Compositions and methods for treatment of ocular disease associated with angiogenesis

Abstract

To provide compositions and methods for treatment of ocular disease associated with angiogenesis.SOLUTION: The present disclosure provides compositions and methods for the treatment of ocular diseases associated with angiogenesis, particularly wet age-related macular degeneration. Ocular vascular diseases such as age-related macular degeneration and diabetic retinopathy are due to abnormal choroidal or retinal neovascularization respectively. Since the retina consists of well-defined layers of neuronal, glial, and vascular elements, relatively small disturbances such as those seen in vascular proliferation or edema can lead to significant loss of a visual function. Inherited retinal degenerations, such as Retinitis Pigmentosa, are also associated with vascular abnormalities, such as arteriolar narrowing and vascular atrophy.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 180,247, filed April 27, 2021, the entire disclosure of which is incorporated herein by reference.

[0002] Submitting sequence listings via EFS-WEB A computer-readable text file entitled "090400-5018-WO-Sequence-Listing", created on or about April 22, 2022, and having a file size of approximately 83 KB, contains the sequence listing for the present application and is incorporated herein by reference in its entirety. [Background technology]

[0003] Background of the Invention Vascular endothelial growth factor (VEGF) proteins and their receptors (VEGFRs) play important roles in vasculogenesis, the development of embryonic vasculature from early differentiating endothelial cells; angiogenesis, the process of forming new blood vessels from pre-existing vessels; and lymphangiogenesis, the process of forming new lymphatic vessels.

[0004] Ocular vascular diseases, such as age-related macular degeneration and diabetic retinopathy, are caused by abnormal choroidal or retinal neovascularization, respectively.Because the retina is composed of well-defined layers of neurons, glia, and vascular elements, relatively small disruptions, such as those seen in vascular proliferation or edema, can lead to significant loss of visual function.Hereditary retinal degeneration, such as retinitis pigmentosa, is also associated with vascular abnormalities, such as arteriolar narrowing and vascular atrophy.

[0005] Strategies to block the function of VEGF have been used. Current standard-of-care treatments include intravitreal (IVT) injections of protein therapies, such as aflibercept, ranibizumab, and brolucizumab, that bind to vascular endothelial growth factor A (VEGF-A) to prevent its binding to its receptor. Anti-VEGF treatment regimens that have been shown to be safe and effective require repeated monthly to bimonthly IVT administrations to maintain vision, and many patients fail to maintain their initial visual gains due to undertreatment. Requiring repeated injections can be a significant burden for patients and caregivers, and some patients require regular anti-VEGF injections despite decades of treatment. Recent studies have shown that in real-world use, many patients receive fewer than the recommended injections and do not receive or maintain the same benefits as those shown in clinical trial settings, with visual gains during the first two years but not maintained at five years.

[0006] Thus, there remains a need for new or improved compounds and therapies for the treatment of neovascular eye diseases, such as wet AMD. Summary of the Invention [Means for solving the problem]

[0007] Summary of the Invention Disclosed are compositions and methods for the treatment of ocular diseases associated with ocular neovascularization, including, but not limited to, wet (neovascular, exudative) age-related macular degeneration; macular edema following retinal vein occlusion; retinal neovascularization resulting from retinal vein occlusion; diabetic macular edema, diabetic retinopathy (including all stages of non-proliferative and proliferative diabetic retinopathy); myopic macular degeneration; branch retinal vein occlusion, hemi-retinal vein occlusion, and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectasia; neovascular glaucoma; vitreous hemorrhage; retinal and choroidal neovascularization secondary to retinal diseases, including, but not limited to, uveitis, trauma, retinal degenerative disorders, inherited retinal and / or choroidal diseases, ocular tumors, and corneal and iris neovascularization. In some embodiments, the neovascular ocular disease is selected from wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; and myopic choroidal neovascularization.

[0008] In some embodiments, a nucleic acid is provided comprising: (i) a nucleotide sequence encoding a first antiangiogenic polypeptide (e.g., aflibercept); and (ii) a nucleotide sequence encoding one or more interfering RNA molecules that reduce the expression of one or more pro-angiogenic target genes. In some aspects, the RNA molecule is a short hairpin RNA (shRNA). In other aspects, the RNA molecule is a primary miRNA molecule. In some aspects, the nucleic acid comprises an expression cassette comprising: (i) a nucleotide sequence encoding a first antiangiogenic polypeptide operably linked to an expression control sequence; and (ii) a nucleotide sequence encoding an interfering RNA molecule that reduces the expression of one or more pro-angiogenic target genes operably linked to an expression control sequence. In some embodiments, the nucleotide sequence encoding the antiangiogenic polypeptide and the nucleotide sequence encoding the interfering RNA molecule are operably linked to separate expression control sequences. In a preferred embodiment, the expression of the antiangiogenic polypeptide and the interfering RNA molecule are driven by a common (i.e., the same) expression control sequence. In some aspects, the expression control sequence(s) comprise a constitutive promoter, e.g., a CAG or CBA promoter. In other aspects, the expression control sequence(s) comprise a cell-specific promoter.

[0009] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an interfering RNA molecule that targets Angiopoietin-2 (also known as Ang2 or Ang-2).Representative human Ang2 sequences can be found, for example, in NCBI Accession No. 015123 and SEQ ID NOs: 517 and 518 of U.S. Patent No. 8,987,420, the contents of which are incorporated herein by reference.In a preferred embodiment, the interfering RNA molecule targets Ang-2 and comprises a sense strand and an antisense strand that comprise, consist essentially of, or consist of a sequence selected from those listed in Table 1 below: [Table 1-1] [Table 1-2] In some embodiments, the interfering RNA molecule comprises a sense strand and an antisense strand, one or both of which comprise, consist essentially of, or consist of a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from those listed in Table 1. In some particularly preferred embodiments, the nucleic acid comprises a nucleotide sequence encoding a first antiangiogenic polypeptide that is aflibercept and a nucleotide sequence encoding an interfering RNA molecule that targets human angiopoietin-2.

[0010] In some preferred embodiments, the nucleic acid comprises a nucleotide sequence encoding an interfering RNA molecule that targets VEGF-C. Representative human VEGF-C sequences can be found, for example, in GenBank accession numbers NM_005429 and X94216. In preferred embodiments, the interfering RNA molecule targets VEGF-C and comprises a sense strand and an antisense strand that comprise, consist essentially of, or consist of a sequence selected from those listed in Table 2 below: [Table 2-1] [Table 2-2] In some embodiments, the interfering RNA molecule comprises a sense strand and an antisense strand, one or both of which comprise, consist essentially of, or consist of a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from those listed in Table 2. Additional interfering RNA molecules that target VEGF-C include those listed in Table 1 of U.S. Patent Application Publication No. 2011 / 0293625 A1, the contents of which are incorporated herein by reference. In some particularly preferred embodiments, the nucleic acid comprises a nucleotide sequence encoding a first antiangiogenic polypeptide that is aflibercept and a nucleotide sequence encoding an interfering RNA molecule that targets human VEGF-C.

[0011] In related embodiments, the nucleic acid comprises a nucleotide sequence encoding a first anti-angiogenic protein (e.g., aflibercept) and further comprises an interfering RNA molecule targeting human VEGF-C and an interfering RNA molecule targeting human Ang-2.

[0012] In other embodiments, the nucleic acid comprises a nucleotide sequence encoding an interfering RNA molecule that targets VEGFR-3. A representative human VEGFR-3 sequence can be found, for example, in GenBank accession number X68203. In a preferred embodiment, the interfering RNA molecule targets VEGFR-3 and comprises a sense strand and an antisense strand that comprise, consist essentially of, or consist of a sequence selected from those listed in Table 3 below: [Table 3-1] [Table 3-2] In some embodiments, the interfering RNA molecule comprises a sense strand and an antisense strand, one or both of which comprise, consist essentially of, or consist of a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from those listed in Table 3. Additional interfering RNA molecules that target VEGFR-3 include those listed in Table 2 of U.S. Patent No. 7,517,864, the contents of which are incorporated herein by reference. In some particularly preferred embodiments, the nucleic acid comprises a nucleotide sequence encoding a first anti-angiogenic polypeptide that is aflibercept and a nucleotide sequence encoding an interfering RNA molecule that targets human VEGFR-3.

[0013] In some embodiments, the synthetic RNA molecule is a small interfering RNA (siRNA). In some embodiments, the interfering RNA is a short hairpin RNA (shRNA). In some aspects, the shRNA has a loop (5' to 3') that comprises the sequence CTCGAG or at least 70% or at least 80% identical thereto.

[0014] In some preferred embodiments, the synthetic RNA molecule is an artificial microRNA (miRNA). In some embodiments, the artificial miRNA comprises the sense and antisense strands described herein embedded in a miRNA "scaffold" derived from miR-30, miR-22, miR-15, miR-16, miR-103 or miR-107. In some preferred embodiments, the artificial miRNA comprises the sense and antisense strands described herein embedded in miR-30:

[0015] [ka] , where (X) n comprises a sense strand from any one of Tables 1 to 3, and (Y) n comprises a sense strand from any one of Tables 1-3.

[0016] In a particularly preferred embodiment, the artificial miRNA comprises the sense and antisense strands described herein embedded in mir-E:

[0017] [ka] , where (X) n comprises a sense strand from any one of Tables 1 to 3, and (Y) n comprises a sense strand from any one of Tables 1-3.

