2024; U.S. Provisional Patent Application Number 63 / 770,299 filed on March 11, 2025; U.S. Provisional Patent Application Number 63 / 721,219 filed on November 15, 2024; and U.S. Provisional Patent Application Number 63 / 770,294 filed on March 11, 2025, each of which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Neovascularization, including vasculogenesis, angiogenesis, and arteriogenesis, is regulated by a wide variety of cell signaling pathways. One of the signaling pathways is regulated by vascular endothelium growth factors (VEGFs). VEGFs are strong mitogens for endothelial cells, inducing proliferation, migration, blood vessel tubing formation, and permeability. As such, increase in VEGF signaling transduction pathway increases neovascularization signal, while decrease or inhibition of VEGF signaling transduction pathway decreases neovascularization signal. VEGF inhibition is one of the most popular treatment options for disease or condition related to neovascularization. For example, Treatment of ocular diseases often involves the use of angiogenesis inhibitors such as VEGF inhibitor.SUMMARY

[0003] Current treatments employing VEGF inhibitors can be cumbersome due to the short halflife of the VEGF inhibitor, which leads to the need for repeated monthly injections for achieving and sustaining suppression of neovascularization. Therefore, there remains a need for a therapeutic for treating ocular diseases. There also remains a need for therapeutics for inhibiting neovascularization by targeting signaling pathways other than or in combination with VEGF signaling pathways.

[0004] Provided herein, in one aspect is an engineered polynucleotide comprising one or more expression cassettes, the one or more expression cassettes encodes a CD59, a complement 3 inhibitor or a C3 degraded fragment, and a C-type natriuretic peptide (CNP). In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is covalently connected to an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a fragment crystallizable (Fc) region. In some embodiments, the CNP is covalently connected to the antibody or fragment thereof. In some embodiments, the CD59 is expressed from a first expression cassette of the one or more expression cassettes, and wherein the complement 3 inhibitor or the C3 degraded fragment and the CNP are expressed from a secondWSGR Docket No. 59561-719.601expression cassette of the one or more expression cassettes. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 234-236 or 331. In some embodiments, the CD59 is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to any one of SEQ ID NOs: 234-236 or 331. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is any one of SEQ ID NOs: 234-236 or 331. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is SEQ ID NO: 231 or SEQ ID NO: 232 In some embodiments, the CNP is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231 In some embodiments, the CNP is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231. In some embodiments, the CNP is encoded from a nucleic acid sequence that is SEQ ID NO: 231. In some embodiments, the CD59 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 244-246. In some embodiments, wherein the CD59 comprises at least 50 contiguous polypeptides that are identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 244-246 In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241 or SEQ ID NO: 242. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises at least 5 contiguous polypeptides that are identical to SEQ ID NO: 241 or SEQ ID NO: 242. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is SEQ ID NO: 241 or SEQ ID NO: 242. In some embodiments, the CNP comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241. In someWSGR Docket No. 59561-719.601embodiments, the CNP comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 241. In some embodiments, the CNP comprises an amino acid sequence that is SEQ ID NO: 241. In some embodiments, the engineered polynucleotide comprises a viral vector. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the AAV vector is an AAV2 vector. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183). In some embodiments, the polypeptide sequence is LALGQTTKPA (SEQ ID NO: 183).

[0005] Provided herein, in one aspect is an engineered polypeptide comprising a CD59, a complement 3 inhibitor or a C3 degraded fragment, and a C-type natriuretic peptide (CNP).

[0006] Provided herein, in one aspect is a vector comprising the engineered polynucleotide disclosed herein, or the engineered polypeptide disclosed herein. In some embodiments, the vector encodes an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183). In some embodiments, the polypeptide sequence is LALGQTTKPA (SEQ ID NO: 183).

[0007] Provided herein, in one aspect is a viral particle comprising the engineered polynucleotide, the engineered polypeptide, or the vector disclosed herein. In some embodiments, the viral particle comprises an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic AcidWSGR Docket No. 59561-719.601(D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183). In some embodiments, the polypeptide sequence is LALGQTTKPA (SEQ ID NO: 183).

[0008] Provided herein, in one aspect is a method comprising: contacting a cell obtained from a subject with the engineered polynucleotide, the engineered polypeptide, the vector, or the viral particle disclosed herein.

[0009] Provided herein, in one aspect is a method of treating a disease or condition in a subject, comprising: administering to the subject the engineered polynucleotide, the engineered polypeptide, the vector, or the viral particle disclosed herein. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring neuroprotective effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring anti-angiogenesis effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring neuroprotective effect and anti-angiogenesis in the subject. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.

[0010] Provided herein, in one aspect is an engineered polynucleotide comprising one or more expression cassettes, the one or more expression cassettes encodes a CD59, a complement 3 inhibitor or a C3 degraded fragment, and an endostatin (ES). In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is covalently connected to an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a fragment crystallizable (Fc) region. In some embodiments, the endostatin is covalently connected to theWSGR Docket No. 59561-719.601antibody or fragment thereof. In some embodiments, the CD59 is expressed from a first expression cassette of the one or more expression cassettes, and wherein the complement 3 inhibitor or the C3 degraded fragment and the endostatin are expressed from a second expression cassette of the one or more expression cassettes. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 234-236 or 332. In some embodiments, the CD59 is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to any one of SEQ ID NOs: 234-236 or 332. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is any one of SEQ ID NOs: 234-236 or 332. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is SEQ ID NO: 231 or SEQ ID NO: 232 In some embodiments, the endostatin is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 233 In some embodiments, the endostatin is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 233. In some embodiments, the endostatin is encoded from a nucleic acid sequence that is SEQ ID NO: 233. In some embodiments, the CD59 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises at least 50 contiguous polypeptides that are identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 244-246. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241 or SEQ ID NO: 242 In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises at least 5 contiguous polypeptides that are identical to SEQ ID NO: 241 or SEQ ID NO: 242. In some embodiments, the complement 3 inhibitor or the C3WSGR Docket No. 59561-719.601degraded fragment comprises an amino acid sequence that is SEQ ID NO: 241 or SEQ ID NO: 242. In some embodiments, the endostatin comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 243. In some embodiments, the endostatin comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 243. In some embodiments, the endostatin comprises an amino acid sequence that is SEQ ID NO: 243. In some embodiments, the engineered polynucleotide comprises a viral vector. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the AAV vector is an AAV2 vector. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is LALGQTTKPA.

[0011] Provided herein, in one aspect is an engineered polypeptide comprising a CD59, a complement 3 inhibitor or a C3 degraded fragment, and an endostatin (ES).

[0012] Provided herein, in one aspect is a vector comprising the engineered polynucleotide, or the engineered polypeptide disclosed herein. In some embodiments, the vector encodes an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is LALGQTTKPA.

[0013] Provided herein, in one aspect is a viral particle comprising the engineered polynucleotide, the engineered polypeptide, or the vector disclosed herein. In some embodiments, the viral particle comprises an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptideWSGR Docket No. 59561-719.601sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-XI -X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is LALGQTTKPA.

[0014] Provided herein, in one aspect is a method comprising: contacting a cell obtained from a subject with the engineered polynucleotide, the engineered polypeptide, the vector, or the viral particle disclosed herein.

[0015] Provided herein, in one aspect is a method of treating a disease or condition in a subject, comprising: administering to the subject the engineered polynucleotide, the engineered polypeptide, the vector, or the viral particle disclosed herein. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the endostatin treats the disease or condition by conferring a neuroprotective effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring anti-angiogenesis effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the endostatin treats the disease or condition by conferring an antiangiogenesis effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the endostatin treats the disease or condition by conferring a neuroprotective effect and anti-angiogenesis in the subject. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.WSGR Docket No. 59561-719.601

[0016] Provided herein, in one aspect is an engineered polynucleotide comprising one or more expression cassettes, the one or more expression cassettes encoding a CD59 and an antibody or fragment thereof flanked by: a complement 3 inhibitor or a C3 degraded fragment; and a C-type natriuretic peptide (CNP) or an endostatin (ES). In some embodiments, the complement 3 inhibitor or the C3 degraded fragment and the CNP or ES are covalently connected to the antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a fragment crystallizable (Fc) region. In some embodiments, the CD59 is expressed from a first expression cassette of the one or more expression cassettes, and wherein the complement 3 inhibitor or the C3 degraded fragment and the CNP or the ES are expressed from a second expression cassette of the one or more expression cassettes. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 234-236, 331, or 332. In some embodiments, the CD59 is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to any one of SEQ ID NOs: 234-236, 331, or 332. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is any one of SEQ ID NOs: 234-236, 331, or 332. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is SEQ ID NO: 231 or SEQ ID NO: 232 In some embodiments, the CNP is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231 In some embodiments, the CNP is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231. In some embodiments, the CNP is encoded from a nucleic acid sequence that is SEQ ID NO: 231. In some embodiments, the endostatin is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 233. In some embodiments, the endostatin is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical toWSGR Docket No. 59561-719.601SEQ ID NO: 233. In some embodiments, the endostatin is encoded from a nucleic acid sequence that is SEQ ID NO: 233 In some embodiments, the CD59 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises at least 50 contiguous polypeptides that are identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 244-246. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241 or SEQ ID NO: 242. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises at least 5 contiguous polypeptides that are identical to SEQ ID NO: 241 or SEQ ID NO: 242. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is SEQ ID NO: 241 or SEQ ID NO: 242. In some embodiments, the CNP comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241. In some embodiments, the CNP comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 241. In some embodiments, the CNP comprises an amino acid sequence that is SEQ ID NO: 241 In some embodiments, the endostatin comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 243 In some embodiments, the endostatin comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 243. In some embodiments, the endostatin comprises an amino acid sequence that is SEQ ID NO: 243. In some embodiments, the engineered polynucleotide comprises a viral vector. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the AAV vector is an AAV2 vector. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183). In some embodiments, the polypeptide sequence is LALGQTTKPA (SEQ ID NO: 183)WSGR Docket No. 59561-719.601

[0017] Provided herein, in one aspect is a vector comprising the engineered polynucleotide disclosed herein.

[0018] Provided herein, in one aspect is a viral particle comprising the engineered polynucleotide or the vector disclosed herein.

[0019] Provided herein, in one aspect is a composition comprising: a CD59; a complement 3 inhibitor or a C3 degraded fragment; and a C-type natriuretic peptide (CNP) or an endostatin (ES). In some embodiments, the composition comprises the CD59, the complement 3 inhibitor or the C3 degraded fragment, and the CNP. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment and the CNP are covalently connected. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment and the CNP are covalently connected by an antibody of fragment thereof. In some embodiments, the composition comprises the CD59, the complement 3 inhibitor or the C3 degraded fragment, and the ES. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment and the ES are covalently connected. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment and the ES are covalently connected by an antibody of fragment thereof. In some embodiments, the antibody or fragment thereof comprises a Fc region.

[0020] Provided herein, in one aspect is a method comprising: contacting a cell obtained from a subject with the engineered polynucleotide, the vector, the viral particle, or the composition disclosed herein.

[0021] Provided herein, in one aspect is a method of treating a disease or condition in a subject, comprising: administering to the subject the engineered polynucleotide, the vector, the viral particle, or the composition.

[0022] Provided herein, in one aspect is a method of contacting a retinal cell of a subject with an engineered polynucleotide encoding a CD59 and an antibody or fragment thereof flanked by: a complement 3 inhibitor or a C3 degraded fragment; and a C-type natriuretic peptide (CNP) or an endostatin (ES).

[0023] Provided herein, in one aspect is a method of treating a disease or condition in a subject, comprising administering to the subject an engineered polynucleotide encoding a CD59 and an antibody or fragment thereof flanked by: a complement 3 inhibitor or a C3 degraded fragment; and a C-type natriuretic peptide (CNP) or an endostatin (ES), wherein administration of the engineered polynucleotide increases therapeutic efficacy compared to administering a comparable complement inhibitor alone or a comparable anti-angiogenic agent alone to the subject. In some embodiments, the engineered polynucleotide encodes the CD59, theWSGR Docket No. 59561-719.601complement 3 inhibitor or the C3 degraded fragment and the CNP. In some embodiments, the engineered polynucleotide encodes the CD59, the complement 3 inhibitor or the C3 degraded fragment and the ES. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP or the ES increases neuroprotective effect in the subject compared to administering the comparable complement inhibitor alone or the comparable anti-angiogenic agent alone to the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP or the ES increases anti-angiogenesis effect in the subject compared to administering the comparable complement inhibitor alone or the comparable anti-angiogenic agent alone to the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP or the ES increases retinal function in the subject compared to administering the comparable complement inhibitor alone or the comparable anti-angiogenic agent alone to the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP or the ES decreases vascular leakage in the subject compared to administering the comparable complement inhibitor alone or the comparable anti -angiogenic agent alone to the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP or the ES decreases inflammation in the subject compared to administering the comparable complement inhibitor alone or the comparable anti-angiogenic agent alone to the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP or the ES decreases neovascularization in the subject compared to administering the comparable complement inhibitor alone or the comparable anti-angiogenic agent alone to the subject. In some embodiments, the comparable completement inhibitor comprises pegcetacoplan. In some embodiments, the comparable anti-angiogenic agent comprises Elyea. In some embodiments, the engineered polynucleotide comprises a viral vector. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the AAV vector is an AAV2 vector. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered polynucleotide is encapsulated in a viral capsid. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabeticWSGR Docket No. 59561-719.601retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.

[0024] Described herein, in some aspects, is an engineered polynucleotide comprising one or more expression cassettes, the one or more expression cassettes encodes a CD59, a complement 3 inhibitor or a C3 degraded fragment, and a C-type natriuretic peptide (CNP). In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is covalently connected to an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a fragment crystallizable (Fc) region. In some embodiments, the CNP is covalently connected to the antibody or fragment thereof. In some embodiments, the CD59 is expressed from a first expression cassette of the one or more expression cassettes, and wherein the complement 3 inhibitor or the C3 degraded fragment and the CNP are expressed from a second expression cassette of the one or more expression cassettes. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 234-236 or 331. In some embodiments, the CD59 is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to any one of SEQ ID NOs: 234-236 or 331. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is any one of SEQ ID NOs: 234-236 or 331. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is SEQ ID NO: 231 or SEQ ID NO: 232 In some embodiments, the CNP is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231 In some embodiments, the CNP is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250WSGR Docket No. 59561-719.601contiguous nucleotides that are identical to SEQ ID NO: 231. In some embodiments, the CNP is encoded from a nucleic acid sequence that is SEQ ID NO: 231. In some embodiments, the CD59 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises at least 50 contiguous polypeptides that are identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises an amino acid sequence that is any one ofSEQ ID NOs: 244-246 In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241 or SEQ ID NO: 242. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises at least 5 contiguous polypeptides that are identical to SEQ ID NO: 241 or SEQ ID NO: 242. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is SEQ ID NO: 241 or SEQ ID NO: 242. In some embodiments, the CNP comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241. In some embodiments, the CNP comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 241 In some embodiments, the CNP comprises an amino acid sequence that is SEQ ID NO: 241 In some embodiments, the engineered polynucleotide comprises a viral vector. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the AAV vector is an AAV2 vector. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183). In some embodiments, the polypeptide sequence is LALGQTTKPA (SEQ ID NO: 183).

[0025] Described herein, in some aspects, is an engineered polypeptide comprising a CD59, a complement 3 inhibitor or a C3 degraded fragment, and a C-type natriuretic peptide (CNP) described herein.

[0026] Described herein, in some aspects, is a vector comprising an engineered described herein or an engineered polypeptide described herein. In some embodiments, the vector encodes anWSGR Docket No. 59561-719.601AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183). In some embodiments, the polypeptide sequence is LALGQTTKPA (SEQ ID NO: 183).

[0027] Described herein, in some aspects, is a viral particle comprising an engineered polynucleotide described herein, an engineered polypeptide described herein, or a vector described herein. In some embodiments, the viral particle comprises an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183). In some embodiments, the polypeptide sequence is LALGQTTKPA (SEQ ID NO: 183).

[0028] Described herein, in some aspects, is a method comprising: contacting a cell obtained from a subject with an engineered polynucleotide described herein, an engineered polypeptide described herein, a vector described herein, or a viral particle described herein.

[0029] Described herein, in some aspects, is a method of treating a disease or condition in a subject, comprising: administering to the subject an engineered polynucleotide described herein, an engineered polypeptide described herein, a vector described herein, or a viral particle described herein. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring neuroprotective effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring anti-angiogenesis effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring neuroprotective effect and anti-angiogenesis in the subject. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, theWSGR Docket No. 59561-719.601ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.

[0030] Described herein, is an engineered polynucleotide comprising one or more expression cassettes, the one or more expression cassettes encoding a CD59, a complement 3 inhibitor or a C3 degraded fragment, and a C-type natriuretic peptide (CNP). In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is covalently connected to an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a fragment crystallizable (Fc) region. In some embodiments, the engineered polynucleotide comprises a viral vector. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the AAV vector is an AAV2 vector. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183). In some embodiments, the polypeptide sequence is LALGQTTKPA (SEQ ID NO: 183).

[0031] Described herein, in some aspects, is an engineered polypeptide comprising a CD59, a complement 3 inhibitor or a C3 degraded fragment, and a C-type natriuretic peptide (CNP). Also described herein, in some aspects, is a vector comprising an engineered polynucleotide described herein, or an engineered polypeptide described herein. In some embodiments, the vector encodes an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-WSGR Docket No. 59561-719.601X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183). In some embodiments, the polypeptide sequence is LALGQTTKPA (SEQ ID NO: 183).

[0032] Described herein, in some aspects, is a viral particle comprising an engineered polynucleotide described herein, an engineered polypeptide described herein, or a vector described herein. In some embodiments, the viral particle comprises an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183). In some embodiments, the polypeptide sequence is LALGQTTKPA (SEQ ID NO: 183).

[0033] Described herein, in some aspects, is a method comprising: contacting a cell obtained from a subject with an engineered polynucleotide described herein, an engineered polypeptide described herein, a vector described herein, or a viral particle described herein.

[0034] Described herein, in some aspects, is a method of treating a disease or condition in a subject, comprising: administering to the subject an engineered polynucleotide described herein, an engineered polypeptide described herein, a vector described herein, or a viral particle described herein. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring neuroprotective effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring anti-angiogenesis effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring neuroprotective effect and anti-angiogenesis in the subject. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macularWSGR Docket No. 59561-719.601degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.

[0035] Described herein, in some aspects, is an engineered polynucleotide comprising one or more expression cassettes, the one or more expression cassettes encodes a CD59, a complement 3 inhibitor or a C3 degraded fragment, and an endostatin (ES). In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is covalently connected to an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a fragment crystallizable (Fc) region. In some embodiments, the endostatin is covalently connected to the antibody or fragment thereof. In some embodiments, the CD59 is expressed from a first expression cassette of the one or more expression cassettes, and wherein the complement 3 inhibitor or the C3 degraded fragment and the endostatin are expressed from a second expression cassette of the one or more expression cassettes. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 234-236 or 332. In some embodiments, the CD59 is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to any one of SEQ ID NOs: 234-236 or 332. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is any one of SEQ ID NOs: 234-236 or 332. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the endostatin is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 233. In some embodiments, the endostatin is encoded from a nucleic acid sequence comprising at least 50, at least 100, at leastWSGR Docket No. 59561-719.601150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 233. In some embodiments, the endostatin is encoded from a nucleic acid sequence that is SEQ ID NO: 233. In some embodiments, the CD59 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises at least 50 contiguous polypeptides that are identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 244-246. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241 or SEQ ID NO: 242 In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises at least 5 contiguous polypeptides that are identical to SEQ ID NO: 241 or SEQ ID NO: 242 In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is SEQ ID NO: 241 or SEQ ID NO: 242. In some embodiments, the endostatin comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 243. In some embodiments, the endostatin comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 243. In some embodiments, the endostatin comprises an amino acid sequence that is SEQ ID NO: 243. In some embodiments, the engineered polynucleotide comprises a viral vector. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the AAV vector is an AAV2 vector. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-XI -X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is LALGQTTKPA.

[0036] Described herein, in some aspects, is an engineered polypeptide comprising a CD59, a complement 3 inhibitor or a C3 degraded fragment, and an endostatin (ES).

[0037] Described herein, in some aspects is a vector comprising an engineered polynucleotide described herein or an engineered polypeptide described herein. In some embodiments, the vector encodes an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid. InWSGR Docket No. 59561-719.601some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is LALGQTTKPA.

[0038] Described herein, in aspects, is a viral particle comprising an engineered polynucleotide described herein, an engineered polypeptide described herein, or a vector described herein. In some embodiments, the viral particle comprises an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is LALGQTTKPA.

[0039] Described herein, in some aspects, is a method comprising: contacting a cell obtained from a subject with an engineered polynucleotide described herein, an engineered polypeptide described herein, a vector described herein, or a viral particle described herein.

[0040] Described herein, in some aspects, is a method of treating a disease or condition in a subject, comprising: administering to the subject an engineered polynucleotide described herein, an engineered polypeptide described herein, a vector described herein, or a viral particle described herein. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the endostatin treats the disease or condition by conferring a neuroprotective effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring anti-angiogenesis effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the endostatin treats the disease or condition by conferring an anti-angiogenesis effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the endostatin treats the disease or condition by conferring a neuroprotective effect and antiangiogenesis in the subject. In some embodiments, the disease or condition comprises an ocularWSGR Docket No. 59561-719.601disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.

[0041] Described herein, in some aspects, is an engineered polynucleotide comprising one or more expression cassettes, the one or more expression cassettes encoding a CD59, a complement 3 inhibitor or a C3 degraded fragment, and an endostatin. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is covalently connected to an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a fragment crystallizable (Fc) region. In some embodiments, the engineered polynucleotide comprises a viral vector. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the AAV vector is an AAV2 vector. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is LALGQTTKPA.

[0042] Described herein, in some aspects, is an engineered polypeptide comprising a CD59, a complement 3 inhibitor or a C3 degraded fragment, and a endostatin. Also described herein is a vector comprising an engineered polynucleotide described herein, or an engineered polypeptide described herein. In some embodiments, the vector encodes an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-XI -X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T),WSGR Docket No. 59561-719.601Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is LALGQTTKPA.

[0043] Described herein, in some aspects, is a viral particle comprising an engineered polynucleotide described herein, an engineered polypeptide described herein, or a vector described herein. In some embodiments, the viral particle comprises an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-XI -X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is LALGQTTKPA.

[0044] Described herein, in some aspects, is a method comprising: contacting a cell obtained from a subject with an engineered polynucleotide described herein, an engineered polypeptide described herein, a vector described herein, or a viral particle described herein.

[0045] Described herein, in some aspects, is a method of treating a disease or condition in a subject, comprising: administering to the subject an engineered polynucleotide described herein, an engineered polypeptide described herein, a vector described herein, or a viral particle described herein. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the endostatin treats the disease or condition by conferring neuroprotective effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the endostatin treats the disease or condition by conferring antiangiogenesis effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the endostatin treats the disease or condition by conferring neuroprotective effect and anti-angiogenesis in the subject. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR),WSGR Docket No. 59561-719.601proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.INCORPORATION BY REFERENCE

[0046] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure can be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein).

[0048] Fig. 1 illustrates vector construct for expressing a complement C3 inhibitor (C3i).

[0049] Fig. 2A and Fig. 2B illustrate vector construct for expressing an C3i operatively coupled to a natriuretic polypeptide.

[0050] Fig. 3 illustrates vector construct for expressing an C3i, an CD59, and a natriuretic polypeptide.

[0051] Fig. 4 illustrates exemplary vectors for expressing the angiogenesis inhibitors described herein.

[0052] Figs. 5A-5C illustrate AAV constructs created and tested in Example 3.

[0053] Fig. 6 illustrates fusion proteins expressed by cells on 6-well plates were detected by SDS-PAGE and Western blot using HRP-conjugated goat anti-human IgGl Fc antibody. Lane content: 1. Vector GAM 3d; 2. Vector EKQ 3d; 3. Vector CME 3d; 4. Non-transfected 3d; 5. Vector GAM 6d; 6. Vector EKQ 6d; 7. Vector CME 6d; 8. Non-transfected 6d.