[0018] In some embodiments, a nucleic acid is provided comprising (i) a nucleotide sequence encoding a first antiangiogenic polypeptide (e.g., aflibercept) and (ii) a nucleotide sequence encoding a second antiangiogenic polypeptide. In some aspects, the nucleic acid comprises an expression cassette comprising (i) a nucleotide sequence encoding the first antiangiogenic polypeptide operably linked to an expression control sequence, and (ii) a nucleotide sequence encoding the second antiangiogenic polypeptide operably linked to an expression control sequence. In some embodiments, the nucleotide sequence encoding the first antiangiogenic polypeptide and the nucleotide sequence encoding the second antiangiogenic polypeptide are operably linked to separate expression control sequences. In preferred embodiments, the expression of the first and second antiangiogenic polypeptides is driven by a common (i.e., the same) expression control sequence. In some aspects, the expression control sequence(s) comprise a constitutive promoter, e.g., a CAG or CBA promoter. In other aspects, the expression control sequence(s) comprise a cell-specific promoter.

[0019] In some embodiments, the first and / or second anti-angiogenic polypeptide (i.e., a polypeptide that inhibits angiogenesis) is selected from endostatin; tumstatin; angiostatin; pigment epithelium-derived factor (PEDF). In some embodiments, the first and / or second anti-angiogenic polypeptide is a "decoy" fusion protein (e.g., a soluble receptor fusion protein) that binds to and inhibits the activity of VEGF (VEGF-A (see, e.g., GenBank Accession No. Q16889), VEGF-B (see, e.g., GenBank Accession No. U48801), VEGF-C (see, e.g., GenBank Accession No. X94216), VEGF-D (see, e.g., GenBank Accession No. AJ000185), and / or placenta growth factor (PIGF; see, e.g., GenBank Accession No. X54936)). Representative examples of soluble VEGFR-1 (also known as Flt-1; see, e.g., GenBank Accession No. X51602) receptor fusion proteins, soluble VEGFR-2 (also known as Flk-1; see, e.g., GenBank Accession No. X59397) receptor fusion proteins, soluble VEGFR-3 (also known as Flt-4; see, e.g., GenBank Accession Nos. X68203 and S66407) receptor fusion proteins, and chimeric soluble receptor fusion proteins containing binding regions from at least two of VEGFR-1, VEGFR-2, and VEGFR-3. VEGF-A binds to VEGFR-1 and VEGFR-2; VEGF-B and PIGF bind to VEGFR-2; and VEGF-C and VEGF-D bind to VEGFR-3.

[0020] In some preferred embodiments, the first antiangiogenic polypeptide is a soluble fusion protein, which comprises the VEGF binding moiety derived from the extracellular domain of VEGFR-1 and VEGFR-2, and is optionally fused with human IgG1 Fc part.In particularly preferred embodiments, the first antiangiogenic polypeptide is aflibercept.Aflibercept is a recombinant fusion protein that is composed of the VEGF binding moiety derived from the extracellular domain of human VEGFR-1 and VEGFR-2, and is fused with human IgG1 Fc part.Aflibercept is indicated for the treatment of neovascular (wet) age-related macular degeneration, macular edema after retinal vein occlusion, diabetic macular edema and diabetic retinopathy.

[0021] In other preferred embodiments, the first and / or second anti-angiogenic polypeptide is a soluble fusion protein comprising one or more VEGF-binding moieties derived from the extracellular domain of VEGFR-3.

[0022] In other embodiments, the first and / or second anti-angiogenic polypeptide is an antibody or antigen-binding fragment thereof that binds to and inhibits the activity of a pro-angiogenic protein, e.g., VEGF and / or angiopoietin (angiopoietin-1 / Ang1 / Ang-1, angiopoietin-2 / Ang2 / Ang-2). In some embodiments, the first and / or second anti-angiogenic polypeptide is an antibody against Ang1 and / or Ang2. In other embodiments, the first and / or second anti-angiogenic polypeptide is an antibody against VEGF-A (e.g., bevacizumab), VEGF-B, VEGF-C, VEGF-D, or PIGF (e.g., TB-403, 16D3) that blocks binding of VEGF to its cognate receptor. In other aspects, the first and / or second anti-angiogenic polypeptide is an antibody against VEGFR-1 (e.g., icrucumab, D16F7, KM1730 / KM1732), an antibody against VEGFR-2 (e.g., ramucirumab), or an antibody against VEGFR-3 that blocks binding of the receptor to VEGF. In some aspects, the antibody is a bifunctional antibody. In some preferred embodiments, the first and / or second anti-angiogenic polypeptide is an antibody or antigen-binding fragment thereof that binds to Ang-2.

[0023] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a first anti-angiogenic polypeptide and a nucleotide sequence encoding a second anti-angiogenic polypeptide, wherein the second anti-angiogenic polypeptide is pigment epithelium-derived factor (PEDF).

[0024] In some embodiments, provided herein are vectors (e.g., adeno-associated virus (AAV) plasmid vectors) comprising a nucleic acid described herein (e.g., comprising a nucleotide sequence encoding a first and / or second antiangiogenic polypeptide and / or an interfering RNA molecule that reduces expression of one or more pro-angiogenic target genes). In preferred embodiments, the vector is a recombinant adeno-associated (rAAV) expression vector. In some embodiments, the rAAV vector comprises a native capsid (e.g., an AAV serotype 2 or AAV serotype 5 or AAV serotype 8 capsid). In other embodiments, the rAAV vector comprises a capsid that is modified compared to a native AAV capsid (e.g., comprises one or more peptide insertions and / or one or more amino acid substitutions (e.g., tyrosine to phenylalanine) and / or amino acid insertions or deletions) (e.g., comprises one or more modifications compared to an AAV capsid of serotype 2, 5, or 8).

[0025] In a preferred embodiment, the rAAV vector comprises a capsid having a variant capsid protein comprising the following amino acid sequence, or a sequence at least 80%, 90%, 95%, or 99% identical thereto: [ka] [ka]

[0026] The variant AAV capsid protein of SEQ ID NO: 48 contains the following modifications compared to the native AAV2 capsid: (i) a proline (P) to alanine (A) mutation at amino acid position 34, located inside the assembled capsid (VP1 protein only), and (ii) an insertion of 10 amino acids (leucine-alanine-isoleucine-serine-aspartic acid-glutamine-threonine-lysine-histidine-alanine / LAISDQTKHA (SEQ ID NO: 49)) at amino acid position 588, present in VP1, VP2, and VP3. In some embodiments, the capsid comprises a variant capsid protein comprising a sequence at least 90%, at least 95%, at least 98%, at least 99% identical to SEQ ID NO: 48, and comprising a P34A substitution and a LAISDQTKHA (SEQ ID NO: 49) peptide insertion between two adjacent amino acids in the GH loop of the capsid, e.g., at a position between amino acids 570 and 611 of VP1, preferably between amino acids 588 and 589 of VP1 (numbering is relative to the native AAV2 VP1 capsid).

[0027] In another embodiment, the present invention provides a host cell comprising the nucleic acid described herein.In some aspects, the host cell is a mammalian cell, including but not limited to CHO cell, HEK293 cell, HeLa cell, BHK21 cell, Vero cell or V27 cell.In other aspects, the host cell is a photoreceptor cell (e.g., rod; cone), retinal ganglion cell (RGC), glial cell (e.g., Müller glial cell, microglial cell), bipolar cell, amacrine cell, horizontal cell or retinal pigment epithelial (RPE) cell.

[0028] In some embodiments, the present disclosure provides a method of treating an ocular disease associated with ocular neovascularization in a subject (e.g., a human subject), comprising administering to the subject a nucleic acid molecule or vector described herein. [Brief explanation of the drawings]

[0029] [Figure 1]Dual transgene constructs. An approach for constructing a multi-mechanistic antiangiogenic gene therapy is shown. Figure 1A: This construct is a representative embodiment of a nucleic acid encoding aflibercept and a second antiangiogenic protein. Figure 1B: This construct is a representative embodiment of a nucleic acid encoding aflibercept and an interfering RNA targeting a pro-angiogenic gene. A ubiquitous promoter (CBA) is used in both constructs.

[0030] [Figure 2] Representative construct depictions. pP141.001 contains a CAG promoter with the miR-E-Ang2 sequence placed within the CAG intron (sufficient after the splice donor site) followed by a codon-optimized sequence encoding aflibercept. pP145.001 is identical to pP141.001, except that the mir-E-VEGF-C sequence is placed within the CAG intron. pP151.001 is identical to pP145.001, except that the miR-E-Ang2 sequence is placed within the 3'UTR of the aflibercept gene. pP151.002 is identical to pP145.001, except that the miR-E-Ang2 sequence is placed within the aflibercept coding sequence. pP153.001 contains the CAG promoter with miR-E-Ang2 and miR-E-VEGF-C sequences (followed by a codon-optimized sequence encoding aflibercept) positioned within the CAG intron. CBA Prom.: ubiquitous chicken b-actin promoter; CBA Ex1 / Int1: chicken b-actin exon 1 / hybrid chicken b-actin and rabbit beta-globin intron; RGB SA / PPT: rabbit beta-globin exon 3 fragment (splice acceptor) / polypyrimidine tract; T2A: self-cleaving peptide; AFLB: aflibercept.

[0031] [Figure 3]Figure 3A is a Western blot using anti-human IgG Fc to detect aflibercept in HEK293T medium after transfection with an AAV plasmid containing a nucleotide sequence encoding aflibercept together with a second polypeptide (PEDF, VEGFR3, anti-Ang2 scFab (LH and HL)), both sequences under the control of a CBA promoter. Figure 3B is a graph illustrating the expression of aflibercept in HEK293T medium after transfection with an AAV plasmid encoding aflibercept and a second polypeptide (PEDF, anti-Ang2 scFab) or aflibercept and an interfering RNA under the control of the indicated promoter. Results are normalized to the expression of aflibercept in HEK293T medium after transfection with an AAV plasmid encoding aflibercept alone under the control of a CBA promoter. Error bars indicate SEM. N=6 biological replicates. Analysis was performed by one-way ANOVA. ****: P<0.0001. Constructs were only statistically weighted relative to their appropriate controls. % indicates mean difference compared to CBA-AFLB.

[0032] [Figure 4] Figure 4 illustrates free active aflibercept (AFLB) in the medium 8 days after transduction of RPE cells with an rAAV comprising a capsid of SEQ ID NO: 48 and a heterologous nucleic acid encoding the indicated transgene at the indicated MOI (N=3 biological replicates per condition; statistics calculated only for matched MOIs).

[0033] [Figure 5A]Figures 5A and 5B illustrate free active aflibercept (AFLB) in the medium 7 days (Figure 5A) and 11 days (Figure 5B) after transduction of RPE cells with an rAAV containing a capsid of SEQ ID NO:48 and a heterologous nucleic acid encoding the indicated transgene at an MOI of 5K and 1K. Figure 5C compares AFLB after transduction of RPE cells with an rAAV containing a construct encoding AFLB alone to an rAAV containing an AFLB plus a construct encoding an RNAi against VEGF-C. [Figure 5B] Same as above. [Figure 5C] Same as above.

[0034] [Figure 6A] Figure 6A-B illustrates a time course comparison of free and active AFLB in the medium of RPE cells after transduction with rAAV containing a construct encoding AFLB alone versus rAAV containing a construct encoding AFLB plus RNAi against VEGF-C. [Figure 6B] Same as above.

[0035] [Figure 7] Figures 7A-B illustrate a comparison of VEGF-A neutralization (assessed by ELISA) in RPE supernatants at days 7 (Figure 7A) and 11 (Figure 7B) after transduction with rAAV containing the capsid protein of SEQ ID NO: 48 and the indicated constructs at the indicated MOI.

[0036] [Figure 8A-C] Figure 8A-D illustrates the expression of functional anti-Ang2 scFab from dual protein constructs (plasmids encoding anti-Ang2 scFab in AFLB+LH or HL configuration) following transfection of HEK293T cells (assessed by competition ELISA and Tie2 receptor competition assay). [Figure 8D] Same as above.

[0037] [Figure 9]Figure 9 illustrates the results of anti-Ang2 scFab (and anti-AFLB) Western blots from the culture medium of RPE cells after transduction with rAAV containing the capsid protein of SEQ ID NO: 48 and heterologous nucleic acid encoding AFLB plus anti-Ang2 scFab (HL and LH conformations) at MOIs of 5000 and 1000, where similar expression levels of the LH and HL forms were observed.

[0038] [Figure 10A] Figures 10A-B illustrate the results of anti-Ang2 functional ELISA (ANG2-coated plates) (Figure 10A) and competitive ELISA (Figure 10B) on media from RPE cells 11 days after transduction with the rAAV described in Figure 9 at MOIs of 5000 and 1000. [Figure 10B] Same as above.

[0039] [Figure 11] FIG. 11 illustrates the binding affinity (assessed by Biacore) of the anti-Ang2 scFab encoded by the rAAV described in FIG.

[0040] [Figure 12] Figures 12A-B illustrate PEDF expression from a dual protein construct (encoding AFLB+PEDF) following plasmid transfection of HEK293T cells.

[0041] [Figure 13] Figures 13A-B illustrate VEGFR3-Fc expression from the dual protein construct (encoding AFLB + VEGFR3-Fc) following plasmid transfection of HEK293T cells. Dramatic loss of aflibercept resulting from heterodimerization is shown by ELISA.

[0042] [Figure 14]Figure 14 illustrates constructs pP143.001 (miRNA-only efficacy control) and pP141.001 encoding Ang2 miR-E (RNAi sense and antisense strands embedded within the miR-E backbone) and RPF657 (pP143.001) or AFLB (pP141.001).

[0043] [Figure 15] Figures 15A-B illustrate the kD of Ang2 (secreted and cellular) by ELISA after transduction of HUVECs with the indicated shAng2 constructs, normalized to control (Figure 15A) or not (Figure 15B).

[0044] [Figure 16] Figure 16A illustrates the results of Ang2 ELISA (secreted and cellular) after transduction of human RMVEC cells with an rAAV containing the capsid protein of SEQ ID NO: 48 and a nucleic acid encoding a miRNA comprising the sense and antisense strands of shRNA#5 (described in Figures 15A-B) embedded within a miR-E backbone. Figure 16B illustrates a significant decrease in Ang2 secretion from RMVEC, as assessed by Ang2 qPCR.

[0045] [Figure 17] Figures 17A-B illustrate Ang2 RNA levels (RT-qPCR; Figure 17A) and protein levels (ELISA; Figure 17B) in human RPE cells 8 days after transduction with the rAAV described in Figure 16 (rAAV encoding AFLB only or GFP, which served as a control) at the indicated MOI.

[0046] [Figure 18] Figures 18A-B illustrate free AFLB protein levels (Figure 18A) and AFLB mRNA levels (Figure 18B) in RMVEC cells 8 days after transduction at the indicated MOI with an rAAV containing the capsid protein of SEQ ID NO: 48 and the indicated nucleic acid construct.

[0047] [Figure 19A] Figures 19A-C illustrate Ang2 secretion (Figure 19A) and Ang2 RNA levels (Figures 19B and 19C) in RMVEC cells 8 days after transduction with an rAAV containing the capsid protein of SEQ ID NO: 48 and the indicated nucleic acid constructs. [Figure 19B-C] Same as above.

[0048] [Figure 20] FIG. 20 illustrates the percentage of VEGF-C RNA levels (normalized to RPL32; RNA analysis by qPCR) in HEK293T cells after transduction with the indicated shVEGF-C constructs.

[0049] [Figure 21A] Figures 21A-C illustrate VEGF-C protein (by ELISA; Figure 21A) and VEGF-C mRNA (by qPCR; Figure 21B) following transduction of human RPE cells at the indicated MOI with an rAAV comprising the capsid protein of SEQ ID NO: 48 and the indicated nucleic acid construct. Figure 21C illustrates a dose-dependent increase in expression of miRNAs targeting VEGF-C in the cells. [Figure 21B-C] Same as above.

[0050] [Figure 22A-B] Figures 22A-C illustrate endogenous VEGF-A neutralization (Figure 22A) and VEGF-C protein (Figure 22B) and mRNA levels (Figure 22C) in RPE cells 8 days after transduction with an rAAV comprising the capsid protein of SEQ ID NO: 48 and the indicated nucleic acid construct at the indicated MOI. [Figure 22C] Same as above.

[0051] [Figure 23]Figure 23 illustrates histopathological staining for colocalization of GFP and CD31 in retinal endothelial cells from NHP eyes after intravitreal injection with 1.2 x 10 vg of rAAV containing the capsid protein of sequence number 48 (R100) and a nucleic acid encoding GFP.

[0052] [Figure 24] Figure 24 illustrates aflibercept (AFLB) expression in the aqueous, vitreous, and retina + choroid of NHPs following intravitreal administration of an rAAV comprising the capsid protein of SEQ ID NO: 48 and nucleic acids encoding aflibercept and miRNAs targeting VEGF-C.

[0053] [Figure 25] Figure 25 illustrates the miRNA copies in the NHP retina (described in Figure 24, left panel) and MiSeq data confirming miRNAs targeting VEGF-C as the predominant miRNA species in the NHP retina.

[0054] [Figure 26] Figure 26 illustrates the protective effect of an rAAV comprising the capsid protein of SEQ ID NO: 48 and nucleic acids encoding aflibercept and miRNAs targeting VEGF-C administered at the indicated doses compared to vehicle controls in an NHP model of choroidal neovascularization.

[0055] [Figure 27] Figure 27 shows cell proliferation (left) and migration (right) of HUVEC cells after electroporation with plasmids encoding (i) aflibercept and miRNA targeting VEGF-C, (ii) aflibercept alone, or (iii) GFP alone.

[0056] [Figure 28]Figure 28 illustrates the concentration of aflibercept (ng / ml) in the aqueous humor of NHPs after a single intravitreal administration at the indicated doses of (i) an rAAV comprising the capsid protein of SEQ ID NO: 48 and nucleic acids encoding aflibercept and an miR targeting VEGF-C (including the sense and antisense strands corresponding to SEQ ID NO: 19 and 20 and the complete construct corresponding to SEQ ID NO: 69), or (ii) an rAAV comprising the capsid protein of SEQ ID NO: 48 and nucleic acid encoding only aflibercept.

[0057] [Figure 29] Figure 29 illustrates intraocular inflammation in NHPs after a single intravitreal administration at the indicated doses of (i) an rAAV comprising the capsid protein of SEQ ID NO: 48 and nucleic acids encoding aflibercept and a miR targeting VEGF-C (including sense and antisense strands corresponding to SEQ ID NO: 19 and 20 and the complete construct corresponding to SEQ ID NO: 69), or (ii) an rAAV comprising the capsid protein of SEQ ID NO: 48 and nucleic acids encoding only aflibercept, as assessed by slit lamp biomicroscopy, compared to vehicle-treated control eyes, at the indicated time points.