[0054] Fig. 7 illustrates fusion proteins expressed by the cells in T125 flasks were detected by SDS-PAGE and Western blot using HRP-conjugated goat anti-human IgGl Fc antibody. LaneWSGR Docket No. 59561-719.601content: 1. Vector GAM 3d; 2. Vector EKQ 3d; 3. Vector CME 3d; 4. non-transfected 3d; 5. Vector GAM 6d; 6. Vector EKQ 6d; 7. Vector CME 6d; 8. non-transfected 6d.

[0055] Fig. 8 illustrates fusion proteins expressed by the cells on 6-well plates were detected by SDS-PAGE and Western blot using rat anti-human CNP antibodies. Lane content: 1. Vector GAM 3d; 2. Vector EKQ 3d; 3. Vector CME 3d; 4. Non-transfected 3d; 5. Vector GAM 6d; 6. Vector EKQ 6d; 7. Vector CME 6d; 8. Non- transfected 6d.

[0056] Fig. 9 illustrates fusion proteins expressed by the cells on T125 flasks were detected by SDS-PAGE and Western blot using rat anti-human CNP antibodies based on Western blot analysis. Lane content: 1. Vector GAM 3d; 2. Vector EKQ 3d; 3. Vector CME 3d; 4. non-transfected 3d; 5. Vector GAM 6d; 6. Vector EKQ 6d; 7. Vector CME 6d; 8. Non-transfected 6d.

[0057] Fig. 10 illustrates membrane-bound CD59 was expressed in transfected cells based on Western blot analysis. Lane content: 1. Vector GTM; 2. Vector GTP; 3. Vector GTQ; 4. Vector GTR; 5. Vector GAT; 6. Vector EKQ; 7. Non-transfected; 8. Purified CD59 protein.

[0058] Fig. 11 illustrates Soluble CD59 (sCD59) protein in the cell culture supernatant at day 3 was detected using HRP-conjugated anti-human CD59 antibody based on Western blot analysis. Lane content: 1. Vector GCK; 2. Vector GCM; 3. Vector GEM; 4. Vector GEP; 5. Vector EKQ; 6. Non-transfected; 7. Purified CD59 protein.

[0059] Fig. 12 illustrates soluble CD59 (sCD59) protein in the cell culture supernatant at day 6 was detected using HRP-conjugated anti-human CD59 antibody based on Western blot analysis. Lane content: 1. Vector GCK; 2. Vector GCM; 3. Vector GEM; 4. Vector GEP; 5. Vector EKQ; 6. Non-transfected; 7. Purified CD59 protein.

[0060] Fig. 13 illustrates schematic AAV vector designs for Example 5.

[0061] Fig. 14 illustrates soluble CD59 (sCD59) protein large scale production in cell culture supernatant at day 6 was shown to be pure using SDS-PAGE gel analysis.

[0062] Fig. 15A and Fig. 15B illustrate graphs depicting the results of a cell lysis inhibition assay. Fig. 15A illustrates a dose-response curve of % cytotoxicity from increasing concentration of normal human serum (NHS). Fig. 15B illustrates a dose-response curve of % max cytotoxicity normalized to BSA control compared to Vector GEM and CP40 cytoprotective effects.

[0063] Fig. 15C illustrates hemolytic inhibition activities of sCD59 and vsCD59 using guinea pig red blood cells (GPRBC) and normal human serum (NHS).

[0064] Fig. 16 illustrates schematic AAV vector designs for Example 6.

[0065] Fig. 17 illustrates fusion proteins expressed by cells detected by SDS-PAGE and Western blot using HRP-conjugated mouse anti-human Fc antibodies. Lane content: M. Pre-stain proteinWSGR Docket No. 59561-719.601marker; 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. Non-transfected cells.

[0066] Fig. 18 illustrates fusion proteins expressed by cells detected by SDS-PAGE and Western blot using Biotin-labeled goat-anti-human IgG antibodies and HRP-Streptavidin conjugate. Lane content: M. Pre-stain 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 proteins.

[0067] Fig. 19 illustrate Vector GGG proteins expressed by the cells HEK293LTV in 6 well plates were shown to be pure using SDS-PAGE gel analysis. Lane content: M. Protein marker; 1. cell culture medium; 2. flowthrough; 3. Wash 1; 4. Wash 2; 5. Elution sample; 6. Proteins in PBS buffer.

[0068] Fig. 20 illustrates Vector GGE proteins expressed by the cells HEK293LTV in 6 well plates were shown to be pure using SDS-PAGE gel analysis. Lane content: M. Protein marker; 1. cell culture medium; 2. flowthrough; 3. Wash 1; 4. Wash 2; 5. Elution sample; 6. Proteins in PBS buffer.

[0069] Figs. 21A-21C illustrate mutant fusion proteins expressed by cells detected by SDS-PAGE and a hemolysis inhibition assay. Fig. 21A illustrates design of mutant proteins derived from Vector GGG. Fig. 21B illustrates protein expressed by Expi293F cells shown to be of high purity using SDS-PAGE gel analysis. Lane content: 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. Fig. 21C illustrates a graph depicting results of a hemolysis inhibition assay for all mutant proteins.

[0070] Fig. 22A and Fig. 22B illustrate fusion proteins expressed by cells detected by SDS-PAGE and results from a hemolysis inhibition assay. Fig. 22A illustrates proteins expressed shown to be pure using SDS-PAGE gel analysis. Lane content: M. Protein marker; 1, Vector GGE; 2, Vector GGG; 3, Vector KAG; 4, Vector KKT; 5, Vector KKA; 6, Vector KPT; 7, Vector KPC. Fig. 22B illustrates a graph depicting results of a hemolysis inhibition assay for all mutant proteins.

[0071] Figs. 23A-23C illustrate graphs depicting results from a binding affinity assay of fusion proteins against C3b, C3c, and C3. Fig. 23A illustrates estimated IC50 values and KD vs C3b of Vector KPT, CP40, Vector KKT, Vector KPC, Vector KKA, Vector GGE, Vector KAG, and Vector GGG. Fig. 23B illustrates estimated IC50 values and KD vs C3c of Vector KPT, CP40, Vector KKT, Vector KPC, Vector KKA, Vector GGE, Vector KAG, and Vector GGG. Fig. 23CWSGR Docket No. 59561-719.601illustrates estimated IC50 values and KD vs C3 of Vector KPT, CP40, Vector KKT, Vector KPC, Vector KKA, Vector GGE, Vector KAG, and Vector GGG.

[0072] Fig. 24 illustrates schematic AAV vector designs for Vector KMR, Vector KKT, and Vector KPP.

[0073] Figs. 25A-25D illustrate protein expression in ARPE-19 cells secreted into cell medium or remaining within the cell. Fig. 25A illustrates expression of CD59 from Vector KMR, Vector KKT, and Vector KPP. Fig. 25B illustrates expression of C3i from Vector KMR, Vector KKT, and Vector KPP. Fig. 25C illustrates expression of Fc-CNP from Vector KKT. Fig. 25D illustrates expression of Endostatin from Vector KPP.

[0074] Fig. 26 illustrates schematic AAV vector designs for Vector KGQ, Vector KGR, Vector KKT, and Vector KKA.

[0075] Figs. 27A-27G illustrate results from AAV in vivo expression in mice eyes and statistical analysis. Fig. 27A illustrates a graph depicting results from the expression of CD59 from Vector KGQ, Vector KGR, Vector KKT, and Vector KKA. Fig. 27B illustrates a graph depicting results from the expression of Fc-CNP from Vector KKT. Fig. 27C illustrates a graph depicting results from the expression of hFc from Vector KGQ, Vector KGR, Vector KKT, and Vector KKA. Fig.27D illustrates a graph depicting results from the expression of endostatin from Vector KKA. Fig. 27E illustrates a graph depicting results from the expression of CD59 and C3i from Vector KGQ and Vector KGR. Fig. 27F illustrates a graph depicting results from the expression of CD59, C3i, and CNP from Vector KKT. Fig. 27G illustrates a graph depicting results from the expression of CD59, C3i, and endostatin from Vector KKA.

[0076] Figs. 28A-28C illustrate results from a ligand binding assay for C3i fusion proteins. Fig.28A illustrates results from an ELISA assay for binding C3b and Vector KMR, Vector KKT, and Vector KPP proteins and estimated Kd values. Fig. 28B illustrates results from an ELISA assay for binding C3c and Vector KMR, Vector KKT, and Vector KPP proteins and estimated Kd values. Fig. 28C illustrates results from an ELISA assay for binding C3 and Vector KMR, Vector KKT, and Vector KPP proteins and estimated Kd values.

[0077] Fig. 29A illustrates a graph depicting results from a ligand binding assay for NPR-b and Vector KKT protein, Fcl-CNP36, and Fc4-CNP36.

[0078] Fig. 29B illustrates binding affinity of vsCD59 to human recombinant C9 protein.

[0079] Fig. 30 illustrates a graph depicting results measuring cGMP production in NIG-ETE cells triggered by Vector CME protein (Fc4-CNP36), Vector KKT protein (C3i-Fc4-CNP36), CNP-22, or Cp40.WSGR Docket No. 59561-719.601

[0080] Fig. 31 illustrates a graph depicting results from an endostatin ligand receptor assay and estimated Kd values for Vector KPP protein.

[0081] Fig. 32 illustrates schematic AAV vector designs for Example 12.

[0082] Fig. 33 illustrates results from a ligand binding assay for wildtype and variant sCD59 proteins.

[0083] Fig. 34A illustrates an example of engineered capsid design and vector design (e.g., Vector CTA, Vector CTC, Vector KPP, Vector KKT, Vector MKT, or Vector MKC for expressing CD59, C3i, and CNP or endostatin).

[0084] Fig. 34B illustrates an example of a mechanism of action of Vector CTA, Vector KKT, Vector MKT, or Vector MKC expression.

[0085] Fig. 34C illustrates an example of engineered capsid design and vector design (e.g., Vector CTC, Vector KPP, Vector KKT, Vector MKT, or Vector MKC for expressing CD59, C3i, and endostatin).

[0086] Fig. 34D illustrates an example of a mechanism of action of Vector CTC, Vector KPP, Vector KKT, Vector MKT, or Vector MKC expression.

[0087] Fig. 35A illustrates inhibition of alternative pathway of complement activation cascade by Vector CTA expression.

[0088] Fig. 35B illustrates inhibition of classical pathway of complement activation cascade by Vector CTA expression.

[0089] Fig. 35C illustrates concentration dependent inhibitions of sheep RBC lysis were observed for Cp40 positive control and C3i-Fc-CNP, but not for BSA. Cp40 was ~20-fold more potent in inhibiting hemolysis than those of C3i-Fc-CNP. C3i-Fc-CNP exhibited higher (21.4-fold) inhibition potency in the factor B depleted serum, with IC50 values of 30.0 and 1.4 pM, in normal vs factor B depleted serum (Fig. 35B), respectively.

[0090] Fig. 35D illustrates inhibition of complement activation by blocking Membrane Attack Complex (MAC) formation by Vector CTA expression.

[0091] Fig. 35E illustrates Inhibition by vsCD59 in the terminal hemolytic pathway.

[0092] Fig. 35F illustrates an example mechanism of action for blocking retina damage by Vector CTA, Vector KKT, Vector MKT, or Vector MKC expression.

[0093] Fig. 36A illustrates gene of interest levels in mouse vitreous humor (VH) post single dose. Vector CTA expressed C3iFc at ~40 pg / mL, which was about 4 times higher above C3 level in patients’ VH.WSGR Docket No. 59561-719.601

[0094] Fig. 36B illustrates decreased angiogenesis using delivered of a vector described herein (Vector CTA) compared to delivery of vehicle or 40 pg / eye Eylea.

[0095] Fig. 36C illustrates decreased average spot leakage score after mouse treatment with expression of a vector described herein (Vector CTA) compared to treatment of vehicle or 40 pg / eye Eylea.

[0096] Fig. 37A illustrates expression of a vector described herein (Vector CTA) protected retinal ganglion cells (RGC) in rodent injury models challenged with N-methyl-D-aspartate (NMDA).

[0097] Fig. 37B illustrates expression of a vector described herein (e.g., Vector CTA) leading to lower retinal ganglion cell (RGC) apoptosis in rat partial optic nerve transection (pONT) using detection of apoptosing retinal cells (DARC).

[0098] Fig. 38A illustrates representative Fundus Fluorescein Angiography (FFA) images demonstrating recovery post sodium iodate (Sl)-induced damage.

[0099] Fig. 38B illustrates ERG results in modeled eyes. Arrow points to injection of sodium iodate by carotid artery (25 mg / kg). Syfovre was IVT dosed at 7.5 mg / eye on the third day (Day -3). Upper left graph illustrates improved function of photoreceptors (rods & cons) post sodium iodate injection via carotid artery in vector (Vector CTA) treated cynomolgus monkeys as compared to vehicle or Syfovre. Upper right graph illustrates protection of photoreceptors impaired by SI induced damage by expression of a vector described herein (Vector CTA) as compared to vehicle or Syfovre. Bottom left graph illustrates improved b-wave (DA 3.0) in the Vector CTA treated group compared to the vehicle control group. Bottom right graph illustrates improved oscillatory potential in the Vector CTA treated group compared to the vehicle control group.

[0100] Fig. 39 illustrates non-limiting examples of the vector designs for Vector KKT, Vector KMR, Vector KPP, Vector MKT, or Vector MKC.

[0101] Figs. 40A-40E illustrate representative images of FFA. Fig. 40A illustrates representative FFA images of mice administered with vehicle. Fig. 40B illustrates representative FFA images of mice administered with Vector KMR. Fig. 40C illustrates representative FFA images of mice administered with Vector KKT. Fig. 40D illustrates representative FFA images of mice administered with Vector KPP. Fig. 40E illustrates representative FFA images of mice administered with Aflibercept (e.g., Eylea).

[0102] Figs. 41A-41E illustrate representative images of FP. Fig. 41 A illustrates representative FP images of mice administered with vehicle. Fig. 41B illustrates representative FP images ofWSGR Docket No. 59561-719.601mice administered with Vector KMR. Fig. 41C illustrates representative FP images of mice administered with Vector. KKT. Fig. 41D illustrates representative FP images of mice administered with Vector KPP. Fig. 41E illustrates representative FP images of mice administered with Aflibercept (e.g., Eylea).

[0103] Fig. 42 illustrates averaged composition of different grades of lesions in each group at one and two weeks post modelling. Compared with the vehicle control, lower rate of grade 3 lesions and higher rate of grade 0 lesions was noticed in test articles and EYLEA® at one and two weeks post modelling, indicating that the inhibitory effect on CNV of test articles and EYLEA® drives the recovery of fluorescence leakage displayed by FFA. Note that statistical differences were not marked in this graph. N=15 for vehicle control and Vector KPP, N=16 for Vector KMR, Vector KKT, or EYLEA®.

[0104] Fig. 43 illustrates the occurrence rates of lesions at grade 3 and averaged CNV scores in each group at one and two weeks post modelling. Compared with the vehicle control, significantly lower rate of grade 3 lesions and averaged CNV scores was observed in test articles and EYLEA® at one and two weeks post modelling, indicating that the inhibitory effect on CNV of test articles and EYLEA® drives the recovery of fluorescence leakage displayed by FFA.Values are Mean with SD. *Compared with the vehicle control, P<0.05.#Compared with the EYLEA®, P<0.05. N=15 for vehicle control and Vector KPP, N=16 for Vector KMR, Vector KPP, or EYLEA®.

[0105] Fig. 44 illustrates individual data of effective spot number and number of each grade laser lesion (one week post modeling) of the efficacy study of Vector KMR, Vector KKT, or Vector KPP in laser-induced CNV model in mice.

[0106] Fig. 45 illustrates individual data of effective spot number and number of each grade laser lesion (one week two modeling) of the efficacy study of Vector KMR, Vector KKT, or Vector KPP in laser-induced CNV model in mice.

[0107] Fig. 46 illustrates individual data of CNV scores and rates (%) of each grade laser lesion (one week post modeling) of the efficacy study of Vector KMR, Vector KKT, or Vector KPP in laser-induced CNV model in mice.

[0108] Fig. 47 illustrates individual data of CNV scores and rates (%) of each grade laser lesion (one week two modeling) of the efficacy study of Vector KMR, Vector KKT, or Vector KPP in laser-induced CNV model in mice.

[0109] Fig. 48 illustrates schematic illustration of Vector CTA and the encoded proteins: A: ITR, B: sC AG promotor; C: TPL1, 2 and 3; D: vsCD59 (T51G); E: WPRE; F: Human growthWSGR Docket No. 59561-719.601hormone polyA sequence; G: CBA promotor; H: C3i; I: IgG4-Fc; J: CNP; K: WPRE; L: poly(A) signal; M: ITR.

[0110] Fig. 49 illustrates Vector CTA functional units detected with specific antibody and cognate binding partner. Abbreviations: C3i-Fc-CNP = complement 3 inhibitory fusion protein to IgG4 Fc fragment-CNP; ELISA = enzyme-linked immunosorbent assay.

[0111] Fig. 50 illustrates C3i-Fc-CNP-GC-cGMP signaling pathway. Abbreviations: C3i-Fc = complement 3 inhibitory fusion protein to IgG4 Fc fragment, C3i-Fc-CNP = complement 3 inhibitory fusion protein to IgG4 Fc fragment-CNP cGMP = cyclic guanosine 3 ',5'-cy clic monophosphate; CNP = C-type natriuretic peptide, GTP = guanosine-5'-triphosphate; mGC = membrane guanylyl cyclase; PKG = GMP-dependent protein kinase.

[0112] Fig. 51 illustrates cGMP production by NIH3T3 cells treated with different CNP constructs. Abbreviations: cGMP = cyclic guanosine 3 ',5'-cy clic monophosphate; CNP = C-type natriuretic peptide; C3i-Fc-CNP = complement 3 inhibitory fusion protein to IgG4 Fc fragment-CNP; Fc-CNP = IgG4 Fc fragment-CNP. Note: Vector CTA spent medium was 8 / concentrated ARPE-19 cell culture transduced with Vector CTA vector.

[0113] Fig. 52 illustrates concentrations of C3i-Fc-CNP fusion protein using two ELIS As.

[0114] Fig. 53 illustrates retinal ganglion cell count of mice from NMDA mouse model, p-values: (*): 0.0358; (**) 0.0089.

[0115] Fig. 54 illustrates DARC counts in the pONT rat models, p-values: (*) 0.0332; (**) 0.0021.

[0116] Fig. 55 illustrates live RGC Counts in the pONT rat models, p-values: (*) 0.0332; (**) 0.0021.

[0117] Fig. 56 illustrates averaged CNV scores in each group at 1- and 2-weeks post-modeling.

[0118] Fig. 57 illustrates distribution of AAV2.N54 capsid across the pig retina after IVT injection. Confocal microscopic image of the pig (no. 26) retina IHC results (left) aligned with human retina H & E staining image (right). Each layer from vitreous humor to choroid was the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer nuclear layer (ONL), inner segment layer (ISL), outer segment layer (OSL), retinal pigment epithelium (RPE).

[0119] Fig. 58 illustrates a non-limiting example of vector design for Vector CTA or Vector CTC.

[0120] Fig. 59 illustrates non-limiting examples of vector designs for Vector ETC, Vector ATR, Vector CPE, Vector TQQ, or Vector CME as examined in Example 18 or Example 19. VectorWSGR Docket No. 59561-719.601ETC and Vector ATR comprise one or more stop codons introduced into the open reading frame (ORF). As such, a gene of interest cannot be expressed, and these vectors are equivalent to the vector only. Vector CPE contains Ig4 Fc fused CNP36 (Fc4-CNP36) under CBA promoter. Vector TQQ is the positive control with Igl fused CNP36 (Fcl-CNP36) under CMV promoter. Vector CME encodes a fusion protein of Ig4 and CNP36 under CMV promoter. The fusion protein was purified from the culture supernatant of the Expi293 cells transfected with Vector CME vectors.

[0121] Fig. 60 illustrates effect on IOP profiles. Data was analyzed by subtracting OD from OS in each animal at each time point. There appears to be a mild upward trend of the TOP profile until day 10 and then down towards the baseline in the AAV-FP condition. A similar pattern of the TOP profile was also seen in the buffer condition. No regular pattern of the profile was observed in the other three conditions (AAV-P, AAV-positive, and A AV-negative).

[0122] Fig. 61 illustrates effect on IOP profiles. Data was analyzed by subtracting OD from OS in each animal at each time point. There appears to be a significant reduction in the TOP (p<0.01) of all FP concentrations, at Day 1 compared to baseline (Day 0) or Day 7 after pONT surgery.

[0123] Fig. 62 illustrates effect on IOP profiles across all groups.

[0124] Fig. 63 illustrates DARC counts in FP groups. Analysis of DARC counts in the FP treatment group are shown at baseline and 7 days after pONT. The FP (H) group appears to significantly reduce (p<0.05) the DARC count compared to buffer at 7 days after pONT.

[0125] Fig. 64 illustrates DARC count analysis. FP condition showed the lowest DARC count. In comparison with FP, the buffer conditions appeared to have the second lowest DARC counts (p=0.51), and the AAV-negative the highest (p=.002). Although still significantly higher, AAV-FP (p=.011) and AAV-positive (p=.01) DARC counts were less elevated than that of AAV-P (p< 001). Note: Based on small n numbers per treatment dose, to improve statistical power, Medium and High concentrations were combined to assess the effect of treatments as single groups. Hence, FP, AAV-P, and AAV-FP consist of both medium and high concentrations only.

[0126] Fig. 65 illustrates DARC count analysis in all groups. FP conditions showed the lowest DARC count with FP (High) concertation significantly lower than AAV-Negative (p<0.01).

[0127] Fig. 66 illustrates Effects of treatments on DARC counts - analysis of three doses of FP, AAV-FP, and AAV-P conditions at separate rime points. At baseline and pre-transection, there were no significant differences in DARC counts comparing all three doses of AAV-P and AAV-WSGR Docket No. 59561-719.601FP. However, at 7 days after pONT, there was a significant reduction in the DARC count in the AAV-FP (M) group compared to the sham (AAV-negative) condition. ((Dunnet’s ANOVA test).

[0128] Fig. 67 illustrates Semi-segmentation of the retinal whole-mount. To assess whether the treatments had a regional difference, the retinal whole-mount was semi-segmented into the superior and inferior halves.

[0129] Fig. 68 illustrates RGC density by retinal regions. RGC density (cell / mm2) was presented in the superior and inferior regions with different treatment conditions in an order of Buffer, FP, AAV-negative, AAV-positive, AAV-FP, and AAV-P.

[0130] Fig. 69 illustrates effects on RGC density. By the subtraction of the superior from the inferior, RGC density appeared to be significantly higher in the AAV-FP condition than the conditions of AAV-P (p =.002), AAV-negative (p =.005), AAV-positive (p =.006). No other comparisons were significant, p values were adjusted for five comparisons. *p<0.05, **p<0.01.

[0131] Fig. 70 illustrates effects on RGC density. By the subtraction of the superior from the inferior, RGC density appeared to be significantly higher in the AAV-FP condition than the conditions of AAV-P (p =.002), AAV-negative (p =.005), AAV-positive (p =.006). No other comparisons were significant, p values were adjusted for five comparisons. *p<0.05, **p<0.01.

[0132] Fig. 71 illustrates effects of treatments on whole retina RGC density - analysis of three doses of FP, AAV-FP, and AAV-P conditions separately. Left panel shows that the whole retina RGC density in all three doses of the FP condition were not significantly different to the buffer only condition. Middle panel shows that the whole retina RGC density in all three doses of the AAV-P condition were not significantly different to the AAV-negative condition. Right panel shows that the whole retina RGC density in all three doses of the AAV-FP condition were not significantly different to the AAV-negative condition. (Dunnet’s ANOVA test).