[0058] [Figure 30] Figure 30 shows the mean total retinal volume and mean central retinal thickness in NHPs from baseline to 22 weeks after a single intravitreal administration of the indicated doses of (i) an rAAV comprising the capsid protein of SEQ ID NO: 48 and nucleic acids encoding aflibercept and an miR targeting VEGF-C (including the sense and antisense strands corresponding to SEQ ID NO: 19 and 20 and the complete construct corresponding to SEQ ID NO: 69), or (ii) an rAAV comprising the capsid protein of SEQ ID NO: 48 and nucleic acids encoding only aflibercept.

[0059] [Figure 31]Figure 31 illustrates the results of full field electroretinography (ffERG) in NHPs at 84 days and 22 weeks after a single intravitreal administration at the indicated doses of (i) an rAAV comprising the capsid protein of SEQ ID NO: 48 and nucleic acids encoding aflibercept and a miR targeting VEGF-C (including the sense and antisense strands corresponding to SEQ ID NO: 19 and 20 and the complete construct corresponding to SEQ ID NO: 69), or (ii) an rAAV comprising the capsid protein of SEQ ID NO: 48 and nucleic acids encoding only aflibercept. DETAILED DESCRIPTION OF THE INVENTION

[0060] Detailed Description of the Invention definition

[0061] "Codon adaptation index," as used herein, refers to a measure of codon usage bias. The codon adaptation index (CAI) measures the deviation of a given protein-coding gene sequence with respect to a reference set of genes (Sharp PM and Li WH, Nucleic Acids Res. 15(3):1281-95 (1987)). The CAI is calculated by determining the geometric mean of the weights associated with each codon over the length (measured in codons) of the gene sequence:

number

number

[0062] The term "isolated" designates biological material (cells, nucleic acids, or proteins) that has been removed from its original environment (the environment in which it naturally occurs). For example, a polynucleotide that occurs in the natural state in a plant or animal is not isolated, but the same polynucleotide separated from the adjacent nucleic acids in which it naturally occurs is considered "isolated."

[0063] As used herein, a "coding region" or "coding sequence" is a portion of a polynucleotide consisting of codons translatable into amino acids. A "stop codon" (TAG, TGA, or TAA), although not typically translated into amino acids, can be considered part of the coding region; however, any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. The boundaries of a coding region are typically determined by a 5'-terminal start codon that encodes the amino terminus of the resulting polypeptide and a 3'-terminal translation stop codon that encodes the carboxyl terminus of the resulting polypeptide. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. It follows that a single vector can contain only a single coding region, or can include two or more coding regions.

[0064] "2A peptide" refers to a "self-cleaving" peptide of approximately 20 amino acids that can be used in place of an IRES element in multicistronic vectors to produce multiple genes at equimolar levels from the same mRNA. Non-limiting examples include the T2A, P2A, E2A, and F2A peptide sequences. In embodiments in which a heterologous nucleic acid comprises a nucleotide sequence encoding multiple gene products, expression of the multiple (e.g., two) gene products can be mediated by multiple (e.g., two) independent promoters, or by a single promoter, with multiple transgenes separated by internal ribosome entry sites (IRES) or 2A peptide sequences.

[0065] As used herein, the term "regulatory region" refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region that influences the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. If the coding region is intended for expression in eukaryotic cells, a polyadenylation signal and transcription termination sequence are usually located 3' of the coding sequence.

[0066] As used herein, the term "nucleic acid" is interchangeable with "polynucleotide" or "nucleic acid molecule" and refers to a polymer of nucleotides.

[0067] A polynucleotide encoding a gene product, e.g., a polypeptide, may contain a promoter and / or other transcriptional or translational control elements operably associated with one or more coding regions. In operably associated states, the coding region of a gene product, e.g., a polypeptide, is associated with one or more regulatory regions in such a manner as to place expression of the gene product under the influence or control of the regulatory region(s). For example, a coding region and a promoter are "operably associated" if induction of promoter function results in transcription of an mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere with the promoter's ability to direct expression of the gene product or the ability of the DNA template to be transcribed. In addition to promoters, other transcriptional control elements, e.g., enhancers, operators, repressors, and transcription termination signals, may also be operably associated with a coding region to direct gene product expression.

[0068] "Transcription control sequence" or "expression control sequence" refers to DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments derived from cytomegalovirus (immediate early promoter in conjunction with intron-A), simian virus 40 (early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit beta-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers and lymphokine-inducible promoters (e.g., promoters inducible by interferon or interleukin).

[0069] The "CAG promoter" is composed of (C) a cytomegalovirus (CMV) early enhancer element, (A) the promoter, first exon and first intron of the chicken beta-actin gene, and (G) the splice acceptor of the rabbit beta-globin gene. See Miyazaki, J., Takaki, S., Araki, K., Tashiro, F., Tominaga, A., Takatsu, K., & Yamamura, K. (1989). Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5. Gene, 79(2), 269-277, the contents of which are incorporated herein by reference.

[0070] Similarly, a variety of translational control elements are known to those skilled in the art, including, but not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites, or IRESs, also called CITE sequences).

[0071] The term "expression," as used herein, refers to the process by which a polynucleotide produces a gene product, such as an RNA or a polypeptide. This includes, but is not limited to, transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), primary miRNA, short hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and translation of mRNA into a polypeptide. Expression results in the production of a "gene product." As used herein, a gene product can be either a nucleic acid, such as a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. The gene products described herein further include nucleic acids with post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides with post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

[0072] A "vector" refers to any vehicle for cloning a nucleic acid and / or transferring a nucleic acid into a host cell. A vector may be a replicon to which another nucleic acid segment can be attached so as to bring about replication of the attached segment. The term "vector" includes both viral and non-viral vehicles for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Numerous vectors, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses, are known and used in the art. Insertion of a polynucleotide into an appropriate vector can be accomplished by ligating a suitable polynucleotide fragment into a chosen vector having complementary cohesive termini.

[0073] A vector can be engineered to encode a selectable marker or reporter that provides for the selection or identification of cells that have incorporated the vector. Expression of the selectable marker or reporter allows for the identification and / or selection of host cells that have incorporated the vector and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc.; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentenyl transferase genes, etc. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ), -glucuronidase (Gus), etc. Selectable markers can also be considered reporters.

[0074] Eukaryotic viral vectors that can be used include, but are not limited to, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, poxviruses such as vaccinia virus vectors, baculovirus vectors, or herpes virus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

[0075] The terms "promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' from the promoter sequence. Promoters may be derived entirely from a native gene, or may be composed of different elements from different promoters found in nature, or even contain synthetic DNA segments. Those skilled in the art will understand that different promoters may direct the expression of genes in different tissues or cell types, at different developmental stages, or in response to different environmental or physiological conditions. A promoter that causes a gene to be expressed in most cell types at most times is generally called a "constitutive promoter." A promoter that causes a gene to be expressed in a specific cell type is generally called a "cell-specific promoter" or "tissue-specific promoter." A promoter that causes a gene to be expressed at a specific developmental or cell differentiation stage is generally called a "development-specific promoter" or "cell differentiation-specific promoter." Promoters that are induced to cause a gene to be expressed following exposure or treatment of cells with a promoter-inducing agent, biological molecule, chemical, ligand, light, etc. are commonly referred to as "inducible promoters" or "regulatable promoters." It is further recognized that because in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0076] The term "plasmid" refers to an extrachromosomal element, often carrying genes that are not part of the cell's central metabolism, usually in the form of a circular double-stranded DNA molecule. Such elements can be linear, circular, or supercoiled, autonomously replicating, genome-integrating, phage, or nucleotide sequences of single- or double-stranded DNA or RNA, from any source, in which several nucleotide sequences are linked or recombined into a unique structure capable of introducing into a cell a promoter fragment and a DNA sequence for a selected gene product, along with appropriate 3' untranslated sequences.

[0077] A polynucleotide or polypeptide has a certain percent "sequence identity" with another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in several different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, Madison, Wis., USA. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc. Alignment programs that allow gaps in sequences are particularly of interest. Smith-Waterman is one type of algorithm that allows gaps in sequence alignment. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[0078] A first anti-angiogenic polypeptide and a nucleic acid encoding an antibody or antigen-binding fragment thereof

[0079] In some embodiments, a nucleic acid is provided comprising a nucleotide sequence encoding a first and a second anti-angiogenic polypeptide, wherein the first anti-angiogenic polypeptide is aflibercept and the second anti-angiogenic polypeptide is an antibody or antigen-binding fragment thereof that binds to and inhibits the activity of a pro-angiogenic protein.