[0133] Fig. 72 illustrates effects of regional retina RGC density - analysis of three doses of FP, AAV-FP, and AAV-P conditions separately. Regional density calculations were done by subtraction of the superior RGC density from the inferior. Left panel shows that RGC density appeared significantly lower (p<0.05) in the FP (L) condition compared to buffer whilst the FP (M) and FP (H) condition showed no significant difference to the buffer condition. Middle panel shows that RGC density in all three doses of AAV-P condition had no significant differences to the vector negative condition. Right panel shows that AAV-FP(L) and AAV-FP (H) conditions both had significantly higher RGC density (p<0.05) to the vector negative condition whilst there was no significant difference between the AAV-FP (M) and vector negative conditions.(Dunnet’s ANOVA test).WSGR Docket No. 59561-719.601

[0134] Fig. 73 illustrates microglial activity. Morphology of Iba-1+ microglia is present as ramified, hyper-ramified, activated, amoeboid, and rod. The percentage of each morphology with different treatment conditions was analyzed. The percentage of ramified microglia appeared to be significantly reduced in FP condition compared to AAV-positive (p<0.05) and AAV-P (p<0.01), which was accompanied by a significant increase in hyper-activated (p<0.05), activated (p<0.05 ~ p<0.01), amoeboid (p<0.05 ~ p<0.01), and rod (p<0.05 ~ p<0.01) morphology compared to other AAV conditions. *p<0.05, **p<0.01

[0135] Fig. 74A and Fig. 74B illustrate percentage of microglia morphotypes in all groups. The graph shows the average percentages of IBA-1 positive microglia morphotypes in grouped experimental groups (Fig. 74A) and also in all treatment groups including low, medium, and high doses (Fig. 74B).

[0136] Fig. 75 illustrates ramified microglia in all groups. The percentage of ramified microglia in FP (H) was significantly lower than in AAV-P (H). No differences were found between other treatment groups.

[0137] Fig. 76A-76C illustrate effects of treatments on the percentage of ramified microglia -analysis of three doses of FP, AAV-FP, and AAV-P conditions separately. Fig. 76A shows that the percentage of ramified microglia in all three doses of the FP condition are not significantly different to the buffer only condition. Fig. 76B shows that the percentage of ramified microglia in all three doses of the AAV-P condition are not significantly different to the AAV-negative condition. Fig. 76C shows that the percentage of ramified microglia in all three doses of the AAV-FP condition are not significantly different to the AAV-negative condition. (Dunnet’s ANOVA test)

[0138] Fig. 77A and Fig. 77B illustrate inflammatory cell OCT analysis. The vitreous inflammatory cells on OCT images were manually counted and presented as time points of one week before (pre-) and one week after (post-, terminal) transection in each treatment condition (Fig. 77 A) and subtracting the pre-transection from the post-terminal timepoint (Fig. 77B). A clear trend of reduction of inflammation scores in terminal time points was observed in all conditions except the FP condition which showed the opposite pattern (Fig. 77A). Subtracting inflammation scores of the pre-transection from the post-terminal timepoint revealed that both the AAV-FP and AAV-P conditions appeared to be more effective in reducing inflammation, compared to the FP condition which had the highest inflammation score (Fig. 77B).WSGR Docket No. 59561-719.601

[0139] Fig. 78 illustrates inflammatory cell OCT analysis in all groups. Inflammation scores in FP (H) was significantly higher than buffer (p<0.01), vector negative (p<0.05), vector positive (p<0.05), AAV-P (M, p<0.05 and H, p<0.05), AAV-FP (L, p<0.05, M, p<0.001, and H, p<0.01).

[0140] Figs. 79A-79C illustrate inflammatory cell OCT analysis - observing three doses of FP, AAV-FP, and AAV-P conditions separately. Fig. 79A shows that the inflammation scores in FP (L), FP (M) and FP (H) conditions were significantly higher (p<0.001) than the buffer condition.Fig. 79B shows that the inflammation scores of all AAV-P dose conditions were not significantly different from the vector negative condition, although AAV-P (M) and AAV-P (H) seemed to have a similar trend in reduced inflammation scores. Fig. 79C shows that the inflammation scores of all AAV-FP dose conditions were also not significantly different from the vector negative condition and all doses followed the same trend as the vector negative condition.(Dunnet’s ANOVA test)

[0141] Fig. 80 illustrates comparison of DARC count with RBPMS+RGC count showing that the DARC count results as bar graphs corresponding to the left y axis and the inferior-superior region RBPMS+RGC cell density as a scatter plot corresponding to the right y axis.

[0142] Fig. 81 illustrates comparison of the percentage of ramified microglia with OCT inflammation showing the percentage of ramified microglia shown as bar graphs which correspond to the left y axis and the OCT inflammation scores (Post-Pre) corresponding to the right y axis. As there was more OCT inflammation, the Post-Pre scores would be elevated and also the ramified percentages to be reduced, except the AAV-FP condition which was the opposite.

[0143] Fig.82A illustrates gene of interest levels in mouse vitreous humor (VH) post single dose. Vector CTC expressed C3iFc at about 40 pg / mL, which was about 4 times higher above C3 level in patients’ VH.

[0144] Fig. 82B illustrates decreased angiogenesis using delivered Vector CTC compared to delivery of vehicle or 40 pg / eye Eylea.

[0145] Fig. 82C illustrates decreased average spot leakage score after mouse treatment with Vector CTC compared to treatment of vehicle or 40 pg / eye Eylea.

[0146] Fig. 83 illustrates representative Fundus Fluorescein Angiography (FFA) images demonstrating recovery post sodium iodate (Sl)-induced damage.

[0147] Fig. 84 illustrates improved function of photoreceptors (rods & cons) post sodium iodate injection via carotid artery in Vector CTC treated cynomolgus monkeys as compared to vehicle or Syfovre (pegcetacoplan).WSGR Docket No. 59561-719.601

[0148] Fig. 85 illustrates protection of photoreceptors impaired by SI induced damage by Vector CTC as compared to vehicle or Syfovre (pegcetacoplan).

[0149] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments.DETAILED DESCRIPTION

[0150] Described herein, in some aspects, is an engineered polynucleotide comprising one or more expression cassettes, the one or more expression cassettes encoding: a CD59; a complement 3 inhibitor (C3i) or a C3 degraded fragment; and a C-type natriuretic peptide (CNP) or an endostatin (ES). In some embodiments, the engineered polynucleotide comprises a viral vector (e.g., Vector KKT, Vector MKT, or Vector MKC as shown in Fig. 39 or Vector CTA or Vector CTC as shown in Fig. 58). In some embodiments, the engineered polynucleotide comprises one or more expression cassettes, where the one or more expression cassettes encodes a CD59, a complement 3 inhibitor or a C3 degraded fragment, a C-type natriuretic peptide (CNP), an endostatin, or a combination thereof. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 231-236. In some embodiments, SEQ ID NOs: 231-236 encode the CD59, the complement 3 inhibitor or the C3 degraded fragment, the CNP, the endostatin, or a combination thereof. In some embodiments, the engineered polynucleotide encodes an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 241-246

[0151] In some embodiments, the engineered polynucleotide is encapsulated in a viral capsid. In some embodiments, the viral capsid can be an engineered capsid described herein. In some embodiments, the engineered capsid can be modified to increase efficiency of delivering the engineered polynucleotide to a cell. Fig. 34A illustrates a non-limiting example of the engineered capsid and the engineered polynucleotide. Fig. 34B illustrates a non-limiting example of mechanism of action for expressing multiple engineered polypeptide described herein (e.g., a CD59, a complement 3 inhibitor or a C3 degraded fragment, a CNP, an ES, or a combination thereof). Figs. 36-38 illustrate the increased therapeutic efficacy of expressing multiple engineered polypeptide described herein (e.g., a CD59, a complement 3 inhibitor or a C3 degraded fragment, a C-type natriuretic peptide (CNP), an endostatin, or a combination thereof).WSGR Docket No. 59561-719.601Fig. 39 illustrates non-limiting examples of the vector designs for Vector KKT, Vector KMR, Vector KPP, Vector MKT, or Vector MKC.

[0152] In some embodiments, the engineered polynucleotide encodes a CD59, a complement 3 inhibitor or a C3 degraded fragment, and a CNP or an ES. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is covalently connected to an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a fragment crystallizable (Fc) region. In some embodiments, the CNP or the ES is covalently connected to the antibody or fragment thereof. In some embodiments, the CD59 is expressed from a first expression cassette of the one or more expression cassettes, and the complement 3 inhibitor or the C3 degraded fragment and the CNP or the ES are expressed from a second expression cassette of the one or more expression cassettes. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 234-236, 331, or 332. In some embodiments, the CD59 is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to any one of SEQ ID NOs: 234-236, 331, or 332. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is any one of SEQ ID NOs: 234-236, 331, or 332. In some embodiments, the CD59 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises at least 50 contiguous polypeptides that are identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 244-246.

[0153] In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241 or SEQ ID NO: 242 In some embodiments, the complement 3 inhibitor or the C3WSGR Docket No. 59561-719.601degraded fragment comprises at least 5 contiguous polypeptide that are identical to SEQ ID NO: 241 or SEQ ID NO: 242 In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is SEQ ID NO: 241 or SEQ ID NO: 242.

[0154] In some embodiments, the CNP is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231. In some embodiments, the CNP is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231. In some embodiments, the CNP is encoded from a nucleic acid sequence that is SEQ ID NO: 231. In some embodiments, the CNP comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241 In some embodiments, the CNP comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 241. In some embodiments, the CNP comprises an amino acid sequence that is SEQ ID NO: 241.

[0155] In some embodiments, the endostatin (ES) is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 233 In some embodiments, the endostatin is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 233. In some embodiments, the endostatin is encoded from a nucleic acid sequence that is SEQ ID NO: 233. In some embodiments, the endostatin comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 243 In some embodiments, the endostatin comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 243 In some embodiments, the endostatin comprises an amino acid sequence that is SEQ ID NO: 243.

[0156] In some embodiments, the engineered polynucleotide described herein encodes a CD59 and a fusion protein comprising two C3i connected with an antibody or fragment thereof. Vector KMR of Fig. 39 illustrates an example of such engineered polynucleotide. In some embodiments, the C3i-C3i fusion is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 232. In some embodiments, C3i-C3i fusion is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 232 In some embodiments, C3i-C3i fusion is encoded from a nucleic acid sequence that is SEQ ID NO: 232. In some embodiments, C3i-C3i fusion comprises an aminoWSGR Docket No. 59561-719.601acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 242 In some embodiments, C3i-C3i fusion comprises at least 5 contiguous polypeptide that are identical to SEQ ID NO: 242. In some embodiments, C3i-C3i fusion comprises an amino acid sequence that is SEQ ID NO: 242.

[0157] In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the AAV vector is an AAV2 vector. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4. In some embodiments, XI 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 embodiments, X3 is E, S, or Q. In some embodiments, X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183). In some embodiments, the engineered capsid comprises a polypeptide sequence. In some embodiments, the polypeptide sequence is inserted into an amino acid sequence of any one of SEQ ID NOs: 221-226 (Table 57). In some embodiments, the polypeptide sequence is inserted in the VP domain of the AAV capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO: 221. In some embodiments, the engineered capsid further comprises a mutation. In some embodiments, the mutation is in a VP1 domain, a VP2 domain, or a VP3 domain. In some embodiments, the mutation is in a GH loop. In some embodiments, the mutation is at a residue at position of 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 221. In some embodiments, the mutation is R to A at position 585 or 588 of SEQ ID NO: 221. In some embodiments, the polypeptide sequence comprises an amino acid sequence of any one of the amino acid sequences in Table 56. In some embodiments, the polypeptide sequence comprises an amino acid sequence that is identical to at least 5, 6, 7, or 8 contiguous amino acids of any one of the amino acid sequences in Table 56. In some embodiments, the engineered capsid comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence of any one of the amino acid sequences in Table 56. In some embodiments, the engineered capsid comprises an amino acid sequence that is any one of the amino acid sequences in Table 56. In some embodiments, the engineered capsid comprises a vector comprising one or more expression cassettes. In some embodiments, the one or more expression cassettes encode one or more transgenes (e.g., one or more therapeutics). In some embodiments, the one or moreWSGR Docket No. 59561-719.601transgenes can treat a disease or condition in a subject. In some embodiments, the engineered capsid comprising the polypeptide sequence increases the expression of the vector in a targeted cell.

[0158] Described herein, in some aspects is an engineered polypeptide comprising a CD59, a complement 3 inhibitor or a C3 degraded fragment, and a C-type natriuretic peptide (CNP) or an endostatin (ES). In some embodiments, the engineered polypeptide is encoded from a vector described herein. In some embodiments, the vector is Vector KKT, Vector MKT, or Vector MKC.

[0159] In some embodiments, described herein is a vector or a viral particle. In some embodiments, the vector encodes an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid described herein. In some embodiments, the viral particle comprising the engineered polynucleotide described herein, the engineered polypeptide described herein, or the vector described herein. In some embodiments, the viral particle comprises an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid described herein.

[0160] Described herein, in some aspects, is a method of contacting a cell obtained from a subject with an engineered polynucleotide described herein, an engineered polypeptide described herein, a vector described herein, or a viral particle described herein. In some embodiments, described herein is a method of treating a disease or condition in a subject by administering to the subject an engineered polynucleotide described herein, an engineered polypeptide described herein, a vector described herein, or a viral particle described herein. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP or the ES treats the disease or condition by conferring neuroprotective effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP or the ES treats the disease or condition by conferring anti-angiogenesis effect in the subject. In some embodiments, a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP and the ES treats the disease or condition by conferring neuroprotective effect and anti-angiogenesis in the subject. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease,WSGR Docket No. 59561-719.601choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.

[0161] Described herein, in some aspects, is an engineered polynucleotide comprising one or more expression cassettes for encoding one or more angiogenesis inhibitors. In some embodiments, the one or more angiogenesis inhibitors are operatively coupled (e.g., covalently connected). In some embodiments, the one or more angiogenesis inhibitors comprise a complement inhibitor, a natriuretic peptide, an inhibitor of a membrane attack complex (MAC), an VEGF inhibitor, or a combination thereof. In some embodiments, the complement inhibitor comprises a complement 3 inhibitor (C3i) or a complement 3 (C3) degraded fragment. In some embodiments, the C3 degraded fragment comprises a C3a, C3b, iC3b, C3f, C3c, C3d, C3g, or a combination thereof. In some embodiments, the engineered polynucleotide comprises a vector such as an AAV vector. In some embodiments, the engineered polynucleotide encodes a first angiogenesis inhibitor and a second angiogenesis inhibitor. In some embodiments, the engineered polynucleotide encodes an angiogenesis inhibitor comprising a complement 3 inhibitor (C3i). For example, Fig. 1 illustrates a vector construct (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) encoding the complement 3 inhibitor. In some embodiments, the engineered polynucleotide encodes a complement 3 inhibitor and a natriuretic peptide (e.g., a C-type natriuretic peptide or an CNP), for example Vector GGE. In some embodiments, the complement 3 inhibitor and the natriuretic peptide are covalently connected. For example, Fig.2A and 2B (Vector GAM and Vector GGE) and Fig. 3 (top panel: Vector GKR sCD59 - EVQL C3i- Fc4-CNP36; and bottom panel: Vector KAR sCD59 -DK C3i- Fc4-CNP36) illustrate vector constructs encoding the complement 3 inhibitor and the CNP. In some embodiments, the complement 3 inhibitor comprises at least one modification compared to a comparable wild-type 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: 1-15.

[0162] In some embodiments, the engineered polynucleotide encodes one or more angiogenesis inhibitors, where one of the angiogenesis inhibitors comprises the CNP. In some embodiments, the CNP is covalently connected to an antibody or fragment thereof (e.g., a fragment crystallizable region). In some embodiments, the natriuretic peptide comprises an amino acidWSGR Docket No. 59561-719.601sequence 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, where one of the angiogenesis inhibitors comprises an inhibitor of a membrane attack complex (MAC). In some embodiments, the inhibitor of the MAC comprises an CD59. For example, Fig. 3 illustrates a vector construct encoding the CD59. In some embodiments, the CD59 comprises 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, where one of the angiogenesis inhibitors comprises a collagen or fragment thereof (e.g., an endostatin or fragment thereof). In some embodiments, the endostatin comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 51. In some embodiments, the engineered polynucleotide encodes one or more angiogenesis inhibitors, where one of the angiogenesis inhibitors comprises an VEGF inhibitor. In some embodiments, the VEGF inhibitor comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 81-92.

[0163] In some embodiments, the engineered polynucleotide comprises a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector comprises an adeno-associated virus (AAV) vector. In some embodiments, the viral vector encodes an engineered viral capsid (e.g., as shown in Table 13 and Table 14). In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered AAV capsid comprises an amino acid sequence of any one 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 In some embodiments, the engineered AAV capsid comprises an amino acid sequence of any one of SEQ ID NOs: 277-303 and SEQ ID NOs: 312-319 and 325-329.

[0164] In some embodiments, the engineered polynucleotide encodes a first angiogenesis inhibitor comprising a complement 3 inhibitor and a second angiogenesis inhibitor comprising a CNP. In some embodiments, the first angiogenesis inhibitor comprises the complement 3 inhibitor, and the second angiogenesis inhibitor comprises a CNP36. In some embodiments, the first angiogenesis inhibitor comprises the complement 3 inhibitor, and the second angiogenesis inhibitor comprises an Fc-CNP36. In some embodiments, the engineered polynucleotide encodes a third angiogenesis inhibitor. In some embodiments, the third angiogenesis inhibitor comprises an inhibitor of a membrane attack complex (MAC).

[0165] In some embodiments, the engineered polynucleotide encodes a first angiogenesis inhibitor comprising an inhibitor of a CD59 and a second angiogenesis inhibitor comprising aWSGR Docket No. 59561-719.601CNP. In some embodiments, the first angiogenesis inhibitor comprises the CD59, and the second angiogenesis inhibitor comprises a CNP36. In some embodiments, the first angiogenesis inhibitor comprises the CD59, and the second angiogenesis inhibitor comprises an Fc-CNP36. In some embodiments, the first angiogenesis inhibitor comprises the CD59, and the second angiogenesis inhibitor comprises a complement 3 inhibitor fused to an Fc-CNP36.

[0166] 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 flanked by the first angiogenesis inhibitor and the second angiogenesis inhibitor. 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 an Fc-CNP36, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a CD59. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises an Fc-CNP36, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a CD59. In some embodiments, the first angiogenesis inhibitor comprises a CD59, and the second angiogenesis inhibitor comprises a complement 3 inhibitor, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a Fc-CNP36. In some embodiments, the first angiogenesis inhibitor comprises a CD59, and the second angiogenesis inhibitor comprises an endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor. In some embodiments, the first angiogenesis inhibitor comprises a CD59, and the second angiogenesis inhibitor comprises a VEGF inhibitor, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises an endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a CD59. In some embodiments, the first angiogenesis inhibitor, the second angiogenesis inhibitor, and the third angiogenesis inhibitor is not a VEGF inhibitor. In some embodiments, the engineered polynucleotide, upon administered to a subject, inhibits neovascularization in the subject. In some embodiments, the first angiogenesis inhibitor or the second angiogenesis inhibitor, upon administered to the subject, exhibits decreased inhibition ofWSGR Docket No. 59561-719.601neovascularization in the subject compared to inhibition of neovascularization caused by a VEGF inhibitor.

[0167] Described herein, in some aspects, is an engineered polypeptide comprising a first angiogenesis inhibitor and a second angiogenesis inhibitor. In some embodiments, the first angiogenesis inhibitor and the second angiogenesis inhibitor are covalently connected 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: 1-15. In some embodiments, the first angiogenesis inhibitor comprises an inhibitor of a membrane attack complex (MAC). In some embodiments, the inhibitor of the MAC comprises CD59 comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 41-45. In some embodiments, the inhibitor of the MAC comprises CD59 comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 312-319 In some embodiments, the inhibitor of the MAC comprises CD59 comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 325-329. In some embodiments, the second angiogenesis inhibitor comprises a natriuretic peptide. In some embodiments, the natriuretic peptide is covalently connected to an antibody or fragment thereof. In some embodiments, the antibody or 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 any one of SEQ ID NOs: 61-72. In some embodiments, the second angiogenesis inhibitor comprises a collagen or fragment thereof (e.g., an endostatin or fragment thereof). In some embodiments, the second angiogenesis inhibitor comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 51. In some embodiments, the second angiogenesis inhibitor comprises a VEGF inhibitor. In some embodiments, the VEGF inhibitor comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 81-92

[0168] In some embodiments, the engineered polynucleotide described herein is a vector described herein (e.g., Vector CTA or Vector CTC as shown in Fig. 58). In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 331 (Table 84). In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 231 (Table 84). In some embodiments, the engineered polynucleotide (e.g., SEQ ID NO: 231) encodes a C3-Fc-CNP fusion described herein. In someWSGR Docket No. 59561-719.601embodiments, the engineered polynucleotide comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 236 (Table 84). In some embodiments, the engineered polynucleotide (e.g., SEQ ID NO: 236) encodes a vsCD59 (T51G) described herein.

[0169] In some embodiments, the engineered polynucleotide described herein encodes an engineered polypeptide described herein. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 241 (Table 84). In some embodiments, the engineered polypeptide is a C3-Fc-CNP fusion described herein. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 246 (Table 84).In some embodiments, the engineered polypeptide is CD59 (vsCD59 T51G) described herein.

[0170] In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 332 (Table 84). In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 233 (Table 84). In some embodiments, the engineered polynucleotide (e.g., SEQ ID NO: 233) encodes a C3-Fc-endostatin fusion described herein. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 236 (Table 84). In some embodiments, the engineered polynucleotide (e.g., SEQ ID NO: 236) encodes a vsCD59 (T51G) described herein.

[0171] In some embodiments, the engineered polynucleotide described herein encodes an engineered polypeptide described herein. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 243 (Table 84). In some embodiments, the engineered polypeptide is a C3-Fc-endostatin fusion described herein. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 246 (Table 84).In some embodiments, the engineered polypeptide is CD59 (vsCD59 T51G) described herein.WSGR Docket No. 59561-719.601Table 84. Non-limiting example of nucleic acid or amino acid sequencesWSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601

[0172] Described herein, in some aspects, is a method for treating a disease or condition in a subject by administering an engineered polynucleotide or an engineered polypeptide to the subject. In some embodiments, the method comprises once of the administering being curative of the disease or condition. In some embodiments, the method does not comprise daily administration. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.Engineered polynucleotideWSGR Docket No. 59561-719.601

[0173] Described herein, in some aspects, is an engineered polynucleotide comprising one or more expression cassettes, the one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor. In some embodiments, the engineered polynucleotide comprises one or more expression cassettes for expressing the one or more angiogenesis inhibitors. In some embodiments, the one or more expression cassettes encode a contiguous polypeptide. In some embodiments, the contiguous polypeptide comprises a protease peptide sequence. In some embodiments, the protease peptide sequence is cleavable by a protease expressed endogenously in a cell. Non-limiting example of the protease can include a serine endoprotease, an aspartic endoprotease, a cysteine thiol endoprotease, a metalloendoprotease, or a glutamic acid and threonine endoprotease. In some embodiments, the protease peptide sequence is cleavable by a serine endoprotease. In some embodiments, the protease peptide sequence is cleavable by Furin. In some embodiments, the contiguous polypeptide comprises a protease cleavable sequence. In some embodiments, the protease cleavable sequence can be cleaved by any one of the proteases described herein. In some embodiments, the protease cleavable sequence can be cleaved by Furin. In some embodiments, the contiguous polypeptide comprises a selfcleaving polypeptide sequence. In some embodiments, the self-cleaving polypeptide sequence comprises a 2A self-cleaving peptide sequence. Non-limiting examples of the 2A self-cleaving peptide sequence can include T2A, P2A, E2A, F2A, or a combination thereof. In some embodiments, the self-cleaving polypeptide sequence comprises a F2A peptide sequence. In some embodiments, the contiguous polypeptide comprises a protease cleavable sequence and a self-cleaving polypeptide sequence. For example, the contiguous polypeptide described herein can comprise a Furin-F2A fusion polypeptide sequence. In some embodiments, the engineered polynucleotide comprises a viral vector such as an AAV vector.