[0080] A preferred nucleotide sequence encoding aflibercept, codon-optimized for expression in humans, is provided below: [ka] In some embodiments, the sequence is at least 80%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 50 and / or includes a stop codon (e.g., TGA) at the end of the sequence. In some embodiments, the aflibercept gene product includes the following amino acid sequence, or a sequence at least 90%, 95%, 97%, 98%, or at least 99% identical thereto:

[0081] [ka]

[0082] In some preferred embodiments, the second anti-angiogenic polypeptide is an antibody or antigen-binding fragment that binds to and inhibits the activity of angiopoietin (angiopoietin-1 / Ang1 / Ang-1, angiopoietin-2 / Ang2 / Ang-2). In some aspects, the second anti-angiogenic polypeptide is an antibody against Ang1 and / or Ang2. In some preferred embodiments, the second anti-angiogenic polypeptide is an antibody or antigen-binding fragment that binds to Ang-2. In particularly preferred embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising heavy chain complementarity determining regions (HCDRs) of HCDR1 = GYYMH (SEQ ID NO: 52); HCDR2 = WINPNSGGTNYAQKFQG (SEQ ID NO: 53) and HCDR3 = SPNPYYYDSSGYYYPGAFDI (SEQ ID NO: 54), and a light chain variable region (LCVR) comprising light chain complementarity determining regions (LCDRs) of LCDR1 = GGNNIGSKSVH (SEQ ID NO: 55), LCDR2 = DDSDRPS (SEQ ID NO: 56) and LCDR3 = QVWDSSSDHWV (SEQ ID NO: 57), or comprises HCDRs and LCDRs at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. In related embodiments, the antibody is a single-chain Fab (scFab) fragment in an LH or HL orientation. In some particularly preferred embodiments, the first anti-angiogenic polypeptide is aflibercept and the second anti-angiogenic polypeptide is an antibody or antigen-binding fragment thereof (e.g., an scFab fragment) that binds to human Ang-2.

[0083] Ang-2 promotes angiogenesis and vascular permeability. Ang-2 expression is particularly elevated in the vitreous of patients with diabetic macular edema (DME), wet age-related macular degeneration (wAMD), and retinal vein occlusion (RVO). High Ang-2 expression correlates with reduced BVCA (best corrected visual acuity) and high central macular thickness (CMT) in wAMD patients.

[0084] Unless otherwise specifically indicated, the term "antibody," as used herein, shall be understood to encompass an antibody molecule comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., a "complete antibody molecule") as well as antigen-binding fragments thereof. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The term "antigen-binding fragment" of an antibody or "antibody fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, CDR-containing fragments, or isolated CDRs. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. Antigen-binding fragments of antibodies can be derived, for example, from intact antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated, for example, chemically or by using molecular biology techniques, to arrange one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, create cysteine residues, modify, add, or delete amino acids, etc.

[0085] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments (e.g., single-chain Fab (scFab) fragments); (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments (single-domain antibodies, i.e., nanobodies or VHH domains); and (vii) hypervariable regions of antibodies (e.g., isolated complementarity-determining regions (CDRs), e.g., CDR3 peptides), or minimal recognition units consisting of amino acid residues that mimic a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs) and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment" as used herein.

[0086] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. In antigen-binding fragments containing a VH domain associated with a VL domain, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be dimeric and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0087] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3, (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3, and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that create a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present invention may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above non-covalently associated with each other and / or with one or more monomeric VH or VL domains (e.g., via disulfide bond(s)).

[0088] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in connection with the antigen-binding fragments of antibodies of the present invention using routine techniques available in the art.

[0089] In some preferred embodiments, there is provided an expression cassette comprising a nucleotide sequence encoding (i) aflibercept + anti-Ang2 HL scFab, (ii) aflibercept + anti-Ang2 LH scFab, (iii) aflibercept + anti-Ang2 HL scFv, or (iv) aflibercept + anti-Ang2 LH scFv, wherein preferably the scFab or scFV comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 52-54 and LCDR1, LCDR2, and LCDR of SEQ ID NOs: 55-57.

[0090] Nucleic acids encoding a first anti-angiogenic polypeptide and a soluble fusion protein

[0091] In some embodiments, a nucleic acid is provided comprising a nucleotide sequence encoding a first and a second anti-angiogenic polypeptide, wherein the first anti-angiogenic polypeptide is aflibercept and the second anti-angiogenic polypeptide is a soluble fusion protein that inhibits the activity of a pro-angiogenic polypeptide.

[0092] In some preferred embodiments, the first antiangiogenic polypeptide is aflibercept and the second antiangiogenic polypeptide is a soluble form of a VEGF receptor (e.g., comprising one or more VEGF-binding domains of VEGFR-1, VEGFR-2, and / or VEGFR-3).

[0093] In some preferred embodiments, the first antiangiogenic polypeptide is aflibercept and the second antiangiogenic polypeptide is a soluble fusion protein comprising one or more VEGF-binding moieties derived from the extracellular domain of VEGFR-3, representative examples of which include the soluble fusion proteins described in U.S. Pat. Nos. 7,034,105, 5,952,199, and 7,422,741, the contents of each of which are incorporated herein by reference.

[0094] VEGF-C promotes angiogenesis and lymphangiogenesis, increasing vascular permeability and leakage. VEGF-C is elevated in the eyes of patients with wAMD after anti-VEGF treatment. Delivery of VEGFR-3-FC, which binds to VEGF-C and VEGF-D, in combination with aflibercept (which binds to VEGF-A, VEGF-B, and PIGF), provides an improved treatment for eye diseases such as wAMD and DME.

[0095] The combination of targeting VEGF-A and blocking VEGFR3 has been effective in preclinical models of choroidal neovascularization (CNV) and is being evaluated in clinical trials. In a Phase 2b clinical trial in subjects with neovascular AMD, the combination of intravitreal ranibizumab and the VEGF C / D antagonist OPT-302 resulted in a 3.4-letter benefit in mean best-corrected visual acuity at 24 weeks compared with treatment with ranibizumab alone (p=0.0107).

[0096] In a particularly preferred embodiment, the second antiangiogenic polypeptide is Opt-302, a soluble form of VEGFR-3 comprising extracellular domains 1-3 of human VEGFR-3 and an Fc fragment of human IgG1 that binds to VEGF-C and VEGF-D and inhibits their activity against endogenous VEGFR-2 and VEGFR-3, as described in U.S. Patent No. 9,745,558, the contents of which are incorporated herein by reference.

[0097] A preferred nucleotide sequence encoding OPT-302, codon-optimized for human expression, is provided below: [ka] [ka]

[0098] A preferred nucleotide sequence encoding a soluble VEGFR-3 containing an alternative IgG2 Fc domain that has been codon-optimized for human expression is provided below: [ka] [ka]

[0099] In some preferred embodiments, an expression cassette is provided that includes a nucleotide sequence encoding (i) aflibercept+VEGFR3-Fc, or (ii) aflibercept+VEGFR3-Fc-IgG2. Preferably, the nucleotide sequence encoding aflibercept includes the sequence of SEQ ID NO: 50 or a sequence at least 90% identical thereto, and / or the nucleotide sequence encoding VEGFR3-Fc includes the sequence of SEQ ID NO: 58, and / or the nucleotide sequence encoding VEGFR3-Fc-IgG2 includes the sequence of SEQ ID NO: 59.

[0100] Nucleic acids encoding first and second anti-angiogenic polypeptides

[0101] In some embodiments, a nucleic acid is provided comprising a nucleotide sequence encoding a first and a second antiangiogenic polypeptide, wherein the first antiangiogenic polypeptide is aflibercept and the second antiangiogenic polypeptide is selected from endostatin; tumstatin; angiostatin; and pigment epithelium-derived factor (PEDF).

[0102] In some preferred embodiments, the nucleic acid comprises the nucleotide sequence encoding aflibercept and the nucleotide sequence encoding PEDF (see, for example, Dawson et al., Science 285:245, 1999; U.S. Patent No. 5,840,686 and International Patent Application WO93 / 24529 and WO99 / 04806, the contents of each of which are incorporated herein by reference).PEDF is a secreted protein that has homology to members of the serpin family of serine protease inhibitors.PEDF is mainly produced by retinal pigment epithelial cells, is expressed in most human tissues, and has anti-angiogenic and neuroprotective properties (see, for example, Dawson DW et al., Science. 1999 July 9; 285(5425):245-8).PEDF prevents photoreceptor degeneration, and PEDF deficiency is associated with angiogenesis diseases such as AMD. Preclinical data point to an inhibitory role for VEGF and FGF in mouse and pig models of choroidal neovascularization (CNV) (see, e.g., Lei XL, Oxid Med Cell Longev.; Vol. 2020, Art. ID 8941057).

[0103] A representative human PEDF sequence is found in GenBank accession P36955 (e.g., P36955.4). In some embodiments, a preferred nucleotide sequence encoding human PEDF that has been codon-optimized for human expression has the following sequence, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto: [ka] [ka]

[0104] In some preferred embodiments, an expression cassette is provided comprising nucleotide sequences encoding aflibercept plus PEDF, preferably wherein the nucleotide sequence encoding aflibercept comprises the sequence of SEQ ID NO:50 and / or the nucleotide sequence encoding PEDF comprises the sequence of SEQ ID NO:60.

[0105] Nucleic acids encoding a first anti-angiogenic polypeptide and an interfering RNA molecule that reduces the expression of angiopoietin

[0106] In some embodiments, a nucleic acid is provided comprising a nucleotide sequence encoding a first antiangiogenic polypeptide and an interfering RNA molecule that targets an angiopoietin, wherein the first antiangiogenic polypeptide is aflibercept.

[0107] In a preferred embodiment, the interfering RNA molecule targets human angiopoietin-1 (also known as Ang1 or Ang-1) and / or human angiopoietin-2 (also known as Ang2 or Ang-2). Representative human Ang2 sequences can be found, for example, in NCBI accession number 015123 and SEQ ID NOs: 517 and 518 of U.S. Patent No. 8,987,420, the contents of which are incorporated herein by reference. Suitable target sequences within the human Ang-2 gene and representative interfering RNA molecules targeting human Ang-2 include those in U.S. Patent No. 7,994,305 (e.g., SEQ ID NOs: 228-427 of U.S. Patent No. 7,994,305) and U.S. Patent No. 8,829,179 (e.g., SEQ ID NOs: 2-69 and 73-104 of U.S. Patent No. 8,829,179), the contents of each of which are incorporated herein by reference. In a particularly preferred embodiment, the interfering RNA molecule targets human Ang-2 and comprises sense and antisense strands according to Table 1.

[0108] In a preferred embodiment, an expression cassette is provided comprising a nucleotide sequence encoding aflibercept and one or more interfering RNAs listed in Table 1. Preferably, the nucleotide sequence encoding aflibercept comprises the sequence of SEQ ID NO:50.

[0109] Nucleic acids encoding a first anti-angiogenic polypeptide and an interfering RNA molecule that reduces expression of VEGF-C and / or VEGF-D.

[0110] In some preferred embodiments, a nucleic acid is provided comprising a nucleotide sequence encoding a first antiangiogenic polypeptide and an interfering RNA molecule that targets VEGF-C and / or VEGF-D, wherein the first antiangiogenic polypeptide is aflibercept.

[0111] In a particularly preferred embodiment, the interfering RNA molecule targets human VEGF-C and / or human VEGF-D. Representative human VEGF-C sequences can be found, for example, in GenBank accession numbers NM_005429 and X94216. Representative human VEGF-D sequences can be found, for example, in GenBank accession number AJ000185.1. Suitable target sequences within the human VEGF-C gene and representative interfering RNA molecules targeting human VEGF-C include those in U.S. Patent No. 7,517,864 (e.g., Table II) and U.S. Patent Application Publication No. 2011 / 0293625 (e.g., SEQ ID NOS: 1-3 and 7-12), the contents of each of which are incorporated herein by reference. In a particularly preferred embodiment, the interfering RNA molecule targets human VEGF-C and comprises a sense strand and an antisense strand according to Table 2.

[0112] The VEGF-C target sequence(s) were selected based on in silico determination of specificity, homology to human and non-human primate (NHP) sequences, and in vitro knockdown of VEGF-C (see Examples). The region of VEGF-C targeted by the RNAi molecule(s) has 100% homology between the human and NHP sequences, but mouse VEGF-C has two point mutations that likely affect the ability of this target sequence to be effective in mice. A sequence alignment of the VEGF-C target region is provided below: [ka]

[0113] Wet AMD (wAMD) is a retinal condition characterized by abnormal, leaky blood vessel growth from the choroid layer through Bruch's membrane into the retina, which can lead to rapid loss of central vision. Current approved treatments include injections of antiangiogenic protein treatments, such as aflibercept, ranibizumab, or brolucizumab, or the aptamer pegaptinib sodium, which blocks VEGF-A-mediated signaling. However, these injected treatments require repeated intravitreal (IVT) administration to maintain vision. Many patients are unable to maintain their initial visual gains due to undertreatment associated with the burdensome frequency of required treatment visits. The nucleic acids described herein, including antiangiogenic polypeptides targeting VEGF-A (e.g., aflibercept) and RNAi molecules targeting VEGF-C, provide improved efficacy to wet AMD patients by reducing the expression of additional angiogenic factors, such as VEGF-C, which are upregulated after the administration of current anti-VEGF treatments.

[0114] In a preferred embodiment, an expression cassette is provided comprising a nucleotide sequence encoding aflibercept and one or more interfering RNAs listed in Table 2. Preferably, the nucleotide sequence encoding aflibercept comprises the sequence of SEQ ID NO:50.

[0115] Nucleic acids encoding a first anti-angiogenic polypeptide and an interfering RNA molecule that reduces the expression of VEGFR-3 In some embodiments, a nucleic acid is provided comprising a nucleotide sequence encoding a first antiangiogenic polypeptide and an interfering RNA molecule that targets VEGFR-3, wherein the first antiangiogenic polypeptide is aflibercept.

[0116] In a preferred embodiment, interfering RNA molecule targets human VEGFR-3.Representative human VEGFR-3 sequence can be found, for example, in GenBank accession number X68203.Suitable target sequence in human VEGFR-3 gene and representative interfering RNA molecule that targets human VEGFR-3 include those listed in table II of US Patent No. 7,517,864, the contents of each of which are incorporated herein by reference.In a particularly preferred embodiment, interfering RNA molecule targets human VEGFR-3 and comprises the sense strand and antisense strand according to table 3.

[0117] In a preferred embodiment, an expression cassette is provided comprising a nucleotide sequence encoding aflibercept and one or more interfering RNAs listed in Table 3. Preferably, the nucleotide sequence encoding aflibercept comprises the sequence of SEQ ID NO:50.

[0118] In embodiments in which the nucleic acid encodes a first antiangiogenic polypeptide and one or more interfering RNAs, the sequence encoding the interfering RNA(s) can be located within a natural or artificial intron (e.g., within a transcriptional regulatory sequence, within the 5' UTR region of a gene, within the coding sequence of a gene, or within an artificial intron within the 3' UTR region of a gene). In some aspects, the interfering RNA is located within a synthetic U2 or U12-based intron or within the interferon regulatory factor 7 intron 4 (IRF7int4; 93 bp).

[0119] In some preferred embodiments, interfering RNA is placed in the artificial intron in transcriptional regulatory sequence.In some preferred embodiments, intron is located in the hybrid chicken β-actin and rabbit β-globin intron of CAG promoter, so that intron is co-transcribed in pre-mRNA by Pol-II and is cut off from pre-mRNA by RNA splicing.The spliced intron containing pre-miRNA structure is further processed into mature miRNA, which can silence angiogenesis-promoting target gene.

[0120] In another embodiment, the interfering RNA is placed within an artificial intron located within the coding sequence of a gene (e.g., encoding aflibercept), whereby the intron is co-transcribed within the pre-mRNA by Pol-II and cleaved from the pre-mRNA by RNA splicing. The spliced intron containing the pre-mRNA structure is further processed into a mature miRNA capable of silencing a pro-angiogenic target gene.

[0121] In other embodiments, the sequence encoding the interfering RNA is located within the 5'UTR or 3'UTR region of the gene, but not within an intron, in which case a portion (e.g., 50%) of the transcribed pre-mRNA is translated into the encoded protein, and a portion (e.g., 50%) of the transcribed pre-mRNA is processed into active shRNA or miRNA. In some preferred embodiments, the interfering RNA is located within the 3'UTR region of the gene.

[0122] Codon-optimized nucleic acid sequences

[0123] In some embodiments, the present invention provides a nucleic acid molecule comprising a nucleotide sequence codon-optimized for human expression. In some embodiments, the nucleotide sequence encodes PEDF and comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 60, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. In another embodiment, the nucleotide sequence encodes OPT-302 and comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 58, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. In another embodiment, the nucleotide sequence encodes VEGFR3-Fc-IgG2 and comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 59, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0124] The term "codon-optimized," when referring to genes or coding regions of a nucleic acid molecule for transformation into various hosts, refers to the alteration of codons in the genes or coding regions of the nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. Such optimization involves replacing at least one, or more than one, or a significant number of codons with one or more codons more frequently used in the genes of that organism.

[0125] Deviations in the nucleotide sequence containing the codons encoding the amino acids of any polypeptide chain allow for variation in the sequence encoding that gene. Because each codon consists of three nucleotides and the nucleotides that make up DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals that terminate translation). The "genetic code," which shows which codons encode which amino acids, is reproduced herein as Table 1. As a result, many amino acids are specified by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six triplets, while tryptophan and methionine are coded for by only one triplet. This degeneracy allows DNA base composition to vary over a wide range without altering the amino acid sequence of the protein coded for by the DNA. [Table 4-1] [Table 4-2]

[0126] Many organisms exhibit biases in the use of specific codons that code for the insertion of specific amino acids in growing peptide chains. Codon preference or codon bias, the difference in codon usage between organisms, is caused by the degeneracy of the genetic code and has been well documented in many organisms. Codon bias often correlates with the efficiency of messenger RNA (mRNA) translation, which is thought to depend, among other things, on the properties of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons most frequently used in peptide synthesis. Therefore, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0127] Given the large number of gene sequences available for a wide variety of animal, plant, and microbial species, the relative frequencies of codon usage have been calculated. Codon usage tables are available, for example, the "Codon Usage Database" available at www.kazusa.or.jp / codon / (accessed June 18, 2012). See Nakamura, Y., et al. Nucl. Acids Res. 28:292 (2000).

[0128] Randomly assigning codons at frequencies optimized for encoding a given polypeptide sequence can be performed manually by calculating the codon frequency for each amino acid and then randomly assigning codons to the polypeptide sequence. Additionally, various algorithms and computer software programs can be used to calculate optimal sequences.

[0129] Non-viral vectors

[0130] In some embodiments, non-viral vector (for example, expression plasmid) is provided, which comprises any nucleotide sequence described herein.In some embodiments, non-viral vector comprises the nucleotide sequence encoding the first anti-angiogenic polypeptide (for example, aflibercept) described herein and one or more interfering RNAs described herein and / or the second anti-angiogenic polypeptide described herein.Preferably, non-viral vector is a plasmid that comprises the expression cassette that comprises the nucleotide sequence described herein.