[0174] In some embodiments, the engineered polynucleotide comprises one or more promoters or internal ribosome entry sites (IRES). In some embodiments, the expression cassette comprises one or more promoters or IRES. In some embodiments, the expression cassette is under expression control of a promoter. In some embodiments, the expression cassette is under expression control of a promoter. In some embodiments, expression cassette can further exert expression control via at least one IRES.

[0175] In some embodiments, the engineered polynucleotide comprises at least two, at least three, at least four, at least five, or more expression cassettes. In some embodiments, the engineered polynucleotide comprises two expression cassettes. In some embodiments, the one orWSGR Docket No. 59561-719.601more angiogenesis inhibitors are each encoded from an expression cassette of the one or more expression cassettes.

[0176] 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 connected 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 48). In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is 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 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) comprises an amino acid sequence that is SEQ ID NO: 1. 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: 2. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is SEQ ID NO: 2. 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: 3. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is SEQ ID NO: 3. 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: 4. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is SEQ ID NO: 4. 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: 5. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is SEQ ID NO: 5. 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 IDWSGR Docket No. 59561-719.601NO: 6. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is SEQ ID NO: 6. 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: 7. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is SEQ ID NO: 7. 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: 8. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is SEQ ID NO: 8. 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: 9. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is SEQ ID NO: 9. 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: 10. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is SEQ ID NO: 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: 11. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is SEQ ID NO: 11. 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: 12. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is SEQ ID NO: 12. 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: 13. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is SEQ ID NO: 13. 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: 14. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acidWSGR Docket No. 59561-719.601sequence that is SEQ ID NO: 14. 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: 15. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is SEQ ID NO: 15.

[0177] In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of any one of SEQ ID NOs: 1-15. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of SEQ ID NO: 1. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of SEQ ID NO: 2 In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of SEQ ID NO: 3. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of SEQ ID NO: 4. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of SEQ ID NO: 5 In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of SEQ ID NO: 6. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of SEQ ID NO: 7. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of SEQ ID NO: 8 In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of SEQ ID NO: 9. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of SEQ ID NO: 10. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguousWSGR Docket No. 59561-719.601amino acids of SEQ ID NO: 11 In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of SEQ ID NO: 12. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of SEQ ID NO: 13. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of SEQ ID NO: 14 In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of SEQ ID NO: 15.Table 48. Exemplary complement 3 inhibitor (C3i) amino acid sequencesWSGR Docket No. 59561-719.601

[0178] 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 49). In some embodiments, the complement 3 inhibitor (C3i) is encoded by a nucleic acid sequence that is any one of SEQ ID NOs: 20-33. In some embodiments, the complement 3 inhibitor (C3i) is encoded by a nucleic acid sequence that is at least 50 contiguous nucleotide bases, at least 60 contiguous nucleotide bases, at least 70 contiguous nucleotide bases, or at least 50 contiguous nucleotide bases of any one of SEQ ID NOs: 20-33Table 49. Exemplary nucleic acid sequence encoding complement 3 inhibitorWSGR Docket No. 59561-719.601

[0179] In some embodiments, the engineered polynucleotide encodes a natriuretic peptide or a natriuretic peptide fusion protein (e.g., a CNP-Fc fusion protein described herein). In some embodiments, the natriuretic peptide is a CNP. In some embodiments, the CNP is covalently connected to a complement 3 inhibitor. In some embodiments, the CNP is covalently connected to a complement 3 inhibitor by a linker. In some embodiments, the natriuretic peptide or the 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 50). In some embodiments, the natriuretic peptide or the 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: 61 In some embodiments, the natriuretic peptide or the 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: 62 In some embodiments, the natriuretic peptide or the 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: 63 In some embodiments, the natriuretic peptide or the 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: 64 In some embodiments, the natriuretic peptide or the 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, atWSGR Docket No. 59561-719.601least 95% identical, or at least 99% identical to SEQ ID NO: 65 In some embodiments, the natriuretic peptide or the 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: 66 In some embodiments, the natriuretic peptide or the 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: 67. In some embodiments, the natriuretic peptide or the 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: 68 In some embodiments, the natriuretic peptide or the 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: 69 In some embodiments, the natriuretic peptide or the 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: 70 In some embodiments, the natriuretic peptide or the 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: 71 In some embodiments, the natriuretic peptide or the 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 the natriuretic peptide fusion protein comprises an amino acid sequence that is any one of SEQ ID NOs: 61-72.Table 50. Exemplary natriuretic peptide or the natriuretic peptide fusion protein amino acid sequencesWSGR Docket No. 59561-719.601<<<<<<<<WSGR Docket No. 59561-719.601

[0180] In some embodiments, the engineered polynucleotide encodes an inhibitor of a membrane attack complex (MAC). In some embodiments, the inhibitor of the MAC comprises CD59. In some embodiments, the 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. (Table 51). In some embodiments, the 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 SEQ ID NO: 41. In some embodiments, the 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 SEQ ID NO: 42 In some embodiments, the 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 SEQ ID NO: 43. In some embodiments, the 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 SEQ ID NO: 44 In some embodiments, the 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 SEQ ID NO: 45. In some embodiments, the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 41-45.Table 51. Exemplary CD59 amino acid sequencesWSGR Docket No. 59561-719.601

[0181] In some embodiments, the engineered polynucleotide encodes an inhibitor of a membrane attack complex (MAC). In some embodiments, the inhibitor of the MAC comprises CD59. In some embodiments, the 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. (Table 52). In some embodiments, the 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 SEQ ID NO: 312. In some embodiments, the 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 SEQ ID NO: 313. In some embodiments, the 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 SEQ ID NO: 314. In some embodiments, the 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 SEQ ID NO: 315 In someWSGR Docket No. 59561-719.601embodiments, the 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 SEQ ID NO: 316. In some embodiments, the 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 SEQ ID NO: 317 In some embodiments, the 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 SEQ ID NO: 318. In some embodiments, the 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 SEQ ID NO: 319. In some embodiments, the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 312-319

[0182] In some embodiments, the engineered polynucleotide encodes an inhibitor of a membrane attack complex (MAC). In some embodiments, the inhibitor of the MAC comprises CD59. In some embodiments, the 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. (Table 52). In some embodiments, the 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 SEQ ID NO: 325. In some embodiments, the 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 SEQ ID NO: 326 In some embodiments, the 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 SEQ ID NO: 327. In some embodiments, the 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 SEQ ID NO: 328. In some embodiments, the 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 SEQ ID NO: 329. In some embodiments, the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 325-329.Table 52. Exemplary CD59 amino acid sequencesWSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601

[0183] In some embodiments, the engineered polynucleotide encodes a collagen or fragment thereof. In some embodiments, the collagen or fragment thereof comprises an endostatin or fragment thereof. In some embodiments, the endostatin or 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:MHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDL YSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQK SVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENWSGR Docket No. 59561-719.601SFMTASK. In some embodiments, the endostatin or fragment thereof comprises an amino acid sequence that is SEQ ID NO: 51.

[0184] In some embodiments, the engineered polynucleotide encodes an VEGF inhibitor. In some embodiments, the VEGF inhibitor comprises an inhibitory RNA for targeting and degrading the VEGF transcript. In some embodiments, the VEGF inhibitor comprises an antibody or a fragment thereof. In some embodiments, the VEGF antibody binds to VEGF to decrease neovascularization signaling comprising the VEGF signaling transduction pathway. In some embodiments, the VEGF antibody binds to VEGF-A, VEGF-B, VEGF-C, VEGF-D, or a combination thereof. In some embodiments, the VEGF antibody binds to one or more isoforms of VEGF-A, including VEGF121, VEGF145, VEGF148, VEGF162, VEGF165, VEGF165b, VEGF183, VEGF189, or VEGF206. In some embodiments, the antibody comprises monovalent Fab’, a divalent Fab2, a F(ab)'3 fragments, a single-chain variable fragment (scFv), a bis-scFv, (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody, or a combination thereof, a binding fragment thereof, or a chemically modified derivative thereof. Non-limiting examples of VEGF antibodies include ranibizumab or bevacizumab. In some embodiments, the VEGF antibody comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to any one of SEQ ID NOs: 81-87 (Table 53), or a combination thereof, or a fragment thereof.Table 53. Exemplary amino acid sequences of VEGF antibodiesWSGR Docket No. 59561-719.601

[0185] In some embodiments, the VEGF inhibitor is not an antibody. For example, the VEGF inhibitor described herein can comprise a VEGF receptor, a combination of VEGF receptors, or a fragment thereof for binding to VEGF for inhibiting or decreasing VEGF signaling transduction pathway. VEGF receptor can include a VEGF receptor 1 (FLT1), a VEGF receptor 2 (KDR / FLK1), a VEGF receptor 3 (FLT4), a fragment thereof, or a combination thereof. In some embodiments, the VEGF receptor can be a soluble VEGF receptor. For example, the soluble VEGF receptor can comprise a soluble VEGFR1, a soluble VEGFR2, a soluble VEGFR3, a soluble fragment thereof, or a combination thereof. In some embodiments, the non-antibody VEGF inhibitor comprises at least one of FLT1, KDR / FLK1, FLT4, a fragment thereof, or a combination thereof. In some embodiments, the non-antibody VEGF inhibitor comprises at least one of soluble FLT1, soluble KDR / FLK1, soluble FLT4, a fragment thereof, or a combination thereof. In some embodiments, the non-antibody inhibitor VEGF comprises a VEGF-Trap. InWSGR Docket No. 59561-719.601some embodiments, the non-antibody VEGF inhibitor comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, is 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 54).Table 54. Exemplary amino acid sequence of non-antibody VEGF inhibitor<<<<<WSGR Docket No. 59561-719.601

[0186] In some embodiments, the engineered polynucleotide comprises one or more expression cassettes, where the one or more expression cassettes encodes a CD59, a complement 3 inhibitor or a C3 degraded fragment, a C-type natriuretic peptide (CNP), an endostatin, or a combination thereof. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 231-236. In some embodiments, SEQ ID NOs: 231-236 encode the CD59, the complement 3 inhibitor or the C3 degraded fragment, the CNP, the endostatin, or a combination thereof. In some embodiments, the engineered polynucleotide encodes an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 241-246.

[0187] In some embodiments, the engineered polynucleotide encodes a CD59, a complement 3 inhibitor or a C3 degraded fragment, and a C-type natriuretic peptide (CNP) or an endostatin (ES). In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is covalently connected to an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a fragment crystallizable (Fc) region. In some embodiments, the CNP or the ES is covalently connected to the antibody or fragment thereof. In some embodiments, the CD59 is expressed from a first expression cassette of the one or more expression cassettes, and the complement 3 inhibitor or the C3 degraded fragment and the CNP or the ES are expressed from a second expression cassette of the one or more expression cassettes. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 234-236, 331, or 332. In some embodiments, the CD59 is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to any one of SEQ ID NOs: 234-236, 331, or 332. In some embodiments, the CD59 isWSGR Docket No. 59561-719.601encoded from a nucleic acid sequence that is any one of SEQ ID NOs: 234-236, 331, or 332. In some embodiments, the CD59 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises at least 50 contiguous polypeptides that are identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 244-246.

[0188] In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241 or SEQ ID NO: 242 In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises at least 5 contiguous polypeptide that are identical to SEQ ID NO: 241 or SEQ ID NO: 242 In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is SEQ ID NO: 241 or SEQ ID NO: 242.

[0189] In some embodiments, the CNP is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231. In some embodiments, the CNP is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231. In some embodiments, the CNP is encoded from a nucleic acid sequence that is SEQ ID NO: 231. In some embodiments, the CNP comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241 In some embodiments, the CNP comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 241. In some embodiments, the CNP comprises an amino acid sequence that is SEQ ID NO: 241.

[0190] In some embodiments, the endostatin is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 233. In some embodiments, the endostatin is encoded from a nucleic acid sequenceWSGR Docket No. 59561-719.601comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 233. In some embodiments, the endostatin is encoded from a nucleic acid sequence that is SEQ ID NO: 233. In some embodiments, the endostatin comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 243 In some embodiments, the endostatin comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 243 In some embodiments, the endostatin comprises an amino acid sequence that is SEQ ID NO: 243.

[0191] In some embodiments, the engineered polynucleotide described herein encodes a CD59 and a fusion protein comprising two C3i connected with an antibody or fragment thereof. Vector KMR of Fig. 39 illustrates an example of such engineered polynucleotide. In some embodiments, In some embodiments, the C3i-C3i fusion is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 232. In some embodiments, C3i-C3i fusion is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 232. In some embodiments, C3i-C3i fusion is encoded from a nucleic acid sequence that is SEQ ID NO: 232. In some embodiments, C3i-C3i fusion comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 242. In some embodiments, C3i-C3i fusion comprises at least 5 contiguous polypeptide that are identical to SEQ ID NO: 242. In some embodiments, C3i-C3i fusion comprises an amino acid sequence that is SEQ ID NO: 242.Table 58. Non-limiting example of nucleic acid sequence for encoding a CD59, a complement 3 inhibitor or a C3 degraded fragment, or a C-type natriuretic peptide (CNP)WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601WSGR Docket No. 59561-719.601Table 59. Amino acid sequence of CD59, C3i, CNP, or a fusion of C3i and CNPWSGR Docket No. 59561-719.601

[0150] In some embodiments, the engineered polynucleotide comprises a viral vector such as an AAV vector comprising one or more expression cassettes for the one or more angiogenesis inhibitors. In some embodiments, the engineered polynucleotide comprises a vector. In some embodiments, the vector is a viral vector. In some embodiments, the engineered polynucleotide comprises an AAV vector. In some embodiments, the engineered polynucleotide comprises an AAV vector encoding an engineered AAV capsid. In some embodiments, the AAV vector comprises one or more expression cassettes for encoding an engineered polypeptide comprising:WSGR Docket No. 59561-719.601a peptide or a fusion protein comprising an antibody or fragment thereof operatively coupled to a peptide.

[0151] In some embodiments, the engineered polynucleotide is a vector. In some embodiments, the engineered polynucleotide is a viral vector comprising an AAV vector. In some embodiments, the engineered polynucleotide is an AAV vector comprising an AAV serotype comprising 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 comprising the AAV2 serotype. In some embodiments, the AAV vector encodes an engineered AAV capsid. In some embodiments, the engineered polynucleotide comprises a viral vector. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the AAV vector comprises an AAV serotype 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. In some embodiments, the engineered AAV capsid comprises an amino acid sequence of any one 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.

[0152] In some embodiments, the engineered polynucleotide 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 of any one of SEQ ID NOs: 101-103 and 121-129. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of any one of SEQ ID NOs: 101-103, and 121-129. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 101. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 102. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 103. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 121 In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 122. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 123. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 124. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 125. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 126. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence ofWSGR Docket No. 59561-719.601SEQ ID NOs: 127 In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 128. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 129. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of any one of SEQ ID NOs: 101-103, and 121-129

[0153] 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 NO: 111-113 and 43-45. In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence that is any one of SEQ ID NO: 111-113 and 43-45

[0154] In some embodiments, the engineered polynucleotide encodes a first angiogenesis inhibitor and a second angiogenesis inhibitor. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises a CNP. In some embodiments, the first angiogenesis inhibitor comprises the complement 3 inhibitor, and the second angiogenesis inhibitor comprises a CNP36. In some embodiments, the first angiogenesis inhibitor comprises the complement 3 inhibitor, and the second angiogenesis inhibitor comprises an Fc region and a CNP36. In some embodiments, the engineered AAV further encodes a third angiogenesis inhibitor. In some embodiments, the third angiogenesis inhibitor comprises an inhibitor of a membrane attack complex (MAC). In some embodiments, the inhibitor of the MAC comprises a CD59. In some embodiments, the engineered polynucleotide encodes a protease site flanked by the second angiogenesis inhibitor and the third angiogenesis inhibitor. In some embodiments, the first angiogenesis inhibitor comprises an inhibitor of a CD59, and the second angiogenesis inhibitor comprises a CNP. In some embodiments, the first angiogenesis inhibitor comprises the CD59, and the second angiogenesis inhibitor comprises a CNP36. In some embodiments, the first angiogenesis inhibitor comprises the CD59, and the second angiogenesis inhibitor comprises an Fc-CNP36. In some embodiments, the first angiogenesis inhibitor comprises the CD59, and the second angiogenesis inhibitor comprises a complement 3 inhibitor fused to an 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, the engineered polynucleotide encodes an Fc region flanked by the first angiogenesis inhibitor and the second angiogenesis inhibitor. In some embodiments, the first angiogenesis inhibitor comprises a VEGF inhibitor, and the secondWSGR Docket No. 59561-719.601angiogenesis inhibitor comprises a complement 3 inhibitor. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises an Fc-CNP36, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a CD59. In some embodiments, the engineered polynucleotide further encodes a protease site flanked by the second angiogenesis inhibitor and the third angiogenesis inhibitor. In some embodiments, the protease site comprises a Furin protease site. In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises an Fc-CNP36, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a CD59. In some embodiments, the first angiogenesis inhibitor comprises a CD59, and the second angiogenesis inhibitor comprises a complement 3 inhibitor, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a Fc-CNP36. In some embodiments, the first angiogenesis inhibitor comprises a CD59, and the second angiogenesis inhibitor comprises an endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor. In some embodiments, the first angiogenesis inhibitor comprises a CD59, and the second angiogenesis inhibitor comprises a VEGF inhibitor, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a complement 3 inhibitor.

[0155] In some embodiments, the first angiogenesis inhibitor comprises a complement 3 inhibitor, and the second angiogenesis inhibitor comprises an endostatin, wherein the engineered polynucleotide further encodes a third angiogenesis inhibitor comprising a CD59. In some embodiments, the first angiogenesis inhibitor, the second angiogenesis inhibitor, and the third angiogenesis inhibitor is not a VEGF inhibitor. In some embodiments, the first angiogenesis inhibitor and the second angiogenesis inhibitor, upon administered to a subject, inhibits neovascularization in the subject. In some embodiments, the first angiogenesis inhibitor or the second angiogenesis inhibitor, upon administered to the subject, exhibits decreased inhibition of neovascularization in the subject compared to inhibition of neovascularization caused by a VEGF inhibitor.

[0156] 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-WSGR Docket No. 59561-719.601furin 2A-C3i-Fc4-CNP36. In some embodiments, the engineered polynucleotide encodes sCD59-furin 2A- endostatin-linker-C3i. In some embodiments, the engineered polynucleotide encodes sCD59-furin 2A- 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-Fcl-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 vsCD59-6xHis. In some embodiments, the engineered polynucleotide encodes vsCD59 N18Q-6xHis. In some embodiments, the engineered polynucleotide encodes vsCD59 Q34E-6xHis. In some embodiments, the engineered polynucleotide encodes vsCD59 K38R-6xHis. In some embodiments, the engineered polynucleotide encodes vsCD59 Q34E, K38R-6xHis. In some embodiments, the engineeredWSGR Docket No. 59561-719.601polynucleotide encodes vsCD59 N18Q, K38R-6xHis. In some embodiments, the engineered polynucleotide encodes vsCD59 N18Q, Q33E, K38R-6*His.

[0157] In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence that is at least 70% identical, that is 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 to of any one of SEQ ID NOs: 250-276, 304-311, and 320-324. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of any one of SEQ ID NOs: 250-276, 304-311, and 320-324. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 250 In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 251. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 252. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 253. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 254. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 255. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 256. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 257. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 258. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 259. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 260. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 261 In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 262. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 263. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 264. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 265. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 266. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 267. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 268. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 269. In some embodiments, the engineeredWSGR Docket No. 59561-719.601polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 270. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 271. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 272. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 273. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 274. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 275. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 276. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 304. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 305. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 306 In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 307. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 308. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 309. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 310. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 311. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 320 In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 321. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 322. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 323. In some embodiments, the engineered polynucleotide comprises a nucleic acid sequence of SEQ ID NOs: 324.

[0158] In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence that is at least 70% identical, that is 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 to any one of SEQ ID NO: 277-303, 312-319, and 325-329. In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence that is any one of SEQ ID NO: 277-303, 312-319, and 325-329.

[0159] In some embodiments, the engineered polynucleotide encodes a sequence as shown in Fig. 1. In some embodiments, the engineered polynucleotide encodes a sequence as shown inWSGR Docket No. 59561-719.601Figs. 2A-B. In some embodiments, the engineered polynucleotide encodes a sequence as shown in Fig. 3. In some embodiments, the engineered polynucleotide encodes a sequence as shown in Fig. 4. In some embodiments, the engineered polynucleotide encodes a sequence as shown in Figs. 5A-C. In some embodiments, the engineered polynucleotide encodes a sequence as shown in Fig. 16. In some embodiments, the engineered polynucleotide encodes a sequence as shown in Fig. 24. In some embodiments, the engineered polynucleotide encodes a sequence as shown in Fig. 26. In some embodiments, the engineered polynucleotide encodes a sequence as shown in Fig. 32. In some cases, the engineered polynucleotide comprises additional features. Additional features can comprise sequences such as tags, signal peptides, intronic sequences, promoters, stuffer sequences, and the like. In some cases, the engineered polynucleotide encodes a signal peptide. A signal peptide is sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide, is a short peptide present at the N-terminus of the majority of newly synthesized proteins that are destined toward the secretory pathway. These proteins include those that reside either inside certain organelles (the endoplasmic reticulum, Golgi or endosomes), secreted from the cell, or inserted into most cellular membranes. In some cases, nucleic acids provided herein can comprise signal peptides. A signal peptide can be of any length but typically from 15-30 amino acids long. A signal peptide can be from about: 10-15, 10-20, 10-30, 15-20, 15-25, 15-30, 20-30, or 25-30 amino acids long. Various signal peptides can be utilized and include but are not limited to: human antibody heavy chain (Vh), human antibody light chain (VI), and aflibercept.

[0160] In some cases, the engineered polynucleotide comprises an intronic sequence. An intron is any nucleotide sequence within a sequence that can be removed by RNA splicing during maturation of the final RNA product. In other words, introns are non-coding regions of an RNA transcript, or the DNA encoding it, that are eliminated by splicing before translation. While introns do not encode protein products, they are players in gene expression regulation. Some introns themselves encode functional RNAs through further processing after splicing to generate noncoding RNA molecules. Alternative splicing is widely used to generate multiple proteins from a single gene. Furthermore, some introns play essential roles in a wide range of gene expression regulatory functions such as nonsense-mediated decay and mRNA export. In an embodiment, an intronic sequence is included in a nucleic acid of the disclosure and can be selected from: hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, hamster EF-1 alpha gene intron 1, intervening sequence intron, human growth hormone intron, and / or human beta globin intron. Any number of intronic sequences are contemplated. In anWSGR Docket No. 59561-719.601embodiment, the intronic sequence is SV40. In some cases, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or up to 10 intronic sequences can be included in a nucleic acid.

[0161] In an embodiment, the engineered polynucleotide comprises an additional feature including a promoter. Promoters are sequences of DNA to which proteins bind that initiate transcription of a single RNA from the DNA downstream of it. This RNA may encode a protein, or can have a function in and of itself, such as tRNA, mRNA, or rRNA. Promoters are located near the transcription start sites of genes, upstream on the DNA (towards the 5' region of the sense strand). Promoters can be about 100-1000 base pairs long. Various promoters are contemplated and can be employed in the engineered polynucleotides of the disclosure. In an embodiment, a promoter is: a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EFla) promoter, a simian vacuolating virus (SV40) promoter, a phosphoglycerate kinase (PGK1) promoter, a ubiquitin C (Ubc) promoter, a human beta actin promoter, a CAG promoter, a Tetracycline response element (TRE) promoter, a UAS promoter, an Actin 5c (Ac5) promoter, a polyhedron promoter, a Ca2+ / calmodulin-dependent protein kinase II (CaMKIIa) promoter, a GALI promoter, a GAL 10 promoter, a TEF1 promoter, a glyceraldehyde 3 -phosphage dehydrogenase (GDS) promoter, an ADH1 promoter, a CaMV35S promoter, aUbi promoter, a human polymerase III RNA (Hl) promoter, a U6 promoter, a polyadenylated construct thereof, and any combination thereof. In some cases, the promoter is the CMV promoter.