[0131] viral vectors

[0132] In some embodiments, a viral vector is provided that comprises the modified (codon-optimized) nucleic acid described herein.In a preferred embodiment, the viral vector comprises the nucleic acid that comprises the nucleotide that encodes the first antiangiogenic polypeptide (such as aflibercept) and the nucleotide sequence that encodes one or more interfering RNAs described herein, and / or the nucleotide sequence that encodes the first and second antiangiogenic polypeptides.Examples of suitable viral vectors include but are not limited to adenovirus, retrovirus, lentivirus, herpesvirus and adeno-associated virus (AAV) vectors.

[0133] In a preferred embodiment, the viral vector comprises an AAV genome in which a portion of the parvovirus genome, for example, the rep and cap genes, has been deleted, and / or the AAV genome has been replaced by an expression cassette containing a sequence encoding a first antiangiogenic polypeptide (e.g., aflibercept) and a nucleotide sequence encoding one or more interfering RNAs described herein, and / or a nucleotide sequence encoding a first and second antiangiogenic polypeptide, and their associated expression control sequences. The expression cassette is typically inserted adjacent to (i.e., flanked by) one or two AAV TR or TR elements suitable for viral replication, in place of the nucleic acid encoding the viral rep and cap proteins (Xiao et al., 1997, J. Virol. 71(2): 941-948). Other regulatory sequences suitable for use in promoting tissue-specific expression in target cells may also be included.

[0134] In some embodiments, the AAV viral vector comprises a nucleic acid comprising: (a) an AAV2 terminal repeat, (b) a transcription control sequence, (c) a nucleotide sequence encoding a first anti-angiogenic polypeptide, (d) a nucleotide sequence(s) encoding an RNAi molecule described herein, (d) a polyadenylation sequence, and (e) an AAV2 terminal repeat.

[0135] In other embodiments, the AAV viral vector comprises a nucleic acid comprising (a) an AAV2 terminal repeat, (b) a transcription control sequence, (c) a nucleotide sequence encoding a first anti-angiogenic polypeptide, (d) a 2A sequence, (e) a nucleotide sequence encoding a second anti-angiogenic polypeptide, (f) a polyadenylation sequence, and (g) an AAV2 terminal repeat. In particularly preferred embodiments, the AAV viral vector comprises a nucleic acid (transgene cassette) comprising any of the sequences of SEQ ID NOs: 64-70, more preferably any of the sequences of SEQ ID NOs: 68-70, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0136] In some embodiments, the 5' ITR has the following sequence: [ka]

[0137] In some embodiments, the 3' ITR has the following sequence: [ka]

[0138] In some embodiments, the SV40 polyadenylation sequence has the following sequence:

[0139] [ka]

[0140] Those skilled in the art will understand that AAV vectors containing a transgene but lacking viral proteins necessary for viral replication (e.g., cap and rep) will be unable to replicate because such proteins are required for viral replication and packaging. Helper viruses typically include adenovirus or herpes simplex virus. Alternatively, as discussed below, helper functions (E1a, E1b, E2a, E4, and VA RNA) can be provided to the packaging cell, including by transfecting the cell with one or more nucleic acids encoding various helper elements, and / or the cell can contain nucleic acids encoding helper proteins. For example, HEK 293 was generated by transforming human cells with adenovirus 5 DNA and currently expresses several adenovirus genes, including, but not limited to, E1 and E3 (see, e.g., Graham et al., 1977, J. Gen. Virol. 36:59-72). Thus, these helper functions can be provided by the HEK 293 packaging cells without the need to supply them to the cells by, for example, plasmids encoding them.

[0141] Viral vectors may be any suitable nucleic acid construct, for example a DNA or RNA construct, which may be single-stranded, double-stranded or duplex (ie self-complementary as described in WO2001 / 92551).

[0142] The viral capsid component of the packaged viral vector can be a parvovirus capsid. AAV Cap and chimeric capsids are preferred. For example, the viral capsid can be an AAV capsid (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAVrh10, AAVrh74, RHM4-1, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAV-LK03, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, and any other AAV now known or later discovered. See, e.g., Fields et al., VIROLOGY, volume 2, chapter 69 (4.sup.th ed., Lippincott-Raven Publishers).

[0143] In some embodiments, the viral capsid component of the packaged viral vector is a variant of a native AAV capsid (i.e., contains one or more modifications compared to a native AAV capsid). In some embodiments, the capsid is a variant of an AAV2, AAV5, or AAV8 capsid. In preferred embodiments, the capsid is a variant of an AAV2 capsid, such as one described in U.S. Patent Application Publication No. 2019 / 0255192 A1 (e.g., containing any of the amino acid sequences of SEQ ID NOs: 42-59), the entire contents of which are incorporated herein by reference. In particularly preferred embodiments, the capsid comprises a VP1 capsid protein comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 48. In certain embodiments, the capsid protein comprises a peptide insertion in the GH loop of the capsid protein compared to the corresponding parent AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence ISDQTKH (SEQ ID NO:74), preferably the peptide insertion comprises the amino acid sequence Y1Y2ISDQTKHY3 (SEQ ID NO:75), wherein each of Y1-Y3 is independently selected from Ala, Leu, Gly, Ser, Thr, and Pro. In a specific embodiment, the peptide insertion comprises the amino acid sequence LAISDQTKHA (SEQ ID NO:49), preferably the site of insertion is between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype. In some embodiments, the capsid protein comprises one or more amino acid substitutions compared to the VP1 capsid of AAV2, or one or more corresponding substitutions in another AAV serotype, preferably the capsid protein comprises a P34A amino acid substitution compared to the VP1 capsid of AAV2, or the corresponding substitution in another AAV serotype.

[0144] A full complement of AAV Cap proteins includes VP1, VP2, and VP3. An ORF containing a nucleotide sequence encoding an AAV VP capsid protein may contain AAV Cap proteins that are not the full complement, or a full complement of AAV Cap proteins may be provided.

[0145] In yet another embodiment, the present invention provides the use of ancestral AAV vectors for therapeutic in vivo gene therapy. Specifically, in silico-derived sequences were de novo synthesized and characterized for biological activity. This effort resulted in the generation of nine putative functional ancestral AAVs and the identification of Anc80, the predicted ancestor of AAV serotypes 1, 2, 8, and 9 (Zinn et al., 2015, Cell Reports 12:1056-1068). The prediction and synthesis of such ancestral sequences, along with assembly into viral particles, can be achieved using the methods described in WO2015 / 054653, the contents of which are incorporated herein by reference. In particular, the use of viral particles assembled from ancestral viral sequences may exhibit reduced susceptibility to existing immunity in today's human populations compared to modern viruses or portions thereof.

[0146] The present invention includes packaging cells encompassed by "host cells" that can be cultured to produce packaged viral vectors of the invention. Packaging cells of the invention generally include cells that have heterologous (1) viral vector function(s), (2) packaging function(s), and (3) helper function(s). Each of these component functions is discussed in the following sections.

[0147] First, vectors can be produced by several methods known to those skilled in the art (see, for example, WO2013 / 063379). A preferred method is described in Grieger, et al. 2015, Molecular Therapy 24(2):287-297, the contents of which are incorporated herein by reference for all purposes. Briefly, efficient transfection of HEK293 cells is used as a starting point, where adherent HEK293 cell lines from a qualified clinical master cell bank are grown in animal-component-free suspension conditions in shake flasks and WAVE bioreactors, which allow for rapid and scalable rAAV production. Using a triple transfection method (e.g., WO96 / 40240), suspension HEK293 cell lines, when harvested 48 hours after transfection, produce 10 5 vector genome-containing particles (vg) / cell or higher than 10 14 vg / L of cell culture. More specifically, triple transfection refers to the fact that packaging cells are transfected with three plasmids: one plasmid encodes the AAV rep and cap genes, another encodes various helper functions (e.g., adenovirus or HSV proteins such as E1a, E1b, E2a, E4, and VA RNA), and another encodes the transgene and its various control elements (e.g., a modified RPGRorf15 gene and the hGRK promoter).

[0148] To achieve the desired yield, several variables are optimized, such as the selection of a suitable serum-free suspension medium that supports both growth and transfection, the choice of transfection reagent, transfection conditions, and cell density. A versatile purification strategy based on ion-exchange chromatography has also been developed, resulting in highly pure vector preparations of AAV serotypes 1-6, 8, and 9, as well as various chimeric capsids. This user-friendly process can be completed within a week and produces a high ratio of filled to empty particles (>90% filled particles), resulting in post-purification yields (>1 x 10) appropriate for clinical application. 13 This scalable manufacturing technology provides high yields (vg / L) and purity, and is universal for all serotypes and chimeric particles. This scalable manufacturing technology has been utilized to produce GMP Phase I clinical AAV vectors for retinal neovascularization (AAV2), hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered to patients. Furthermore, implementing a perfusion method entails harvesting rAAV from the culture medium at multiple time points post-transfection, resulting in a minimum 5-fold increase in overall vector production.

[0149] The packaging cell contains a viral vector function along with a packaging function and a vector function. The viral vector function typically includes an AAV genome in which a portion of the parvovirus genome, e.g., rep and cap, has been deleted and replaced with a first antiangiogenic polypeptide sequence that inhibits VEGF-A activity and at least one synthetic RNA molecule or a second antiangiogenic polypeptide sequence and its associated expression control sequences. The viral vector function includes expression control sequences sufficient to cause replication of the viral vector for packaging. Typically, the viral vector includes an AAV genome in which a portion of the parvovirus genome, e.g., rep and cap, has been deleted and replaced with a transgene and its associated expression control sequences. The transgene is typically flanked by two AAV TRs in place of the deleted viral rep and cap ORFs. Suitable expression control sequences, e.g., a tissue-specific promoter and other regulatory sequences suitable for use in promoting tissue-specific expression of the transgene in target cells, are included. The transgene is typically a nucleic acid sequence that can be expressed to produce a therapeutic or marker polypeptide.