[0162] Any of the provided the engineered polynucleotide can comprise viral vector sequences. A viral vector can be, without limitation, a lentivirus, a retrovirus, or an adeno-associated virus. A viral vector can be an adeno-associated viral (AAV) vector. In some cases, a viral vector is an adeno-associated viral vector. Many serotypes of AAV vectors are contemplated and include but are not limited to: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and / or AAV12. Based on these initial serotypes, AAV capsid of each serotype can be engineered to make them better suited for biological functions, tissue or cell selection. In some embodiments, an AAV vector is AAV2 and variants AAV2.N53 and AAV2.N54. Chimeric AAV vectors are also contemplated that may contain at least 2 AAV serotypes. In some cases, at least 3, at least 4, at least 5, at least 6, at least 7, or up to 8 different serotypes are combined in a chimeric AAV vector. In some cases, only a portion of the AAV is chimeric. For example, suitable portions can include the capsid, VP1, VP2, or VP3 domains and / or Rep. In some cases, at least one of VP1, VP2, and VP3 has at least one amino acid substitution compared to an otherwise comparable wild-type AAV capsid protein. In some cases, a mutation can occur inWSGR Docket No. 59561-719.601VP1 and VP2, in VP1 and VP3, in VP2 and VP3, or in VP1, VP2, and VP3. In some embodiments, at least one of VP1, VP2, and VP3 has from one to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3, e.g., from about one to about 5, from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, or from about 20 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3. In some cases, a VP can be removed. For example, in some embodiments a mutant AAV does not comprise at least one of VP1, VP2, or VP3.

[0163] In some cases, an AAV vector can be modified. For example, an AAV vector can comprise a modification such as an insertion, deletion, chemical alteration, or synthetic modification. In some cases, a single nucleotide is inserted into an AAV vector. In other cases, multiple nucleotides are inserted into a vector.Codon optimization

[0164] In an embodiment, the engineered polynucleotide described herein comprises a modification that confers enhanced expression of the one or more angiogenesis inhibitors described herein. For example, the one or more angiogenesis inhibitors are derived from natural gene sequences and contain unmodified sequences that are not optimized for introduction and expression in target cells. In an embodiment, an isolated, engineered polynucleotide is codon optimized. Codon optimization can be specific for cell type-specific codon usage. Different organisms and cell types exhibit bias towards use of certain codons over others for the same amino acid. Some species are known to avoid certain codons almost entirely. Similarly, certain cell types are biased toward use of certain codons over others for the same amino acid. In an embodiment, a method of optimizing a codon of an engineered polynucleotide can comprise reassigning codon usage based on the frequencies of each codon’s usage in a target cell. In some cases, a target cell can be of a certain tissue or organ. In some cases, a modification is performed to increase guanine and / or cytosine content.

[0165] In an embodiment, an engineered nucleic acid sequence can be modified to replace at least one codon with another codon coding for an identical amino acid. In some cases, a codon is modified within a coding region of a sequence. In some cases, a codon is modified within a noncoding region of a sequence. In some cases, a codon is modified within about 100, about 50, about 25, about 15, or about 5 bases from a termination codon. E-CAI can be utilized to estimate a value of a codon adaptation index.

[0166] Various modifications are contemplated herein. In some cases, codons can be interchanged. For example, a sequence can be modified to replace AGA with AGG. In otherWSGR Docket No. 59561-719.601cases, CCC is replaced with CCT. In other cases, AGC is replaced with TCC. In other cases, CCC is replaced with CCG. Any of the non-limiting replacements provided in Table 55 can be applied to modify a nucleic acid. Any number of codons can be interchanged in a nucleic acid. In some cases, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 42, at least 44, at least 46, at least 48, or up to 50 codons can be replaced. In an embodiment, an engineered polynucleotide comprises 3 codon modifications. In an embodiment, an engineered polynucleotide comprises 16 codon modifications. In an embodiment, an engineered polynucleotide comprises 3-5, 5-10, 5-15, 10-15, 10-20, 15-20, 1-20, 12-20, 12-25, 15-30, or 15-25 codon modifications. In an embodiment, an engineered polynucleotide comprises two codon modifications that are: AGA to AGG and at least one of: CCT to CCC, AGC to TCC, or CCC to CCG. In an embodiment, an engineered polynucleotide comprises three codon modifications that are: AGA to AGG and at least two of: CCT to CCC, AGC to TCC, or CCC to CCG. In an embodiment, an engineered polynucleotide comprises four codon modifications that are: AGA to AGG, CCT to CCC, AGC to TCC, and CCC to CCG. Additional modifications can comprise any of the codon modifications provided in Table 55 in combination with any of the above codons and / or any additional modifications possible from Table 55. In an embodiment, a nucleic acid is modified such that AGA is replaced with AGG and CCT is replaced with CCC. In an embodiment, a nucleic acid is modified such that AGA is replaced with AGG and AGC is replaced with TCC. In an embodiment, a nucleic acid is modified such that AGA is replaced with AGG and CCC is replaced with CCG.Table 55. Non-limiting exemplary codons that can be interchanged for modification of nucleic acids. Thymine can be replaced with uracil in the below codonsWSGR Docket No. 59561-719.601

[0167] In some embodiments, an engineered nucleic acid sequence can comprise a viral vector sequence. In some embodiments, a viral vector sequence can be a scAAV vector sequence. In some embodiments, a AAV vector sequence can be of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, an AAV vector sequence can be of the AAV2 serotype. In some embodiments, a viral vector sequence can comprise sequences of at least 2 AAV serotypes. In some embodiments, at least two serotypes can be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV11, and AAV12.

[0168] In some cases, a modification can also comprise a chemical modification. Modified nucleic acids can comprise modifications of their backbones, sugars, or nucleobases, and even novel bases or base pairs. Modified nucleic acids can have improved chemical and / or biological stability. Decoration with diverse chemical substituents (e.g., hydrophobic groups) can also yield improved properties and functionalities such as new structural motifs and enhanced target binding.

[0169] Exemplary chemical modification includes but are not limited to: 2’F, 2’ -fluoro; 2’OMe, 2’-O-methyl; LNA, locked nucleic acid; FANA, 2'-fluoro arabinose nucleic acid; HNA, hexitol nucleic acid; 2M0E, 2 ’-O-m ethoxy ethyl; ribuloNA, (r-3’)-P-L-ribulo nucleic acid; TNA, a-L-threose nucleic acid; tPhoNA, 3’-2’ phosphonomethyl-threosyl nucleic acid; dXNA, 2’-deoxyxylonucleic acid; PS, phosphorothioate; phNA, alkyl phosphonate nucleic acid; PNA, and peptide nucleic acid.Engineered capsid

[0170] Provided herein are engineered adeno-associated virus (AAV) capsid-containing compositions and methods of using the same. An engineered AAV capsid can comprise exogenous sequences as compared to an otherwise comparable unmodified AAV capsid.Exogenous sequences can refer to exogenous polypeptide sequences. AAV capsids can beWSGR Docket No. 59561-719.601engineered to confer upon them, and any compositions and / or methods in which they are utilized, improved functionality thereby resulting in better therapeutics, particularly for ocular use.

[0171] 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 seems to code for a protein with supportive function in genome replication. Significantly more information is available for rep and cap. The rep gene is located in the first half of the AAV WT genome and codes for a family of non- structural proteins (Rep proteins) required for viral transcription control and replication as well as packaging of viral genomes into the newly produced, preassembled capsids. The second half of the AAV genome contains the cap gene, which codes for the viral proteins (VPs) VP1, VP2, and VP3, and the assembly-activating protein (AAP).Transcription of all VPs, which are the capsid monomers, is controlled by a single promoter (p40 in case of AAV2) resulting in a single mRNA. Splicing (VP1) and an unusual translational start codon (VP2) are responsible for an approximately 10 times lower presence of VP1 and VP2 compared with 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 also VP2 and VP1 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 23 kDa protein encoded in an alternative cap ORF. It is used for stabilizing and transporting newly produced VP proteins from the cytoplasm into the cell nucleus. While AAV serotypes 1-3, 6-9, and rhlO failed to produce capsids in the absence of AAP, a low but detectable capsid production was reported for AAV4 and AAV5.

[0172] In an aspect, an AAV can comprise a modification. A modification can be of a rep, cap, and / or X coding polypeptide sequence of an AAV. In some cases, the modification can be of a cap polypeptide. A cap polypeptide can be modified in any one of the VP domains, for example VP1, VP2, and / or VP3. In some cases, VP1 is modified. In some cases, VP2 is modified. In some cases, VP3 is modified. In some aspects, two or all of the VP domains can be modified. In some cases, VP1 and VP2 are modified. In some cases, VP1 and VP3 are modified. Additionally, VP2 and VP3 can be modified or VP1, VP2, and VP3 are modified. Other combinations are contemplated, such as modifications in Rep and Cap, Cap and X, Rep and X, and / or Rep, Cap, and X. Any combination of domains can be modified such as any one of the aforementioned VP modifications in conjunction with a Rep and / or X modification. In some cases, Rep and VP1 and / or VP2 are modified. In some aspects, a subject Rep is modified. A rep modification canWSGR Docket No. 59561-719.601comprise a modification as provided herein and can be in at least one of Rep 78, Rep 68, Rep 52 or Rep 40. In some cases, a Rep is of a different AAV serotype than a subject capsid.

[0173] In some cases, a modification is of an AAV capsid. Capsids of AAV serotypes are assembled from 60 VP monomers with approximately 50 copies of VP3, 5 copies of VP2, and 5 copies of VP1. Topological prominent capsid surface structures are pores or “channel -like-structures” at each fivefold, depressions at each twofold, and three protrusions surrounding each threefold axis of symmetry. The pores allow exchange between the capsid interior and the outside. The depressions, more precisely the floor at each twofold axis, are the thinnest part of the viral capsid. The protrusions around the threefold axis harbor five of the nine so-called variable regions (VRs). Specifically, VR-IV, -V, and -VIII form loops (loop 1-4) at the top of the protrusions, while VR-VI and -VII are found at their base. VRs differ between serotypes and are responsible for serotype-specific variations in antibody and receptor binding. Because of their exposed positions and their function in receptor binding, VRs forming loops of the protrusions are ideal positions for capsid modifications aiming to re-direct or expand AAV tropism (cell surface targeting). While a re-directed tropism (vector re-targeting) combines ablation of natural receptor binding, for example by site-directed mutagenesis, with insertion of a ligand that mediates transduction through a novel non-natural AAV receptor, AAV vectors with tropism expansion gain the ability to transduce cells through an extra receptor while maintaining their natural receptor binding abilities.

[0174] In some aspects, a modification of an AAV capsid, can refer to an insertion of an exogenous polypeptide sequence. In other aspects, a modification can refer to a deletion in a polypeptide sequence. A modification can also refer to a modification of at least one amino acid residue, canonical or non-canonical, in a polypeptide sequence.

[0175] An insertion can comprise inserting at least 1 exogenous amino acid residue into a sequence coding an AAV capsid. The amino acid can refer to a canonical amino acid or a non-canonical amino acid. Any number of amino acid residues can be inserted. In some cases, an insertion site can be in the GH loop, or loop IV, of the AAV capsid protein, e.g., in a solvent-accessible portion of the GH loop, or loop IV, of the AAV capsid protein.

[0176] In some cases, a modification comprises insertion of an exogenous polypeptide sequence that comprises a sequence of Formula 1: X0-X1-X2-X1-X3-X1-X1-X4. In some cases, X0 is Valine (V), Isoleucine (I), Leucine (L), Phenylalanine (F), Tryptophan (W), Tyrosine (Y) or Methionine (M). In some cases, XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H).WSGR Docket No. 59561-719.601In some cases, X2 is V, I, L, or M, where X3 is E, S, or Q. In some cases, X4 is K, R, E, or A. In some cases, Formula 1 further comprises X5. X5 can be Proline (P) or R.

[0177] In some cases, Formula 1 comprises: L-A-L-G-X3-X1-X1-X4 (SEQ ID NO: 401), L-K-L-G- X3-X1-X1-X4 (SEQ ID NO: 402), or V-K-L-G-X3-X1-X1-X4 (SEQ ID NO: 403). In some cases, Formula 1 comprises V-K-L-G-X3-X1-X1-X4 (SEQ ID NO: 404). In some cases, an exogenous polypeptide is V-K-L-G-X3-X1-T-X4 (SEQ ID NO: 405) and / or V-K-L-G-X3-Xl-Xl-K (SEQ ID NO: 406). In some cases, an exogenous polypeptide comprises L-A-L-G-X3-X1-X1-X4 (SEQ ID NO: 407). In some cases, an exogenous polypeptide comprises L-A-L-G-X3-X1-T-X4 (SEQ ID NO: 408) and / or L-A-L-G-X3-X1-S-X4 (SEQ ID NO: 409). In some cases, an exogenous polypeptide comprises: L-A-L-G- X3-X1-T-R (SEQ ID NO: 410), L-A-L-G-X3-X1-T-K (SEQ ID NO: 411), L-A-L-G-X3-X1-T-E (SEQ ID NO: 412), and / or L-A-L-G-X3-X1-T-A (SEQ ID NO: 413). In some cases, an exogenous polypeptide comprises L-A-L-G-X3-X1-S-K (SEQ ID NO: 414). In some cases, an exogenous polypeptide comprises L-K-L-G-X3-X1-X1-X4 (SEQ ID NO: 415). In some cases, an exogenous polypeptide comprises: L-K-L-G-X3-X1-T-X4 (SEQ ID NO: 416). In some cases, an exogenous polypeptide comprises: L-K-L-G-X3-X1-T-K (SEQ ID NO: 419)

[0178] In some cases, an exogenous polypeptide comprises a sequence of Formula 1. In some cases, a sequence of Formula I comprises a polypeptide sequence having at least 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or up to about 100% identity with a sequence of Table 56. In some cases, an exogenous polypeptide is one of Table 56 with 0-2 modifications to a residue.

[0179] In some embodiments, the engineered capsid comprises an engineered AAV1 capsid (e.g., AV1.N54), where LALGQTTKPA (SEQ ID NO: 183) is inserted after S588 amino acid residue of the AAV1 VP1 capsid. In some embodiments, the engineered capsid comprises an engineered AAV2 capsid (e.g., AAV2.N54), where LALGQTTKPA (SEQ ID NO: 183) is inserted after N587 amino acid residue of the AAV2 VP1 capsid (e.g., as illustrated in SEQ ID NO: 221). In some embodiments, the engineered capsid comprises an engineered AAV6 capsid (AAV6.N54), where LALGQTTKPA (SEQ ID NO: 183) is inserted after N587 amino acid residue of the AAV6 VP1 capsid (e.g., as illustrated in SEQ ID NO: 224).

[0180] In some cases, at least 2 of the exogenous polypeptides, such as those described by Formula 1, are inserted into a capsid sequence of an AAV provided herein. The at least 2 exogenous polypeptides can be inserted into the same location or at different locations. In an aspect, any one of the exogenous polypeptide sequences provided in Table 56 can be insertedWSGR Docket No. 59561-719.601into an unmodified AAV capsid sequence, such as those wildtype sequences provided in Table 57, to generate an engineered AAV capsid.Table 56. Exemplary exogenous polypeptide sequences that can be inserted into AAV capsids (exemplary insertion sites are shown for AAV2 but comparable locations of other AAV serotypes are also contemplated)WSGR Docket No. 59561-719.601<<<< <<<<<<<<<< <<<<<<<<<<WSGR Docket No. 59561-719.601<<<<<< <<<<<<<<<<<<<<WSGR Docket No. 59561-719.601<<<< <<<<<<<<<< <<<<<<WSGR Docket No. 59561-719.601<<<< <<<<<<<<<< <<<<<<WSGR Docket No. 59561-719.601<<<< <<<<<<<<<< <<<<<<WSGR Docket No. 59561-719.601<<<< <<<<<<<<<< <<<<<<WSGR Docket No. 59561-719.601<<<< <<<<<<<<<< <<<<<<WSGR Docket No. 59561-719.601<<<< <<<<<<<<<< <<<<<<WSGR Docket No. 59561-719.601<<<< <<<<<<<<<< <<<<<<WSGR Docket No. 59561-719.601<<<< <<<<<<Table 57. Exemplary wild type AAV capsid polypeptide sequences<<<<<<<WSGR Docket No. 59561-719.601<<<<<<<<WSGR Docket No. 59561-719.601<<<

[0181] Similarly, a deletion can comprise deleting at least 1 amino acid residue in a sequence that codes for an AAV capsid. Any number of amino acids can be deleted. In some cases, at least, or at most: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or up to about 50 exogenous amino acid residues can be inserted and / or deleted in a polypeptide sequenceWSGR Docket No. 59561-719.601that codes for an AAV capsid. In some cases, at least or at most: 1-5, 5-10, 10-15, 15-20, or combinations thereof of exogenous amino acid residues can be inserted and / or deleted in a polypeptide sequence that codes for an AAV capsid. In some cases, from about or up to about: 5 amino acids to about 11 amino acids are inserted in an insertion site in the GH loop or loop IV of the capsid protein relative to a corresponding unmodified AAV capsid protein. For example, the insertion site can be between amino acids 587 and 588 of AAV2, or the corresponding positions of the capsid subunit of another AAV serotype. It should be noted that the insertion site 587-588 is based on an AAV2 capsid protein. From about 5 amino acids to about 11 amino acids can be inserted in a corresponding site in an AAV serotype other than AAV2 (e.g., AAV5, AAV6, AAV8, AAV9, etc.).

[0182] In some embodiments, the insertion site is a single insertion site between two adjacent amino acids located between amino acids 570-614 of VP1 of any AAV serotype, e.g., the insertion site is between two adjacent amino acids located in amino acids 570-610, amino acids am-600, amino acids 570-575, amino acids 575-580, amino acids 580-585, amino acids 585-590, amino acids 590-600, or amino acids 600-614, of VP1 of any AAV serotype or variant. For example, the insertion site can be between amino acids 580 and 581, amino acids 581 and 582, amino acids 583 and 584, amino acids 584 and 585, amino acids 585 and 586, amino acids 586 and 587, amino acids 587 and 588, amino acids 588 and 589, or amino acids 589 and 590. The insertion site can be between amino acids 575 and 576, amino acids 576 and 577, amino acids 577 and 578, amino acids 578 and 579, or amino acids 579 and 580. The insertion site can be between amino acids 590 and 591, amino acids 591 and 592, amino acids 592 and 593, amino acids 593 and 594, amino acids 594 and 595, amino acids 595 and 596, amino acids 596 and 597, amino acids 597 and 598, amino acids 598 and 599, or amino acids 599 and 600.

[0183] In some aspects, an insertion site can be between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 588 and 589 of AAV10.

[0184] As another example, the insertion site can be between amino acids 450 and 460 of an AAV capsid protein, as shown in Table 57. For example, the insertion site can be at (e.g., immediately N-terminal to) amino acid 453 of AAV2, at amino acid 454 of AAV1, at amino acid 454 of AAV6, at amino acid 456 of AAV7, at amino acid 456 of AAV8, at amino acid 454 of AAV9, or at amino acid 456 of AAV10.WSGR Docket No. 59561-719.601

[0185] In some embodiments, a subject capsid protein includes a GH loop comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence set forth in Table 57. Those skilled in the art would know, based on a comparison of the amino acid sequences of capsid proteins of various AAV serotypes, where an insertion site “corresponding to amino acids 587-588 of AAV2” would be in a capsid protein of any given AAV serotype.

[0186] In some cases, an exogenous polypeptide can have from 0 to 4 spacer amino acids (Yi-Y4) at the amino terminus and / or at the carboxyl terminus of any one of the exemplary polypeptides of Table 56 or Formula 1. Suitable spacer amino acids include, but are not limited to, leucine, alanine, glycine, and / or serine.

[0187] A modification of an AAV capsid can comprise a modification of at least one amino acid residue in a polypeptide sequence. In some cases, a modification can be made at any AAV capsid position, as described herein, and can include any number of modifications. In some cases, a modification can comprise a mutation. A mutation can comprise: a point mutation, missense mutation, nonsense mutation, deletion, duplication, frameshift, and / or repeat expansion.

[0188] In an aspect, an amino acid can be a non-polar, aliphatic residue such as glycine, alanine, valine, leucine, methionine, isoleucine, or proline. In an aspect, an amino acid residue is aromatic and is phenylalanine, tyrosine, or tryptophan. In an aspect, an amino acid residue is polar, noncharged and is serine, threonine, cysteine, asparagine, or glutamine. In an aspect, an amino acid is positively charged and is lysine, arginine, or histidine. In an aspect, an amino acid is negatively charged and is aspartate or glutamate.

[0189] In some cases, a mutation is a point mutation. A point mutation comprises a change from a charged amino acid residue to a polar or non-polar amino acid residue. In some cases, the charged amino acid is positively charged. In some cases, the charged amino acid is negatively charged.

[0190] A point mutation can be a conservative mutation. Non-limiting examples of conservative mutations comprise: a nonpolar aliphatic amino acid to a nonpolar aliphatic amino acid, a polar amino acid to a polar amino acid, a positively charged amino acid to a positively charged amino acid, a negatively charged amino acid to a negatively charged amino acid, and an aromatic amino acid to an aromatic amino acid. For example, 20 naturally occurring amino acids can share similar characteristics. Aliphatic amino acids can be glycine, alanine, valine, leucine, or isoleucine. Hydroxyl or sulfur / selenium-containing amino acids can be Serine, cysteine, selenocysteine, threonine, or methionine. A cyclic amino acid can be proline. An aromatic aminoWSGR Docket No. 59561-719.601acid can be phenylalanine, tyrosine, or tryptophan. A basic amino acid can be histidine, lysine, and arginine. An acidic amino acid can be aspartate, glutamate, asparagine, or glutamine. A conservative mutation can be, serine to glycine, serine to alanine, serine to serine, serine to threonine, serine to proline. A conservative mutation can be arginine to asparagine, arginine to lysine, arginine to glutamine, arginine to arginine, arginine to histidine. A conservative mutation can be Leucine to phenylalanine, leucine to isoleucine, leucine to valine, leucine to leucine, leucine to methionine. A conservative mutation can be proline to glycine, proline to alanine, proline to serine, proline to threonine, proline to proline. A conservative mutation can be threonine to glycine, threonine to alanine, threonine to serine, threonine to threonine, threonine to proline. A conservative mutation can be alanine to glycine, alanine to threonine, alanine to proline, alanine to alanine, alanine to serine. A conservative mutation can be valine to methionine, valine to phenylalanine, valine to isoleucine, valine to leucine, valine to valine. A conservative mutation can be glycine to alanine, glycine to threonine, glycine to proline, glycine to serine, glycine to glycine. A conservative mutation can be Isoleucine to phenylalanine, isoleucine to isoleucine, isoleucine to valine, isoleucine to leucine, isoleucine to methionine. A conservative mutation can be phenylalanine to tryptophan, phenylalanine to phenylalanine, phenylalanine to tyrosine. A conservative mutation can be tyrosine to tryptophan, tyrosine to phenylalanine, tyrosine to tyrosine. A conservative mutation can be cysteine to serine, cysteine to threonine, cysteine to cysteine. A conservative mutation can be histidine to asparagine, histidine to lysine, histidine to glutamine, histidine to arginine, histidine to histidine. A conservative mutation can be glutamine to glutamic acid, glutamine to asparagine, glutamine to aspartic acid, glutamine to glutamine. A conservative mutation can be asparagine to glutamic acid, asparagine to asparagine, asparagine to aspartic acid, asparagine to glutamine. A conservative mutation can be lysine to asparagine, lysine to lysine, lysine to glutamine, lysine to arginine, lysine to histidine. A conservative mutation can be aspartic acid to glutamic acid, aspartic acid to asparagine, aspartic acid to aspartic acid, aspartic acid to glutamine. A conservative mutation can be glutamine to glutamine, glutamine to asparagine, glutamine to aspartic acid, glutamine to glutamine. A conservative mutation can be methionine to phenylalanine, methionine to isoleucine, methionine to valine, methionine to leucine, methionine to methionine. A conservative mutation can be tryptophan to tryptophan, tryptophan to phenylalanine, tryptophan to tyrosine.

[0191] Non-limiting examples of additional amino acid mutations can be: A to R, A to N, A to D, A to C, A to Q, A to E, A to G, A to H, A to I, A to L, A to K, A to M, A to F, A to P, A to S,WSGR Docket No. 59561-719.601A 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, Gto L, Gto K, Gto M, GtoF, Gto P, Gto S, Gto T, Gto W, Gto Y, Gto V, Hto I, Hto L, H to K, H to M, H to F, H to P, H to S, H to T, H to W, H to Y, H to V, I to L, I to K, I to M, I to F, I to P, I to S, I to T, I to W, I to Y, I to V, L to K, L to M, L to F, L to P, L to S, L to T, L to W, L to Y, L to V, K to M, K to F, K to P, K to S, K to T, K to W, K to Y, K to V, M to F, M to P, M to S, M to T, M to W, M to Y, M to V, F to P, F to S, F to T, F to W, F to Y, F to V, P to S, P to T, P to W, P to Y, P to V, S to T, S to W, S to Y, S to V, T to W, T to Y, T to V, W to Y, W to V, Y to V, and any of the previously described mutations in reverse.

[0192] Any one of the aforementioned modifications, insertions, deletions, and / or mutations, can be made at any residue in an AAV sequence. The sequence may be a capsid sequence. In other cases, the sequence may not be a capsid sequence but rather a Rep and / or X sequence. The sequence may be in a VP1, VP2, and / or VP3 as previously described. In some cases, the sequence modification is of a loop of a capsid sequence, such as loop 3 and / or loop 4. In some cases, the modification is of a residue of a sequence in Table 57.

[0193] In some cases, a modification, such as insertion, deletion, and / or mutation is of a residue of a capsid polypeptide sequence in Table 57. In some cases, a modification is from 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, or combinations thereof. In some cases, a modification is in a residue at position 200-300, 300-400, 400-500, 500-600 or combinations thereof. In some cases, a modification is in a residue at position 300-500 or combinations thereof. In an aspect, an insertion site is in the GH loop, or loop IV, of the AAV capsid protein, e.g., in a solvent-accessible portion of the GH loop, or loop IV, of the AAV capsid protein. For example, the insertion site is within amino acids 570-611 of AAV2, within amino acids 571-612 of AAV1, within amino acids 560-601 of AAV5, within amino acids 571 to 612 of AAV6, within amino acids 572 to 613 of AAV7, within amino acids 573 to 614 of AAV8, within amino acids 571 to 612 of AAV9, or within amino acids 573 to 614 of AAV10.WSGR Docket No. 59561-719.601

[0194] For example, the insertion site can be between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 589 and 590 of AAV10. In some cases, a modification is at position 452, 453, 466, 467, 468, 471, 585, 586, 587, and / or 588 of AAV2. In some cases, a modification is at position 452 or 453 of AAV2. In some cases, a modification is at position 587 or 588 of AAV2. In some cases, a modification is an insertion at position 452, 453, 466, 467, 468, 471, 585, 586, 587, and / or 588 of any one of SEQ ID NOs: 221-226. In some cases, a modification is an insertion at position 452, 453, 466, 467, 468, 471, 585, 586, 587, and / or 588 of SEQ ID NO: 221. In some cases, a modification is a mutation, and the mutation is R585A or R588A of any one ofSEQ ID NOs: 221-226 In some cases, a modification is a mutation, and the mutation is R585A orR588A of SEQ ID NO: 221.

[0195] In some embodiments, a subject engineered AAV capsid does not include any other amino acid modifications mutations, substitutions, insertions, or deletions, other than an insertion of from about 5 amino acids to about 11 amino acids in a loop (loop 3 and / or 4) relative to a corresponding unmodified AAV capsid protein. In other embodiments, a subject variant AAV capsid includes from 1 to about 25 amino acid insertions, deletions, or substitutions, compared to an unmodified AAV capsid protein, in addition to an insertion of from about 5 amino acids to about 11 amino acids in the loop 3 and / or loop 4 relative to an unmodified AAV capsid protein. In an embodiment, a subject AAV virion capsid does not include any other amino acid substitutions, insertions, or deletions, other than an insertion of from about 7 amino acids to about 10 amino acids in a GH loop or loop IV relative to a corresponding parental AAV capsid protein. In other embodiments, a subject AAV virion capsid includes from 1 to about 25 amino acid insertions, deletions, or substitutions, compared to the parental AAV capsid protein, in addition to an insertion of from about 7 amino acids to about 10 amino acids in the GH loop or loop IV relative to a corresponding parental AAV capsid protein. For example, in some embodiments, a subject AAV virion capsid includes from 1 to about 5, from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, or from about 20 to about 25 amino acid insertions, deletions, or substitutions, compared to the parental AAV capsid protein, in addition to an insertion of from about 7 amino acids to about 10 amino acids in the GH loop or loop IV relative to a corresponding parental AAV capsid protein.WSGR Docket No. 59561-719.601

[0196] In some cases, a chimeric AAV capsid is provided herein. A chimeric capsid comprises a polypeptide sequence from at least 2 AAV serotypes. A chimeric capsid can comprise a mix of sequences selected from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and / or AAV12. In some cases, the chimeric serotypes are different between VP1, VP2, and / or VP3. In some cases, a chimeric capsid comprises sequences from at least 2 serotypes selected from: AAV4 and AAV6, AAV5 and AAV6, AAV11 and AAV6, AAV12 and AAV6, and any combination thereof. In some cases, a first AAV serotype can be AAV4 and a second serotype can be AAV6. In some cases, a first AAV serotype and a second AAV serotype of a chimeric AAV vector can be AAV11 and AAV6. In some cases, a first AAV serotype and a second AAV serotype of a chimeric AAV vector can be AAV12 and AAV6. In some cases, a chimeric capsid comprises sequences from: AAV2 and AAV5 or AAV2 and AAV6. In some cases, a chimeric capsid comprises sequences from: AAV2 and AAV5, AAV2 and AAV6, AAV2 and AAV8, AAV2 and AAV9, AAV2 and AAV1, and AAV2 and AAV12.

[0197] The modifications to an AAV provided herein can confer enhanced activity to the engineered AAV as compared to an otherwise unmodified or wildtype AAV. Modifications provided herein can improve cell transduction, tropism, and / or reduce immunogenicity associated with the capsid.

[0198] In some cases, a modification provided herein enhances cellular transduction. Cellular transduction can refer to the ability of an AAV to infect a cell (in vivo or in vitro) and / or deliver a transgene into the cell.

[0199] In some cases, a modification provided herein enhances tropism. Enhanced tropism refers to gaining the ability to transduce cells through an extra receptor, as compared to an otherwise unmodified AAV. In some aspects, enhanced tropism can improve infectivity of an ocular cell, thereby improving gene therapy by way utilization of the engineered AAV. In some cases, a modification provided herein can improve tropism to an ocular cell selected from: bipolar, retinal ganglion, horizontal, amacrine, epithelial, retinal pigment, photoreceptor, or any combination thereof. In some cases, a modification improves tropism to a retinal cell.

[0200] Also provided herein are AAV vectors. AAV vectors comprise inverted terminal repeats (ITRs), Rep, Cap, AAP, and X sequences. Typically, the AAV viral genome is flanked by the ITRs, which serve as packaging signal and origin of replication. The rep gene encodes a family of multifunctional proteins (Rep proteins) responsible for controlling viral transcription, replication, packaging, and integration in AAVS1. For AAV2, four Rep proteins are described. Expression of Rep78 and Rep68 is controlled by the AAV2-specific p5 promoter, while p!9WSGR Docket No. 59561-719.601controls expression of the smaller Rep proteins (Rep52 and Rep40). Rep68 and Rep40 are splice variants of Rep78 and Rep52, respectively. Numbers indicate the molecular weight. Expression of AAP and the viral capsid proteins VP1 (90 kDa), VP2 (72 kDa), and VP3 (60 kDa), all encoded in the cap gene, is controlled by the p40 promoter. The X gene is located 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 seems to enhance viral replication, AAP is essential for capsid assembly. The three different VPs contribute in a 1 (VP1):1 (VP2):10 (VP3) ratio to the icosahedral AAV2 capsid.

[0201] An engineered capsid protein disclosed herein can be isolated, e.g., purified. In some embodiments, an engineered capsid disclosed herein is included in an AAV vector or an AAV virion (for example recombinant AAV virion, rAAV, or an AAV viral particle). In other embodiments, such engineered AAV vectors and / or AAV variant virions are used in an in vivo or ex vivo method of treating ocular disease in a primate retina, for example human retina.

[0202] Provided herein are also vectors that comprise engineered AAV capsids. Any one of the previously described modifications can be encompassed in a vector provided herein. In some cases, an AAV vector comprises an engineered capsid that comprises an exogenous sequence in at least two loops of a VP domain as compared to an otherwise comparable AAV capsid sequence that lacks the exogenous sequence. In some aspects, vectors provided herein can further comprise a transgene sequence.Engineered polypeptide

[0203] Described herein, in some aspects, is an engineered polypeptide. In some embodiments, the engineered polypeptide is encoded by an engineered polynucleotide described herein. In some embodiments, the engineered polypeptide comprises a first angiogenesis inhibitor and a second angiogenesis inhibitor described herein. In some embodiments, the engineered polypeptide comprises a third angiogenesis inhibitor. In some embodiments, the engineered polypeptide comprises two or more angiogenesis inhibitors, where the two or more angiogenesis inhibitors are covalently connected by an antibody (e.g., an Fc region described herein) or a linker.

[0204] In some embodiments, the engineered polypeptide comprises 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 inhibitorWSGR Docket No. 59561-719.601(C3i) comprises an amino acid sequence that is any one of SEQ ID NOs: 1-15. In some embodiments, the complement 3 inhibitor (C3i) comprises an amino acid sequence that is at 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 12 contiguous amino acids of any one of SEQ ID NOs: 1-15.

[0205] 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 connected to a complement 3 inhibitor. In some embodiments, the CNP is covalently connected to a complement 3 inhibitor by a linker. In some embodiments, the natriuretic peptide or the 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 the natriuretic peptide fusion protein comprises an amino acid sequence that is any one of SEQ ID NOs: 61-72.

[0206] In some embodiments, the engineered polypeptide comprises an inhibitor of a membrane attack complex (MAC) and at least one additional angiogenesis inhibitor. In some embodiments, the inhibitor of the MAC comprises CD59. In some embodiments, the 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, the 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, the 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, the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 41-45. In some embodiments, the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 312-319. In some embodiments, the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 325-329.

[0207] In some embodiments, the engineered polypeptide comprises a collagen or fragment thereof. In some embodiments, the collagen or fragment thereof comprises an endostatin or fragment thereof. In some embodiments, the endostatin or 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 someWSGR Docket No. 59561-719.601embodiments, the endostatin or fragment thereof comprises an amino acid sequence that is SEQ ID NO: 51

[0208] In some embodiments, the engineered polypeptide comprises an 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 any 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 any one of SEQ ID NOs: 81-87 or SEQ ID NOs: 88-92, or a combination thereof, or a fragment thereof.

[0209] 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 connected to an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a fragment crystallizable (Fc) region. In some embodiments, the engineered polypeptide further comprises a CD59, an endostatin, a VEGF inhibitor, or a combination thereof. In some embodiments, the engineered polypeptide further comprises a CD59, an endostatin, and a VEGF inhibitor. In some embodiments, the engineered polypeptide further comprises a 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 a CD59 and a VEGF inhibitor. In some embodiments, the engineered polypeptide further comprises a CD59, and an endostatin. In some embodiments, the engineered polypeptide comprises a complement 3 inhibitor and an inhibitor of a membrane attack complex (MAC). In some embodiments, the inhibitor of the MAC comprises CD59. In some embodiments, the engineered polypeptide comprises a natriuretic peptide, an endostatin, a VEGF inhibitor, or a combination thereof. In some embodiments, the engineered polypeptide comprises a natriuretic peptide, an endostatin, and a VEGF inhibitor. In some embodiments, the engineered polypeptide comprises a complement 3 inhibitor and a collagen or fragment thereof. In some embodiments, the collagen or fragment thereof comprises an endostatin or fragment thereof. In some embodiments, the engineered polypeptide further comprises a natriuretic peptide, a CD59, a VEGF inhibitor, or a combination thereof. In some embodiments, the engineered polypeptide further comprises aWSGR Docket No. 59561-719.601natriuretic peptide. In some embodiments, the engineered polypeptide further comprises a 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.

[0210] In some embodiments, the engineered polypeptide comprises a 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 connected to an antibody or fragment thereof. In some embodiments, the antibody or 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.

[0211] In some embodiments, the engineered polypeptide comprises a CD59 and a collagen or fragment thereof. In some embodiments, the collagen or fragment thereof comprises an endostatin or fragment thereof. In some embodiments, the collagen or fragment thereof comprises an 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.

[0212] In some embodiments, the engineered polypeptide comprises a CD59 and a VEGF inhibitor.

[0213] In some embodiments, the engineered polypeptide comprises a natriuretic peptide, a complement 3 inhibitor, an endostatin, or a combination thereof. In some embodiments, the engineered polypeptide comprises a natriuretic peptide, a complement 3 inhibitor, and an endostatin.

[0214] 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-furin 2A-C3i-Fc4-CNP36. In some embodiments, the engineered polypeptide comprises sCD59-furin 2A- endostatin-linker-C3i. InWSGR Docket No. 59561-719.601some embodiments, the engineered polypeptide comprises sCD59-furin 2A- Aflibercept-linker-C3i. In some embodiments, the engineered polypeptide comprises C3i-Fc4-CNP36-Furin-mCD59. In some embodiments, the engineered polypeptide comprises C3i-Fc4-endostatin-Furin sCD59. In some embodiments, the engineered polypeptide comprises a sequence as shown in Fig. 1. In some embodiments, the engineered polypeptide comprises a sequence as shown in Figs. 2A-B. In some embodiments, the engineered polypeptide comprises a sequence as shown in Fig. 3. In some embodiments, the engineered polypeptide comprises a sequence as shown in Fig.4. In some embodiments, the engineered polypeptide comprises a sequence as shown in Figs. 5A-C. In some embodiments, the engineered polynucleotide encodes a sequence as shown in Fig.16. In some embodiments, the engineered polynucleotide encodes a sequence as shown in Fig. 24. In some embodiments, the engineered polynucleotide encodes a sequence as shown in Fig. 26. In some embodiments, the engineered polynucleotide encodes a sequence as shown in Fig. 32.

[0215] In some embodiments, the engineered polypeptide can be administered to a subject to treat a disease or condition. In some embodiments, the engineered polypeptide can be formulated into a pharmaceutical composition to be administered to a subject to treat a disease or condition. In some embodiments, the engineered polypeptide can increase activity or signal cascade associated with complement pathway. In some embodiments, the engineered polypeptide can increase activity or signal cascade associated with a natriuretic peptide receptor (NPR). In some embodiments, the engineered polypeptide can increase activity or signal cascade associated with a cyclic GMP (cGMP) signaling pathway. In some embodiments, the engineered polypeptide can increase activity or signal cascade associated with CD59. In some embodiments, the engineered polypeptide can increase activity or signal cascade associated with endostatin. In some embodiments, the engineered polypeptide can decrease activity or signal cascade associated with VEGF.

[0216] In some embodiments, the engineered polypeptide can be administered to a subject to treat a disease or condition by increasing activity or signal cascade associated with complement pathway. In some embodiments, the engineered polypeptide can be administered to a subject to treat a disease or condition by increasing activity or signal cascade associated with a natriuretic peptide receptor (NPR). In some embodiments, the engineered polypeptide can be administered to a subject to treat a disease or condition by increasing activity or signal cascade associated with a cGMP signaling pathway. In some embodiments, the engineered polypeptide can be administered to a subject to treat a disease or condition by increasing activity or signal cascadeWSGR Docket No. 59561-719.601associated with CD59. In some embodiments, the engineered polypeptide can be administered to a subject to treat a disease or condition by increasing activity or signal cascade associated with endostatin. In some embodiments, the engineered polypeptide can be administered to a subject to treat a disease or condition by decreasing activity or signal cascade associated with VEGF.

[0217] In some embodiments, the engineered polypeptide described herein comprises a CD59 described herein. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 234-236, 331, or 332. In some embodiments, the CD59 is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to any one of SEQ ID NOs: 234-236, 331, or 332. In some embodiments, the CD59 is encoded from a nucleic acid sequence that is any one of SEQ ID NOs: 234-236, 331, or 332. In some embodiments, the CD59 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises at least 50 contiguous polypeptides that are identical to any one of SEQ ID NOs: 244-246. In some embodiments, the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 244-246

[0218] In some embodiments, the engineered polypeptide described herein comprises a complement 3 inhibitor or a C3 degraded fragment described herein, a C-type natriuretic peptide (CNP), or an endostatin (ES) described herein. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment described herein, and the C-type natriuretic peptide (CNP) described herein can be a fusion protein. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment described herein, and the endostatin (ES) described herein can be a fusion protein. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is SEQ ID NO: 231 or SEQ ID NO: 232. In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQWSGR Docket No. 59561-719.601ID NO: 241 or SEQ ID NO: 242 In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises at least 5 contiguous polypeptide that are identical to SEQ ID NO: 241 or SEQ ID NO: 242 In some embodiments, the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is SEQ ID NO: 241 or SEQ ID NO: 242.

[0219] In some embodiments, the CNP is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231. In some embodiments, the CNP is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231. In some embodiments, the CNP is encoded from a nucleic acid sequence that is SEQ ID NO: 231. In some embodiments, the CNP comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241 In some embodiments, the CNP comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 241. In some embodiments, the CNP comprises an amino acid sequence that is SEQ ID NO: 241.

[0220] In some embodiments, the endostatin is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 233. In some embodiments, the endostatin is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 233. In some embodiments, the endostatin is encoded from a nucleic acid sequence that is SEQ ID NO: 233. In some embodiments, the endostatin comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 243 In some embodiments, the endostatin comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 243 In some embodiments, the endostatin comprises an amino acid sequence that is SEQ ID NO: 243.Pharmaceutical composition

[0221] Described herein are pharmaceutical compositions comprising an engineered polynucleotide, an AAV vector comprising the engineered polynucleotide, an engineered polypeptide, a cell transduced by an AAV vector comprising an engineered polynucleotide, a viral particle comprising the engineered polynucleotide, or a combination thereof. In some embodiments, the pharmaceutical composition further comprises as pharmaceutically acceptable: carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition comprises two or more active agents as disclosed herein. In some embodiments, the pharmaceuticalWSGR Docket No. 59561-719.601composition comprising the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, or the AAV vector comprising the engineered polynucleotide treats a disease or condition described herein. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the disease or condition comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, traumatic glaucoma, Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), bluecone monochromacy, or a combination thereof.

[0222] For in vivo delivery, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the engineered polypeptide, the cell transduced by an AAV vector comprising the engineered polynucleotide, or a combination thereof can be formulated into pharmaceutical compositions and can generally be administered intravitreally or parenterally (e.g., administered via an intramuscular, subcutaneous, intratumoral, transdermal, intrathecal, etc., route of administration). In some embodiments, the pharmaceutical composition is formulated for administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or a combination thereof to a subject in need thereof to a subject in need thereof.

[0223] In some aspects, a pharmaceutical composition can be used to treat a subject such as a human or mammal, in need thereof. In some cases, a subject can be diagnosed with a disease, e.g., ocular disease. In some aspects, subject pharmaceutical compositions are co-administered with secondary therapies. A secondary therapy can comprise any therapy for ocular use. In some cases, a secondary therapy comprises nutritional therapy, vitamins, laser treatment, such as laser photocoagulation, photodynamic therapy, Visudyne, anti-VEGF therapy, eyewear, eye drops, numbing agents, Orthoptic vision therapy, Behavioral / perceptual vision therapy, and the like. In some aspects, any of the previously described biologies can be considered a secondary therapy.WSGR Docket No. 59561-719.601

[0224] In some embodiments, an effective amount of the pharmaceutical composition results in a decrease in the rate of loss of retinal function, anatomical integrity, or retinal health, e.g., a 2-fold, 3-fold, 4-fold, or 5-fold or more decrease in the rate of loss and hence progression of disease, for example, a 10-fold decrease or more in the rate of loss and hence progression of disease.

[0225] In some embodiments, an effective amount of the pharmaceutical composition decreases neovascularization signaling in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more compared to neovascularization signaling in a cell that is not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition decreases neovascularization in a subject in need thereof at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more compared to neovascularization in the subject if the subject is not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition decreases blood vessel leakage in a subject in need thereof 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 blood vessel leakage in the subject if the subject is not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition decreases inflammation in a subject in need thereof at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more compared to inflammation in the subject if the subject is not treated with the pharmaceutical composition.

[0226] In some embodiments, the effective amount of the subject rAAV virion results in a gain in visual function, retinal function, an improvement in retinal anatomy or health, and / or an improvement in ocular motility and / or improvement in neurological function, e.g. a 2-fold, 3-fold, 4- fold or 5 -fold improvement or more in retinal function, retinal anatomy or health, and / or improvement in ocular motility, e.g. a 10-fold improvement or more in retinal function, retinal anatomy or health, and / or improvement in ocular motility. As can be readily appreciated by the ordinarily skilled artisan, the dose required to achieve the desired treatment effect can typically be in the range of 1 x 108to about 1 x 1015recombinant virions, typically referred to by the ordinarily skilled artisan as 1 x 108to about 1 x 1015“vector genomes”.WSGR Docket No. 59561-719.601

[0227] In some aspects, compositions provided herein, such as pharmaceutical compositions are administered to a subject in need thereof. In some cases, an administration comprises delivering a dosage of an AAV of about vector 0.5 x 109vg, 1.0 x 109 vg, 1.0 x 1010, 1.0 x 1011vg, 3.0 x 1011vg, 6 x 1011vg, 8.0 x 1011vg, 1.0 x 1012vg, 1.0 x 1013vg, 1.0 x 1014vg, 1.0 x 1015vg, 1.5 x 1015vg. For example, for in vivo injection, e.g., injection directly into the eye, a therapeutically effective dose can be on the order of from about 106to about 1015of subject AAV virions, e.g., from about 108to 1012engineered AAV virions. For in vitro transduction, an effective amount of engineered AAV virions to be delivered to cells can be on the order of from about 108to about 1013of the engineered AAV virions. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.

[0228] Administrations can be repeated for any amount of time. In some aspects, administering is performed: twice daily, every other day, twice a week, bimonthly, trimonthly, once a month, every other month, semiannually, annually, or biannually.

[0229] Dosage treatment may be a single dose schedule or a multiple dose schedule. Moreover, the subject may be administered as many doses as appropriate. One of skill in the art can readily determine an appropriate number of doses. In some aspects, a pharmaceutical composition is administered via intravitreal injection, subretinal injection, microinjection, or supraocular injection.

[0230] In practicing the methods of treatment or use provided herein, therapeutically effective amounts of the pharmaceutical composition described herein are administered to a mammal having a disease, disorder, or condition to be treated, e.g., cancer. In some embodiments, the mammal is a human. A therapeutically effective amount 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 cases, compositions described herein, may be used singly or in combination with one or more therapeutic agents as components of mixtures.

[0231] The pharmaceutical composition described herein may be administered to a subject by appropriate administration routes, including but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration routes. The composition described herein may include, but not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills,WSGR Docket No. 59561-719.601delayed release formulations, extended-release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[0232] The pharmaceutical composition may be manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping or compression processes.

[0233] In certain embodiments, the pharmaceutical composition provided herein includes one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0234] In some embodiments, the pharmaceutical composition described herein is formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations. In one aspect, a therapeutic agent as discussed herein, e.g., therapeutic agent is formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection. In one aspect, formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for rehydration into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the growth of microorganisms may be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some cases, it is desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectableWSGR Docket No. 59561-719.601pharmaceutical form may be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

[0235] In another aspect, dosage forms include microencapsulated formulations. In some embodiments, one or more other compatible materials are present in the microencapsulation material. Non-limiting example of materials includes pH modifiers, erosion facilitators, antifoaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0236] Liquid formulation dosage forms for oral administration are optionally aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. In addition to therapeutic agent the liquid dosage forms optionally include additives, such as: (a) disintegrating agents; (b) dispersing agents; (c) wetting agents; (d) at least one preservative, (e) viscosity enhancing agents, (f) at least one sweetening agent, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersions\ further includes a crystal-forming inhibitor.

[0237] In some embodiments, the pharmaceutical composition described herein is selfemulsifying drug delivery systems (SEDDS). Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are created by vigorous mechanical dispersion. SEDDS, as opposed to emulsions or microemulsions, spontaneously form emulsions when added to an excess of water without any external mechanical dispersion or agitation. An advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Additionally, water or the aqueous phase is optionally added just prior to administration, which ensures stability of an unstable or hydrophobic active ingredient. Thus, the SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provides improvements in the bioavailability of hydrophobic active ingredients.

[0238] Furthermore, the pharmaceutical composition optionally includes one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.WSGR Docket No. 59561-719.601

[0239] Additionally, the pharmaceutical composition optionally includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate. Kit

[0240] Disclosed herein, in some embodiments, are kits for using comprising an engineered polynucleotide, an AAV comprising the engineered polynucleotide, an engineered polypeptide, a cell transduced by an AAV vector comprising an engineered polynucleotide, a viral particle comprising the engineered polynucleotide, a pharmaceutical composition, or a combination thereof described herein. In some embodiments, the kit disclosed herein may be used to treat a disease or condition in a subject. In some embodiments, the kit comprises an assemblage of materials or components apart from comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the engineered polypeptide, the cell transduced by an AAV vector comprising an engineered polynucleotide, or the pharmaceutical composition.

[0241] In some embodiments, the kit described herein comprises components for selecting for a homogenous population of AAV containing the engineered polynucleotide described herein. In some embodiments, the kit comprises the components for assaying the number of units of a biomolecule (e.g., the AAV) synthesized, and / or released or expressed on the surface by a host cell. In some embodiments, the kit comprises components for performing assays such as enzyme-linked immunosorbent assay (ELISA). The exact nature of the components configured in the kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating a disease or condition disclosed herein (e.g., cancer) in a subject. In some embodiments, the kit is configured particularly for the purpose of treating mammalian subjects. In some embodiments, the kit is configured particularly for the purpose of treating human subjects.

[0242] Instructions for use may be included in the kit. In some embodiments, the kit comprises instructions for administering the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the engineered polypeptide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the pharmaceutical composition, or a combination thereof to a subject in need thereof. In some embodiments, the kit comprises instructions for further engineering a cell to express a biomolecule (e.g., the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the engineeredWSGR Docket No. 59561-719.601polypeptide, the AAV comprising the engineered polynucleotide, or the cell transduced with the AAV vector). In some embodiments, the kit comprises instructions for thawing or otherwise restoring biological activity of the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, AAV comprising the engineered polynucleotide, which may have been cryopreserved or lyophilized during storage or transportation. In some embodiments, the kit comprises instructions for measuring the viability of the restored the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, AAV comprising the engineered polynucleotide to ensure efficacy for its intended purpose (e.g., therapeutic efficacy if used for treating a subject).

[0243] Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia. The materials or components assembled in the kit may be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example, the components may be in dissolved, dehydrated, or lyophilized form; they may be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s).Method of delivery

[0244] The engineered polynucleotide can be readily introduced into a host cell, e.g., a mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the engineered polynucleotide can be transferred into a host cell by physical, chemical, or biological means. In some embodiments, the engineered polynucleotide can be delivered to a host cell by encapsulating the engineered polynucleotide in a viral particle such as an AAV particle. In some embodiments, the engineered polynucleotide can be delivered into the cell via physical methods such as calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, electroporation, and the like.

[0245] Physical methods for introducing the engineered polynucleotide encoding into the cell can include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, electroporation, and the like. One method for the introduction of the engineered polynucleotide a host cell is calcium phosphate transfection.

[0246] Chemical means for introducing the engineered polynucleotide encoding the non-naturally into the cell can include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, spherical nucleic acid (SNA), liposomes, or lipidWSGR Docket No. 59561-719.601nanoparticles. An example colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of engineered polynucleotide or vector encoding the engineered polynucleotide with targeted nanoparticles.

[0247] In the case where a non-viral delivery system is utilized, an example delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the engineered polynucleotide or vector encoding the engineered polynucleotide into a cell (in vitro, ex vivo, or in vivo). In another aspect, the vector can be associated with a lipid. The vector associated with a lipid can be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the engineered polynucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, in some embodiments, they are present in a bilayer structure, as micelles, or with a “collapsed” structure. Alternately, they are simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which are, in some embodiments, naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0248] Lipids suitable for use are obtained from commercial sources. Stock solutions of lipids in chloroform or chloroform / methanol are often stored at about -20 °C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes are often characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids, inWSGR Docket No. 59561-719.601some embodiments, assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0249] In some cases, non-viral delivery method comprises lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, exosomes, polycation or lipid: cargo conjugates (or aggregates), naked polypeptide (e.g., recombinant polypeptides), naked DNA, artificial virions, and agent-enhanced uptake of polypeptide or DNA. In some embodiments, the delivery method comprises conjugating or encapsulating the compositions or the engineered polynucleotides described herein with at least one polymer such as natural polymer or synthetic materials. The polymer can be biocompatible or biodegradable. Nonlimiting examples of suitable biocompatible, biodegradable synthetic polymers can include aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylenes oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, and poly(anhydrides). Such synthetic polymers can be homopolymers or copolymers (e.g., random, block, segmented, graft) of a plurality of different monomers, e.g., two or more of lactic acid, lactide, glycolic acid, glycolide, epsilon-caprolactone, trimethylene carbonate, p-dioxanone, etc. In an example, the scaffold can be comprised of a polymer comprising glycolic acid and lactic acid, such as those with a ratio of glycolic acid to lactic acid of 90 / 10 or 5 / 95. Non-limiting examples of naturally occurring biocompatible, biodegradable polymers can include glycoproteins, proteoglycans, polysaccharides, glycosamineoglycan (GAG) and fragment(s) derived from these components, elastin, laminins, decrorin, fibrinogen / fibrin, fibronectins, osteopontin, tenascins, hyaluronic acid, collagen, chondroitin sulfate, heparin, heparan sulfate, ORC, carboxymethyl cellulose, and chitin.

[0250] In some cases, the engineered polynucleotide described herein can be packaged and delivered to the cell via extracellular vesicles. The extracellular vesicles can be any membranebound particles. In some embodiments, the extracellular vesicles can be any membrane-bound particles secreted by at least one cell. In some instances, the extracellular vesicles can be any membrane-bound particles synthesized in vitro. In some instances, the extracellular vesicles can be any membrane-bound particles synthesized without a cell. In some cases, the extracellular vesicles can be exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptosomes, oncosomes, exophers, enveloped viruses, exomeres, or other very large extracellular vesicles.

[0251] In some embodiments, the engineered polynucleotide can be delivered into the cell via biological methods such as the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes intoWSGR Docket No. 59561-719.601mammalian, e.g., human cells. Other viral vectors, in some embodiments, are derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. Exemplary viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV vectors), pox vectors, parvoviral vectors, baculovirus vectors, measles viral vectors, or herpes simplex virus vectors (HSVs). In some instances, the retroviral vectors include gamma-retroviral vectors such as vectors derived from the Moloney Murine Keukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Steam cell Virus (MSCV) genome. In some instances, the retroviral vectors also include lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome. In some instances, AAV comprises a serotype, including AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a combination thereof. Based on these initial serotypes, AAV capsid of each serotype can be engineered to make them better suited for biological functions, tissue or cell selection. In some embodiments, an AAV is AAV2 and variants AAV2.N53 and AAV2.N54 which are used in the examples of the present disclosure. Chimeric AAVs are also contemplated that may contain at least 2 AAV serotypes. In some cases, at least 3, at least 4, at least 5, at least 6, at least 7, or up to 8 different serotypes are combined in a chimeric AAV. In some cases, only a portion of the AAV is chimeric. For example, suitable portions can include the capsid, VP1, VP2, or VP3 domains and / or Rep. In some cases, at least one of VP1, VP2, and VP3 has at least one amino acid substitution compared to an otherwise comparable wild-type AAV capsid protein. In some cases, a mutation can occur in VP1 and VP2, in VP1 and VP3, in VP2 and VP3, or in VP1, VP2, and VP3. In some embodiments, at least one of VP1, VP2, and VP3 has from one to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3, e.g., from about one to about 5, from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, or from about 20 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3. In some cases, a VP can be removed. For example, in some embodiments a mutant AAV does not comprise at least one of VP1, VP2, or VP3.Method of modifying cell

[0252] In an aspect, provided herein are also methods of modifying cells to thereby generate engineered cells. Cells can refer to primary cells, recombinant cells, or cell lines. In some cases, a cell is a packaging cell. A packaging cell can be any one of: HEK 293 cells, HeLa cells, and Vero cells to name a few. An engineered cell can be a primary cell. In some cases, an engineered cell can be an ocular cell. Suitable ocular cells include but are not limited to a: photoreceptor, ganglion cell, RPE cell, amacrine cell, horizontal cell, muller cell, and the like.WSGR Docket No. 59561-719.601

[0253] In some cases, a cell is a packaging cell utilized to generate viral particles. To generate AAV virions or viral particles, an AAV vector is introduced into a suitable host cell using known techniques, such as by transfection. In some cases, transfection techniques are used, e.g., CaPO4 transfection or electroporation, and / or infection by hybrid adenovirus / AAV vectors into cell lines such as the human embryonic kidney cell line HEK 293 (a human kidney cell line containing functional adenovirus El genes which provides trans-acting El proteins). Suitable transfection methods include calcium phosphate co-precipitation, direct micro-injection, electroporation, liposome mediated gene transfer, and nucleic acid delivery using high velocity microprojectiles, which are known in the art.

[0254] To engineer a cell, a plurality of cells may be contacted with an isolated engineered polynucleotide. Contacting can comprise any length of time and may include from about 5 min to about 5 days. Contacting can last from about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, or about 60 minutes. In some cases, the contacting can last from 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.

[0255] In some cases, supernatant of the packaging cell line is treated by PEG precipitation for concentrating the virus. In other cases, a centrifugation step can be used to concentrate a virus. For example, a column can be used to concentration a virus during a centrifugation. In some embodiments, a precipitation occurs at no more than about 4 °C. (for example about 3 °C, about 2 °C, about 1 °C, or about 1 °C) for at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours, or at least about 24 hours. In some embodiments, the recombinant AAV is isolated from the PEG-precipitated supernatant by low-speed centrifugation followed by CsCl gradient. The low-speed centrifugation can be to can be 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-precipitated supernatant by centrifugation at about 5000 RPM for about 30 minutes followed by CsCl gradient. In some cases, CsCl purification can be replaced with IDX gradient ultracentrifugation. Supernatant can be collected at about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, or a time between any of these two time points after a transfection. Supernatant can also be purified, concentrated, or a combination thereof. For example, a concentration or viral titer can be determined by qPCR or silver stain.WSGR Docket No. 59561-719.601

[0256] In an aspect, provided is also a plurality of AAV particles (containing the engineered polynucleotide described herein) isolated from an engineered cell. A viral titer can be from about 102vp / mL, about 103vp / mL, about 104vp / mL, about 105vp / mL, about 106vp / mL, about 107vp / mL, about 108vp / mL, or up to about 109vp / mL. A viral titer can be from about 102GC / mL, about 103GC / mL, about 104GC / mL, about 105GC / mL, about 106GC / mL, about 107GC / mL, about 108GC / mL, or up to about 109GC / mL. In some cases, a viral titer can be from about 102TU / mL, about 103TU / mL, about 104TU / mL, about 105TU / mL, about 106TU / mL, about 107TU / mL, about 108TU / mL, or up to about 109TU / mL. An optimal viral titer can vary depending on cell type to be transduced. A range of virus can be from about 1000 MOI to about 2000 MOI, from about 1500 MOI to about 2500 MOI, from about 2000 MOI to about 3000 MOI, from about 3000 MOI to about 4000 MOI, from about 4000 MOI to about 5000 MOI, from about 5000 MOI to about 6000 MOI, from about 6000 MOI to about 7000 MOI, from about 7000 MOI to about 8000 MOI, from about 8000 MOI to about 9000 MOI, from about 9000 MOI to about 10,000 MOI. For example, to infect 1 million cells using a MOI of 10,000, one can need 10,000 x 1,000,000 = 1010GC.

[0257] In some cases, a plurality of AAV particles can be formulated into unit dose form.Various formulations are contemplated for adult or pediatric delivery and include but are not limited to: 0.5 x 109vg, 1.0 x 109vg, 1.0 x 1010, 1.0 x 1011vg, 3.0 x 1011vg, 6 x 1011vg, 8.0 x 1011vg, 1.0 x 1012vg, 1.0 x 1013vg, 1.0 x 1014vg, 1.0 x 1015vg, or up to 1.5 x 1015vg.Compositions of viral particles can be cryopreserved or otherwise stored in suitable containers.

[0258] Provided compositions and methods herein can be sufficient to enhance delivery and / or expression of subject biologic by at least about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or up to 100% more than an otherwise comparable unmodified nucleic acid. In some cases, the otherwise comparable unmodified nucleic acid is one that encodes VEGF-Trap. In some cases, modifications can be sufficient to enhance delivery and / or expression of subject biologies by at least about 1-fold, about 6-fold, about 11 -fold, about 16-fold, about 21 -fold, about 26-fold, about 31-fold, about 36-fold, about 41-fold, about 46-fold, about 51-fold, about 56-fold, about 61-fold, about 66-fold, about 71-fold, about 76-fold, about 81-fold, about 86-fold, about 91-fold, about 96-fold, about 101-fold, about 106-fold, about 111-fold, about 116-fold, about 121-fold, about 126-fold, about 131-fold, about 136-fold, about 141 -fold, about 146-fold, about 151-fold, aboutWSGR Docket No. 59561-719.601156-fold, about 161-fold, about 166-fold, about 171-fold, about 176-fold, about 181-fold, about 186-fold, about 191-fold, about 196-fold, about 201 -fold, about 206-fold, about 211-fold, about 216-fold, about 221-fold, about 226-fold, about 231-fold, about 236-fold, about 241-fold, about 246-fold, about 251-fold, about 256-fold, about 261-fold, about 266-fold, about 271-fold, about 276-fold, about 281-fold, about 286-fold, about 291-fold, about 296-fold, about 301-fold, about 306-fold, about 311-fold, about 316-fold, about 321-fold, about 326-fold, about 331-fold, about 336-fold, about 341-fold, about 346-fold, or about 350-fold more than an otherwise comparable unmodified nucleic acid. In an embodiment, increased expression comprises at least a 5-fold, at least a 10-fold, at least a 20-fold, at least a 50-fold, at least a 100-fold, at least a 200-fold, or at least a 500-fold increase as determined by in in vitro assay. Suitable in vitro assays include ELISA, western blot, Luminex, microscopy, imaging, and / or flow cytometry.

[0259] A subject AAV virion can exhibit at least 1-fold, at least 6-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold, increased infectivity of a retinal cell, compared to the infectivity of the retinal cell (photoreceptor, ganglion cell, RPE cell, amacrine cell, horizontal cell, muller cell, and the like) by an AAV virion comprising an otherwise comparable WT AAV capsid protein.Method of treatment

[0260] Provided herein are methods of treating a disease or condition described here. In some aspects, the method confers protection against the disease or condition. A method of treatment can comprise introducing to a subject in need an engineered polynucleotide, an engineered polypeptide, an AAV vector comprising the engineered polynucleotide, an AAV comprising the engineered polynucleotide, a cell transduced with an AAV vector, a viral particle comprising the engineered polynucleotide, a pharmaceutical composition, or a combination thereof. Also provided is a method of treating disease or condition that comprises administering a pharmaceutical composition to a subject in need thereof. A pharmaceutical composition can comprise a sequence that encodes a biologic that comprises the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, a viral particle comprising the engineered polynucleotide, or a combination thereof. In some embodiments, administration is by any suitable mode of administration, including systemic administration (e.g., intravenous, intravitreal, subretinal, or etc.). In some embodiments, the subject is human.

[0261] In some embodiments, the method comprises treating a disease or condition in a subject in need thereof by administering to the subject a therapeutically effective amount of anWSGR Docket No. 59561-719.601engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or pharmaceutical composition described herein. In some embodiments, the method treats a disease or condition, where once of the administering of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or pharmaceutical composition described herein is curative of the disease or condition. In some embodiments, the method treats a disease or condition, where the administering of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or pharmaceutical composition described herein does not comprise daily administration. In some embodiments, the disease or condition comprises an ocular disease. Non-limiting example of the ocular disease can include ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, traumatic glaucoma, Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof. In some embodiments, the disease or condition is neovascular glaucoma (NG). In some embodiments, the disease or condition is glaucoma. In some embodiments, the disease or condition is traumatic glaucoma.

[0262] In another aspect, provided herein is a pharmaceutical composition comprising an engineered polynucleotide disclosed herein, an engineered polypeptide disclosed herein, a vector disclosed herein, a viral particle disclosed herein, a cell disclosed herein, or a composition disclosed herein. In some embodiments, the pharmaceutical composition is formulated for administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or a combination thereof to a subject in need thereof. In some embodiments, the pharmaceutical composition is formulated for administering intrathecally. In some embodiments, the pharmaceutical composition is formulated for administering retinally. In some embodiments, the pharmaceutical composition is formulated for administering intraocularly. In some embodiments,WSGR Docket No. 59561-719.601the pharmaceutical composition is formulated for administering intravitreally, subretinally, or suprachoroidally.

[0263] In another aspect, provided herein is a method comprising contacting a cell obtained from a subject with an engineered polynucleotide disclosed herein, an engineered polypeptide disclosed herein, a vector disclosed herein, a viral particle disclosed herein, a cell disclosed herein, a composition disclosed herein, or a pharmaceutical composition disclosed herein.

[0264] In another aspect, provided herein is a method of treating a disease or condition in a subject, comprising administering to the subject an engineered polynucleotide disclosed herein, an engineered polypeptide disclosed herein, a vector disclosed herein, a viral particle disclosed herein, a cell disclosed herein, a composition disclosed herein, or a pharmaceutical composition disclosed herein. In some embodiments, the administering is curative of the disease or condition. In some embodiments, the administering does not comprise daily administration. In some embodiments, the administering comprises a weekly administration, a bi-weekly administration, a monthly administration, a bi-month administration, a semiannual administration, an annual administration, or a combination thereof. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Conerod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof. In some embodiments, the ocular disease comprises GA or dAMD.

[0265] In another aspect, provided herein is a method of treating a disease or condition in a subject, the method comprising administering to the subject an engineered polynucleotide comprising one or more expression cassettes, the one or more expression cassettes encoding a first angiogenesis inhibitor and a second angiogenesis inhibitor. In some embodiments, the first angiogenesis inhibitor and the second angiogenesis inhibitor are each encoded by an expression cassette of the one or more expression cassettes. In some embodiments, the first angiogenesis inhibitor and the second angiogenesis inhibitor are operatively coupled. In some embodiments,WSGR Docket No. 59561-719.601the first angiogenesis inhibitor and the second angiogenesis inhibitor are covalently connected by a linker. In some embodiments, the first angiogenesis inhibitor comprises a complement inhibitor such as a complement 3 inhibitor. In some embodiments, the complement 3 inhibitor comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1-15. 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: 20-33. In some embodiments, the first angiogenesis inhibitor comprises an inhibitor of a membrane attack complex (MAC) such as CD59. In some embodiments, the CD59 comprises an amino acid sequence that is at least 80% identical to any one ofSEQ ID NOs: 41-45 In some embodiments, the second angiogenesis inhibitor comprises a natriuretic peptide. In some embodiments, the natriuretic peptide comprises 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 comprises a collagen or fragment thereof. In some embodiments, the second angiogenesis inhibitor comprises an endostatin or fragment thereof. In some embodiments, the second angiogenesis inhibitor comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 51 In some embodiments, the second angiogenesis inhibitor comprises a VEGF inhibitor. In some embodiments, the VEGF inhibitor comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 81-92.

[0266] 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, where the engineered AAV capsid comprises an amino acid sequence of any one 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.

[0267] In some embodiments, the method comprises once of the administering being curative of the disease or condition. In some embodiments, the administering does not comprise daily administration. In some embodiments, the administering comprises a weekly administration, a biweekly administration, a monthly administration, a bi-month administration, a semiannual administration, an annual administration, or a combination thereof. In some embodiments, the disease or condition comprises an ocular disease such as ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dryWSGR Docket No. 59561-719.601age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof. In some embodiments, the ocular disease comprises GA or dAMD. In some embodiments, the first angiogenesis inhibitor and the second angiogenesis inhibitor, upon administered to a subject, inhibits neovascularization in the subject.

[0268] In some embodiments, the first angiogenesis inhibitor or the second angiogenesis inhibitor, upon administered to the subject, exhibits decreased inhibition of neovascularization in the subject compared to inhibition of neovascularization caused by a VEGF inhibitor. In such case, the decreased inhibition of neovascularization induced by the first angiogenesis inhibitor or the second angiogenesis can be more therapeutically effective in treating the disease or condition. For example, the decreased inhibition of neovascularization allows presence of blood vessels for transporting and delivering the first angiogenesis inhibitor or the second angiogenesis to a site associated with the disease or condition.

[0269] In some embodiments, administering a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or pharmaceutical composition described herein to a subject protects the subjection from the disease or condition. For example, administering a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or pharmaceutical composition can protect the subject from developing disease or condition stemmed from injury. In some embodiments, administering a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or pharmaceutical composition protects or promotes survival of cells in a subject. In some embodiments, administering a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide, or pharmaceutical composition protects or promotes survival of ocular cells in a subject. In some embodiments, administering a therapeutically effective amount of an engineered polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide or, pharmaceutical composition protects or promotes survival of retinal ganglion cells in a subject. In some embodiments, administering a therapeutically effective amount of an engineeredWSGR Docket No. 59561-719.601polynucleotide, an engineered polypeptide, a cell transduced with an engineered polynucleotide or, pharmaceutical composition decreases intraocular pressure in a subject.

[0270] In some embodiments, the engineered polynucleotide, the engineered polypeptide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, or the pharmaceutical composition is administered at least once during a period of time (e.g., every 2 days, twice a week, once a week, every week, three times per month, two times per month, one time per month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, once a year). In some embodiments, the composition is administered two or more times (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) during a period of time. In some embodiments, the administration described herein comprises a single administration. In some embodiments, the administration described herein does not include daily administration.

[0271] In some embodiments, the method comprises administering the engineered polynucleotide, the engineered polypeptide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, or the pharmaceutical composition in a therapeutically effective amount by various forms and routes including, for example, oral, or topical administration. In some embodiments, a composition may be administered by intravitreal, subretinal, suprachoroidal, parenteral, intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, subarachnoid, intraocular, intrasternal, ophthalmic, endothelial, local, intranasal, intrapulmonary, rectal, intraarterial, intrathecal, inhalation, intralesional, intradermal, epidural, intracapsular, subcapsular, intracardiac, transtracheal, subcuticular, subarachnoid, or intraspinal administration, e.g., injection or infusion. In some embodiments, a composition may be administered by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa administration). In some embodiments, the composition is delivered via multiple administration routes.

[0272] In some embodiments, the method comprises administering the engineered polynucleotide, the engineered polypeptide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof by intravenous infusion. In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAVWSGR Docket No. 59561-719.601comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof is administered by slow continuous infusion over a long period, such as more than 24 hours. In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof is administered as an intravenous injection or a short infusion. In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof is administered via vitreous route. In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof may be administered in a local manner, for example, via injection of the agent directly into an organ, optionally in a depot or sustained release formulation or implant.

[0273] In some embodiments, the engineered polynucleotide, the engineered polypeptide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof may be administered in conjunction with other therapies, for example, an antiviral therapy, a chemotherapy, an antibiotic, a cell therapy, a cytokine therapy, or an anti-inflammatory agent. In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof may be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the composition containing a therapeutic agent may vary. In some cases, the composition may be used as a prophylactic and may be administered continuously to subjects (e.g., the subject for immunization or the subject for treatment) with a susceptibility to a coronavirus or a propensity to a condition or disease associated with a coronavirus. Prophylactic administration may lessen a likelihood of theWSGR Docket No. 59561-719.601occurrence of the infection, disease or condition, or may reduce the severity of the infection, disease or condition.

[0274] The engineered polynucleotide, the engineered polypeptide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof may be administered to a subject before the onset of the symptoms. In some embodiments, the engineered polynucleotide, the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof may be administered to a subject (e.g., the subject for immunization or the subject for treatment) after (e.g., as soon as possible after) a test result, for example, a test result that provides a diagnosis, a test that shows the presence of a coronavirus in a subject (e.g., the subject for immunization or the subject for treatment), or a test showing progress of a condition, e.g., a decreased blood oxygen levels. A therapeutic agent may be administered after (e.g., as soon as is practicable after) the onset of a disease or condition is detected or suspected. A therapeutic agent may be administered after (e.g., as soon as is practicable after) a potential exposure to a coronavirus, for example, after a subject (e.g., the subject for immunization or the subject for treatment) has contact with an infected subject or learns they had contact with an infected subject that may be contagious.

[0275] Actual dosage levels of an agent of the disclosure (e.g., the engineered polynucleotide or a pharmaceutical composition) may be varied so as to obtain an amount of the agent to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject (e.g., the subject for immunization or the subject for treatment). The selected dosage level may depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0276] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a 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 reducedWSGR Docket No. 59561-719.601or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects (e.g., the subjects for immunization or the subjects for treatment); each unit contains a predetermined quantity of active agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure may be determined by and directly dependent on (a) the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active agent for the treatment of sensitivity in individuals. A dose may be determined by reference to a plasma concentration or a local concentration of the circular polyribonucleotide or antibody or antigen-binding fragment thereof. A dose may be determined by reference to a plasma concentration or a local concentration of the linear polyribonucleotide or antibody or antigenbinding fragment thereof.

[0277] The engineered polynucleotide, the engineered polypeptide the AAV vector comprising the engineered polynucleotide, the AAV comprising the engineered polynucleotide, the cell transduced with the AAV vector, the viral particle comprising the engineered polynucleotide, the pharmaceutical composition, or a combination thereof described herein may be in a unit dosage form suitab...

Claims

WSGR Docket No. 59561-719.601CLAIMS WHAT IS CLAIMED IS:

1. An engineered polynucleotide comprising one or more expression cassettes, the one or more expression cassettes encodes a CD59, a complement 3 inhibitor or a C3 degraded fragment, and a C-type natriuretic peptide (CNP).

2. The engineered polynucleotide of claim 1, wherein the complement 3 inhibitor or the C3 degraded fragment is covalently connected to an antibody or fragment thereof.

3. The engineered polynucleotide of claim 2, wherein the antibody or fragment thereof comprises a fragment crystallizable (Fc) region.

4. The engineered polynucleotide of claim 2 or 3, wherein the CNP is covalently connected to the antibody or fragment thereof.

5. The engineered polynucleotide of claim 4, wherein the CD59 is expressed from a first expression cassette of the one or more expression cassettes, and wherein the complement 3 inhibitor or the C3 degraded fragment and the CNP are expressed from a second expression cassette of the one or more expression cassettes.

6. The engineered polynucleotide of any one of claims 1-5, wherein the CD59 is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 234-236 or 331.

7. The engineered polynucleotide of any one of claims 1-5, wherein the CD59 is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to any one of SEQ ID NOs: 234-236 or 331.

8. The engineered polynucleotide of any one of claims 1-5, wherein the CD59 is encoded from a nucleic acid sequence that is any one of SEQ ID NOs: 234-236 or 331.

9. The engineered polynucleotide of any one of claims 1-8, wherein the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231 or SEQ ID NO: 23210. The engineered polynucleotide of any one of claims 1-8, wherein the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231 or SEQ ID NO: 232WSGR Docket No. 59561-719.60111. The engineered polynucleotide of any one of claims 1-8, wherein the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is SEQ ID NO: 231 or SEQ ID NO: 23212. The engineered polynucleotide of any one of claims 1-11, wherein the CNP is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231.

13. The engineered polynucleotide of any one of claims 1-11, wherein the CNP is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231.

14. The engineered polynucleotide of any one of claims 1-11, wherein the CNP is encoded from a nucleic acid sequence that is SEQ ID NO: 231.

15. The engineered polynucleotide of any one of claims 1-14, wherein the CD59 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 244-246.

16. The engineered polynucleotide of any one of claims 1-14, wherein the CD59 comprises at least 50 contiguous polypeptides that are identical to any one of SEQ ID NOs: 244-246.

17. The engineered polynucleotide of any one of claims 1-14, wherein the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 244-246.

18. The engineered polynucleotide of any one of claims 1-17, wherein the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241 or SEQ ID NO: 24219. The engineered polynucleotide of any one of claims 1-17, wherein the complement 3 inhibitor or the C3 degraded fragment comprises at least 5 contiguous polypeptides that are identical to SEQ ID NO: 241 or SEQ ID NO: 242.

20. The engineered polynucleotide of any one of claims 1-17, wherein the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is SEQ ID NO: 241 or SEQ ID NO: 24221. The engineered polynucleotide of any one of claims 1-20, wherein the CNP comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241.WSGR Docket No. 59561-719.60122. The engineered polynucleotide of any one of claims 1-20, wherein the CNP comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 241.

23. The engineered polynucleotide of any one of claims 1-20, wherein the CNP comprises an amino acid sequence that is SEQ ID NO: 241.

24. The engineered polynucleotide of any one of claims 1-23, wherein the engineered polynucleotide comprises a viral vector.

25. The engineered polynucleotide of claim 24, wherein the viral vector comprises an AAV vector.

26. The engineered polynucleotide of claim 25, wherein the AAV vector is an AAV2 vector.

27. The engineered polynucleotide of claim 26, wherein the AAV vector encodes an engineered AAV capsid.

28. The engineered polynucleotide of claim 27, wherein the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A.

29. The engineered polynucleotide of claim 28, wherein the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183).

30. The engineered polynucleotide of claim 29, wherein the polypeptide sequence is LALGQTTKPA (SEQ ID NO: 183).

31. An engineered polypeptide comprising a CD59, a complement 3 inhibitor or a C3 degraded fragment, and a C-type natriuretic peptide (CNP).

32. A vector comprising the engineered polynucleotide of any one of claims 1-30, or the engineered polypeptide of claim 31.

33. The vector of claim 32, wherein the vector encodes an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid.

34. The vector of claim 33, wherein the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), AsparticWSGR Docket No. 59561-719.601Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A.

35. The vector of claim 34, wherein the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183).

36. The vector of claim 35, wherein the polypeptide sequence is LALGQTTKPA (SEQ ID NO: 183).

37. A viral particle comprising the engineered polynucleotide of any one of claims 1-30, the engineered polypeptide of claim 31, or the vector of any one of claims 32-36.

38. The viral particle of claim 37, wherein the viral particle comprises an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid.

39. The viral particle of claim 38, wherein the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A.

40. The viral particle of claim 39, wherein the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183).

41. The viral particle of claim 40, wherein the polypeptide sequence is LALGQTTKPA (SEQ ID NO: 183)42. A method comprising: contacting a cell obtained from a subject with the engineered polynucleotide of any one of claims 1-30, the engineered polypeptide of claim 31, the vector of any one of claims 32-36, or the viral particle of any one of claims 37-41.

43. A method of treating a disease or condition in a subject, comprising: administering to the subject the engineered polynucleotide of any one of claims 1-30, the engineered polypeptide of claim 31, the vector of any one of claims 32-36, or the viral particle of any one of claims 37-41.

44. The method of claim 43, wherein a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring neuroprotective effect in the subject.

45. The method of claim 43, wherein a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring antiangiogenesis effect in the subject.WSGR Docket No. 59561-719.60146. The method of claim 43, wherein a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring neuroprotective effect and anti-angiogenesis in the subject.

47. The method of any one of claims 43-46, wherein the disease or condition comprises an ocular disease.

48. The method of claim 47, wherein the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.

49. An engineered polynucleotide comprising one or more expression cassettes, the one or more expression cassettes encodes a CD59, a complement 3 inhibitor or a C3 degraded fragment, and an endostatin (ES).

50. The engineered polynucleotide of claim 49, wherein the complement 3 inhibitor or the C3 degraded fragment is covalently connected to an antibody or fragment thereof.

51. The engineered polynucleotide of claim 50, wherein the antibody or fragment thereof comprises a fragment crystallizable (Fc) region.

52. The engineered polynucleotide of claim 50 or 51, wherein the endostatin is covalently connected to the antibody or fragment thereof.

53. The engineered polynucleotide of claim 52, wherein the CD59 is expressed from a first expression cassette of the one or more expression cassettes, and wherein the complement 3 inhibitor or the C3 degraded fragment and the endostatin are expressed from a second expression cassette of the one or more expression cassettes.

54. The engineered polynucleotide of any one of claims 49-53, wherein the CD59 is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 234-236 or 332.WSGR Docket No. 59561-719.60155. The engineered polynucleotide of any one of claims 49-53, wherein the CD59 is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to any one of SEQ ID NOs: 234-236 or 332.

56. The engineered polynucleotide of any one of claims 49-53, wherein the CD59 is encoded from a nucleic acid sequence that is any one of SEQ ID NOs: 234-236 or 332.

57. The engineered polynucleotide of any one of claims 49-56, wherein the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231 or SEQ ID NO: 23258. The engineered polynucleotide of any one of claims 49-56, wherein the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231 or SEQ ID NO: 23259. The engineered polynucleotide of any one of claims 49-56, wherein the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is SEQ ID NO: 231 or SEQ ID NO: 23260. The engineered polynucleotide of any one of claims 49-59, wherein the endostatin is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 233.

61. The engineered polynucleotide of any one of claims 49-59, wherein the endostatin is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 233.

62. The engineered polynucleotide of any one of claims 49-59, wherein the endostatin is encoded from a nucleic acid sequence that is SEQ ID NO: 233.

63. The engineered polynucleotide of any one of claims 49-62, wherein the CD59 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 244-246.

64. The engineered polynucleotide of any one of claims 49-62, wherein the CD59 comprises at least 50 contiguous polypeptides that are identical to any one of SEQ ID NOs: 244-246.

65. The engineered polynucleotide of any one of claims 49-62, wherein the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 244-246.

66. The engineered polynucleotide of any one of claims 49-65, wherein the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is at least 70%, atWSGR Docket No. 59561-719.601least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241 or SEQ ID NO: 24267. The engineered polynucleotide of any one of claims 49-65, wherein the complement 3 inhibitor or the C3 degraded fragment comprises at least 5 contiguous polypeptides that are identical to SEQ ID NO: 241 or SEQ ID NO: 242.

68. The engineered polynucleotide of any one of claims 49-65, wherein the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is SEQ ID NO: 241 or SEQ ID NO: 24269. The engineered polynucleotide of any one of claims 49-68, wherein the endostatin comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 243.

70. The engineered polynucleotide of any one of claims 49-68, wherein the endostatin comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 243.

71. The engineered polynucleotide of any one of claims 49-69, wherein the endostatin comprises an amino acid sequence that is SEQ ID NO: 243.

72. The engineered polynucleotide of any one of claims 49-71, wherein the engineered polynucleotide comprises a viral vector.

73. The engineered polynucleotide of claim 72, wherein the viral vector comprises an AAV vector.

74. The engineered polynucleotide of claim 73, wherein the AAV vector is an AAV2 vector.

75. The engineered polynucleotide of claim 74, wherein the AAV vector encodes an engineered AAV capsid.

76. The engineered polynucleotide of claim 75, wherein the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A.

77. The engineered polynucleotide of claim 76, wherein the polypeptide sequence comprises an amino acid sequence of LALGQTTKP A.

78. The engineered polynucleotide of claim 77, wherein the polypeptide sequence isLALGQTTKPA.WSGR Docket No. 59561-719.60179. An engineered polypeptide comprising a CD59, a complement 3 inhibitor or a C3 degraded fragment, and an endostatin (ES).

80. A vector comprising the engineered polynucleotide of any one of claims 49-78, or the engineered polypeptide of claim 79.

81. The vector of claim 80, wherein the vector encodes an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid.

82. The vector of claim 81, wherein the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A.

83. The vector of claim 82, wherein the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA.

84. The vector of claim 83, wherein the polypeptide sequence is LALGQTTKPA.

85. A viral particle comprising the engineered polynucleotide of any one of claims 49-78, the engineered polypeptide of claim 79, or the vector of any one of claims 80-84.

86. The viral particle of claim 85, wherein the viral particle comprises an AAV capsid, and wherein the AAV capsid comprises an engineered AAV capsid.

87. The viral particle of claim 86, wherein the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A.

88. The viral particle of claim 87, wherein the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA.

89. The viral particle of claim 88, wherein the polypeptide sequence is LALGQTTKPA.

90. A method comprising: contacting a cell obtained from a subject with the engineered polynucleotide of any one of claims 49-78, the engineered polypeptide of claim 79, the vector of any one of claims 80-84, or the viral particle of any one of claims 85-89.

91. A method of treating a disease or condition in a subject, comprising: administering to the subject the engineered polynucleotide of any one of claims 49-78, the engineered polypeptide of claim 79, the vector of any one of claims 80-84, or the viral particle of any one of claims 85-89.WSGR Docket No. 59561-719.60192. The method of claim 91, wherein a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the endostatin treats the disease or condition by conferring a neuroprotective effect in the subject.

93. The method of claim 91, wherein a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP treats the disease or condition by conferring antiangiogenesis effect in the subject.

94. The method of claim 91, wherein a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the endostatin treats the disease or condition by conferring an antiangiogenesis effect in the subject.

95. The method of claim 91, wherein a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the endostatin treats the disease or condition by conferring a neuroprotective effect and anti-angiogenesis in the subject.

96. The method of any one of claims 91-95, wherein the disease or condition comprises an ocular disease.

97. The method of claim 96, wherein the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.

98. An engineered polynucleotide comprising one or more expression cassettes, the one or more expression cassettes encoding a CD59 and an antibody or fragment thereof flanked by: a complement 3 inhibitor or a C3 degraded fragment; and a C-type natriuretic peptide (CNP) or an endostatin (ES).

99. The engineered polynucleotide of claim 98, wherein the complement 3 inhibitor or the C3 degraded fragment and the CNP or ES are covalently connected to the antibody or fragment thereof.

100. The engineered polynucleotide of claim 99, wherein the antibody or fragment thereof comprises a fragment crystallizable (Fc) region.WSGR Docket No. 59561-719.601101. The engineered polynucleotide of any one of claims 98-100, wherein the CD59 is expressed from a first expression cassette of the one or more expression cassettes, and wherein the complement 3 inhibitor or the C3 degraded fragment and the CNP or the ES are expressed from a second expression cassette of the one or more expression cassettes.

102. The engineered polynucleotide of any one of claims 98-101, wherein the CD59 is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 234-236, 331, or 332.

103. The engineered polynucleotide of any one of claims 98-102, wherein the CD59 is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to any one of SEQ ID NOs: 234-236, 331, or 332.

104. The engineered polynucleotide of any one of claims 98-103, wherein the CD59 is encoded from a nucleic acid sequence that is any one of SEQ ID NOs: 234-236, 331, or 332.

105. The engineered polynucleotide of any one of claims 98-104, wherein the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231 or SEQ ID NO: 232106. The engineered polynucleotide of any one of claims 98-104, wherein the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231 or SEQ ID NO: 232107. The engineered polynucleotide of any one of claims 98-104, wherein the complement 3 inhibitor or the C3 degraded fragment is encoded from a nucleic acid sequence that is SEQ ID NO: 231 or SEQ ID NO: 232108. The engineered polynucleotide of any one of claims 98-107, wherein the CNP is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 231.

109. The engineered polynucleotide of any one of claims 98-107, wherein the CNP is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 231.

110. The engineered polynucleotide of any one of claims 98-107, wherein the CNP is encoded from a nucleic acid sequence that is SEQ ID NO: 231.WSGR Docket No. 59561-719.601111. The engineered polynucleotide of any one of claims 98-107, wherein the endostatin is encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 233.

112. The engineered polynucleotide of any one of claims 98-107, wherein the endostatin is encoded from a nucleic acid sequence comprising at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous nucleotides that are identical to SEQ ID NO: 233.

113. The engineered polynucleotide of any one of claims 98-107, wherein the endostatin is encoded from a nucleic acid sequence that is SEQ ID NO: 233.

114. The engineered polynucleotide of any one of claims 98-113, wherein the CD59 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 244-246.

115. The engineered polynucleotide of any one of claims 98-113, wherein the CD59 comprises at least 50 contiguous polypeptides that are identical to any one of SEQ ID NOs: 244-246.

116. The engineered polynucleotide of any one of claims 98-113, wherein the CD59 comprises an amino acid sequence that is any one of SEQ ID NOs: 244-246.

117. The engineered polynucleotide of any one of claims 98-116, wherein the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241 or SEQ ID NO: 242118. The engineered polynucleotide of any one of claims 98-116, wherein the complement 3 inhibitor or the C3 degraded fragment comprises at least 5 contiguous polypeptides that are identical to SEQ ID NO: 241 or SEQ ID NO: 242.

119. The engineered polynucleotide of any one of claims 98-116, wherein the complement 3 inhibitor or the C3 degraded fragment comprises an amino acid sequence that is SEQ ID NO: 241 or SEQ ID NO: 242120. The engineered polynucleotide of any one of claims 98-119, wherein the CNP comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 241.

121. The engineered polynucleotide of any one of claims 98-119, wherein the CNP comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 241.

122. The engineered polynucleotide of any one of claims 98-119, wherein the CNP comprises an amino acid sequence that is SEQ ID NO: 241.WSGR Docket No. 59561-719.601123. The engineered polynucleotide of any one of claims 98-119, wherein the endostatin comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 243.

124. The engineered polynucleotide of any one of claims 98-119, wherein the endostatin comprises an amino acid sequence that is at least 10 contiguous polypeptides that are identical to SEQ ID NO: 243.

125. The engineered polynucleotide of any one of claims 98-119, wherein the endostatin comprises an amino acid sequence that is SEQ ID NO: 243.

126. The engineered polynucleotide of any one of claims 98-125, wherein the engineered polynucleotide comprises a viral vector.

127. The engineered polynucleotide of claim 126, wherein the viral vector comprises an AAV vector.

128. The engineered polynucleotide of claim 127, wherein the AAV vector is an AAV2 vector.

129. The engineered polynucleotide of claim 128, wherein the AAV vector encodes an engineered AAV capsid.

130. The engineered polynucleotide of claim 129, wherein the engineered AAV capsid comprises a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A.

131. The engineered polynucleotide of claim 130, wherein the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183).

132. The engineered polynucleotide of claim 131, wherein the polypeptide sequence is LALGQTTKPA (SEQ ID NO: 183).

133. A vector comprising the engineered polynucleotide of any one of claims 98-132.

134. A viral particle comprising the engineered polynucleotide of any one of claims 98-132 or the vector of claim 133.

135. A composition comprising: a CD59; a complement 3 inhibitor or a C3 degraded fragment; and a C-type natriuretic peptide (CNP) or an endostatin (ES).

136. The composition of claim 135, comprising the CD59, the complement 3 inhibitor or the C3 degraded fragment, and the CNP.WSGR Docket No. 59561-719.601137. The composition of claim 136, wherein the complement 3 inhibitor or the C3 degraded fragment and the CNP are covalently connected.

138. The composition of claim 137, wherein the complement 3 inhibitor or the C3 degraded fragment and the CNP are covalently connected by an antibody of fragment thereof.

139. The composition of claim 135, comprising the CD59, the complement 3 inhibitor or the C3 degraded fragment, and the ES.

140. The composition of claim 139, wherein the complement 3 inhibitor or the C3 degraded fragment and the ES are covalently connected.

141. The composition of claim 140, wherein the complement 3 inhibitor or the C3 degraded fragment and the ES are covalently connected by an antibody of fragment thereof.

142. The composition of claim 138 or 141, wherein the antibody or fragment thereof comprises a Fc region.

143. A method comprising: contacting a cell obtained from a subject with the engineered polynucleotide of any one of claims 98-132, the vector of claim 133, the viral particle of claim 134, or the composition of any one of claims 135-142.

144. A method of treating a disease or condition in a subject, comprising: administering to the subject the engineered polynucleotide of any one of claims 98-132, the vector of claim 133, the viral particle of claim 134, or the composition of any one of claims 135-142.

145. A method of contacting a retinal cell of a subject with an engineered polynucleotide encoding a CD59 and an antibody or fragment thereof flanked by: a complement 3 inhibitor or a C3 degraded fragment; and a C-type natriuretic peptide (CNP) or an endostatin (ES).

146. A method of treating a disease or condition in a subject, comprising administering to the subject an engineered polynucleotide encoding a CD59 and an antibody or fragment thereof flanked by: a complement 3 inhibitor or a C3 degraded fragment; and a C-type natriuretic peptide (CNP) or an endostatin (ES), wherein administration of the engineered polynucleotide increases therapeutic efficacy compared to administering a comparable complement inhibitor alone or a comparable anti-angiogenic agent alone to the subject.

147. The method of claim 145 or 146, wherein the engineered polynucleotide encodes the CD59, the complement 3 inhibitor or the C3 degraded fragment and the CNP.

148. The method of claim 145 or 146 wherein the engineered polynucleotide encodes the CD59, the complement 3 inhibitor or the C3 degraded fragment and the ES.

149. The method of any one of claims 144-148, wherein a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP or the ES increasesWSGR Docket No. 59561-719.601neuroprotective effect in the subject compared to administering the comparable complement inhibitor alone or the comparable anti-angiogenic agent alone to the subject.

150. The method of any one of claims 144-149, wherein a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP or the ES increases antiangiogenesis effect in the subject compared to administering the comparable complement inhibitor alone or the comparable anti-angiogenic agent alone to the subject.

151. The method of any one of claims 144-150, wherein a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP or the ES increases retinal function in the subject compared to administering the comparable complement inhibitor alone or the comparable anti -angiogenic agent alone to the subject.

152. The method of any one of claims 144-151, wherein a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP or the ES decreases vascular leakage in the subject compared to administering the comparable complement inhibitor alone or the comparable anti -angiogenic agent alone to the subject.

153. The method of any one of claims 144-152, wherein a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP or the ES decreases inflammation in the subject compared to administering the comparable complement inhibitor alone or the comparable anti -angiogenic agent alone to the subject.

154. The method of any one of claims 144-153, wherein a combination of the CD59, the complement 3 inhibitor or a C3 degraded fragment, and the CNP or the ES decreases neovascularization in the subject compared to administering the comparable complement inhibitor alone or the comparable anti-angiogenic agent alone to the subject.

155. The method of any one of claims 146-154, wherein the comparable completement inhibitor comprises pegcetacoplan.

156. The method of any one of claims 146-154, wherein the comparable anti-angiogenic agent comprises Elyea.

157. The method of any one of claims 145-156, wherein the engineered polynucleotide comprises a viral vector.

158. The method of claim 157, wherein the viral vector comprises an AAV vector.

159. The method of claim 158, wherein the AAV vector is an AAV2 vector.

160. The method of claim 159, wherein the AAV vector encodes an engineered AAV capsid.

161. The method of any one of claims 145-160, wherein the engineered polynucleotide is encapsulated in a viral capsid.WSGR Docket No. 59561-719.601162. The method of any one of claims 145-161, wherein the disease or condition comprises an ocular disease.

163. The method of claim 162, wherein the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.

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