[0150] The terminal repeats (TR(s)) (separable and non-separable) selected for use in the viral vector are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5, and 6 being preferred. Separable AAV TRs need not have wild-type TR sequences (e.g., the wild-type sequence may be altered by insertion, deletion, truncation, or missense mutation), so long as the TR mediates the desired function, such as viral packaging, integration, and / or proviral rescue. A TR can be a synthetic sequence that functions as an AAV inverted terminal repeat, such as the "double-D sequence" described in U.S. Pat. No. 5,478,745 to Samulski et al., the entire disclosure of which is incorporated herein by reference in its entirety. Typically, but not necessarily, the TRs are derived from the same parvovirus, e.g., both TR sequences are derived from AAV2.

[0151] The packaging function includes a capsid component. The capsid component is preferably derived from a parvovirus capsid, such as an AAV capsid or a chimeric AAV capsid function. Examples of suitable parvovirus viral capsid components are those derived from Parvoviridae, such as autonomous parvoviruses or dependoviruses. For example, the capsid component can be an AAV capsid, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrhlO, AAVrhl74, RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAV The capsid components may be selected from Hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9, AAV2i8G9, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, and AAV-LK03, as well as other novel capsids that have not yet been identified or are derived from non-human primate sources. The capsid components may comprise components derived from two or more AAV capsids.

[0152] Packaged viral vectors generally contain sequences encoding one or more anti-angiogenic polypeptides and / or interfering RNAs described herein flanked by TR elements and corresponding expression control sequence(s), referred to herein as a "transgene" or "transgene expression cassette," sufficient to result in packaging of the vector DNA and subsequent expression of the interfering RNA and / or gene sequences in transduced cells (e.g., photoreceptors). Viral vector functions can be supplied to cells, for example, as components of a plasmid or amplicon. Viral vector functions can reside extrachromosomally within the cell line and / or can be integrated into the chromosomal DNA of the cell.

[0153] Any method of introducing a nucleotide sequence carrying viral vector function into a cellular host for replication and packaging may be used, including, but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal. In embodiments in which viral vector function is provided by transfection using a viral vector, standard methods for causing viral infection may be used.

[0154] Packaging functions include genes for replication and packaging of viral vectors. Thus, for example, packaging functions may include functions necessary for viral gene expression, viral vector replication, rescue of viral vectors from integrated states, viral gene expression, and packaging of viral vectors into viral particles, as needed. Packaging functions can be provided to packaging cells together or separately using gene constructs, such as plasmids or amplicons, baculoviruses, or HSV helper constructs. Packaging functions can be present extrachromosomally in packaging cells, but are preferably integrated into the chromosomal DNA of the cell. Examples include the genes encoding AAV Rep and Cap proteins.

[0155] Helper functions include helper virus elements necessary to establish active infection of packaging cells required to initiate viral vector packaging. Examples include functions derived from adenovirus, baculovirus, and / or herpesvirus sufficient to cause viral vector packaging. For example, adenovirus helper functions typically include the adenovirus components E1a, E1b, E2a, E4, and VA RNA. Packaging functions can be supplied by infection of packaging cells with the required virus. Packaging functions can be supplied to packaging cells together or separately using genetic constructs, such as plasmids or amplicons. See, for example, the pXR helper plasmid described in Rabinowitz et al., 2002, J. Virol. 76:791, and the pDG plasmid described in Grimmet et al., 1998, Human Gene Therapy 9:2745-2760. The packaging functions can be present extrachromosomally in the packaging cell, but are preferably integrated into the chromosomal DNA of the cell (eg, E1 or E3 in HEK293 cells).

[0156] Any suitable helper virus function can be used.For example, when packaging cell is insect cell, baculovirus can function as helper virus.Herpesvirus can also be used as helper virus in AAV packaging method.Hybrid herpesvirus encoding AAV Rep protein can advantageously promote more scalable AAV vector production scheme.

[0157] Any method of introducing a nucleotide sequence carrying helper functions into a cellular host for replication and packaging can be used, including, but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal. In embodiments in which helper functions are provided by transfection using a viral vector or infection using a helper virus, standard methods for causing viral infection can be used.

[0158] Any suitable permissive or packaging cell known in the art can be used in producing packaged viral vectors. Mammalian or insect cells are preferred. Examples of cells useful for producing packaging cells in the practice of the present invention include, for example, human cell lines such as VERO, WI38, MRC5, A549, HEK 293 cells (which express functional adenovirus E1 under the control of a constitutive promoter), B-50 or any other HeLa cell, HepG2, Saos-2, HuH7, and HT1080 cell lines. In one aspect, the packaging cells can be grown in suspension culture, and more preferably, the cells can be grown in serum-free culture. In one embodiment, the packaging cells are HEK293 cells grown in suspension in serum-free medium. In another embodiment, the packaging cells are HEK293 cells described in U.S. Patent No. 9,441,206 and deposited under ATCC No. PTA 13274. Numerous rAAV packaging cell lines are known in the art, including, but not limited to, those disclosed in WO2002 / 46359. In another embodiment, the packaging cells are cultured in the form of cell stacks (e.g., 10-layer cell stacks seeded with HEK293 cells).

[0159] The cell line for use as packaging cell includes insect cell line.Any insect cell that allows AAV replication and can be maintained in culture can be used according to the present invention.Examples include Spodoptera frugiperda, such as Sf9 or Sf21 cell line, Drosophila spp. cell line, or mosquito cell line, such as Aedes albopictus cell line.Preferred cell line is Spodoptera frugiperda Sf9 cell line. The following references are incorporated herein for their teachings regarding the use of insect cells for expression of heterologous polypeptides, methods for introducing nucleic acids into such cells, and methods for maintaining such cells in culture: Methods in Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., 1989, J. Virol. 63:3822-3828; Kajigaya et al., 1991, Proc. Nat'l. Acad. Sci. USA 88: 4646-4650; Ruffing et al., 1992, J. Virol. 66:6922-6930; Kimbauer et al., 1996, Virol. 219:37-44; Zhao et al., 2000, Virol. 272:382-393; and Samulski et al., U.S. Patent No. 6,204,059.

[0160] Viral capsids according to the invention can be produced using any method known in the art, for example, by expression from baculovirus (Brown et al., (1994) Virology 198:477-488). As a further alternative, viral vectors of the invention can be produced in insect cells using a baculovirus vector to deliver the rep / cap genes and rAAV template, as described, for example, by Urabeetal., 2002, Human Gene Therapy 13:1935-1943.

[0161] In another aspect, the present invention provides a method for rAAV production in insect cells, in which a baculovirus packaging system or vector can be constructed to carry the AAV Rep and Cap coding regions by engineering these genes into the polyhedrin coding region of the baculovirus vector and producing a viral recombinant by transfection into host cells. In particular, when using baculovirus production for AAV, the AAV DNA vector product is preferably a self-complementary AAV-like molecule without mutations to the AAV ITRs. This appears to be a by-product of inefficient AAV Rep nicking in insect cells, which results in a self-complementary DNA molecule due to the lack of functional Rep enzyme activity. The host cell can be a baculovirus-infected cell, or have additional nucleic acid encoding or containing baculovirus helper functions introduced therein. These baculovirus viruses can express AAV components and subsequently promote capsid production.

[0162] During production, packaging cells generally contain one or more viral vector functions, along with helper and packaging functions sufficient to effect replication and packaging of the viral vector. These various functions can be supplied to the packaging cell together or separately using genetic constructs, e.g., plasmids or amplicons, and can be present extrachromosomally within the cell line or integrated into the cell's chromosome.

[0163] Cells can be supplied with any one or more of the mentioned functions already incorporated, such as cell lines with one or more vector functions either extrachromosomally integrated or integrated into the cell's chromosomal DNA, cell lines with one or more packaging functions either extrachromosomally integrated or integrated into the cell's chromosomal DNA, or cell lines with helper functions either extrachromosomally integrated or integrated into the cell's chromosomal DNA.

[0164] rAAV vectors can be purified by standard methods in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors are known in the art, including those described in Clark et al., 1999, Human Gene Therapy 10(6):1031-1039; Schenpp and Clark, 2002, Methods Mol. Med. 69:427-443; U.S. Patent No. 6,566,118 and WO98 / 09657.

[0165] Treatment method

[0166] In some embodiments, the nucleic acid described herein - or a pharmaceutical composition comprising such a nucleic acid and a pharmaceutically acceptable excipient - is administered to a subject (e.g., a human) intraocularly, preferably by subretinal, suprachoroidal, or intravitreal injection. In some preferred embodiments, the nucleic acid or pharmaceutical composition is administered via intravitreal and / or subretinal injection, more preferably by a single intravitreal injection, to treat VEGF-related ocular diseases. In some embodiments, the VEGF-related ocular disease is a VEGF-A-related ocular disease. In other embodiments, the nucleic acid or pharmaceutical composition is administered topically or intracamerally. In some embodiments, the VEGF-related ocular disease is selected from wet (neovascular, exudative) age-related macular degeneration; macular edema following retinal vein occlusion; retinal neovascularization resulting from retinal vein occlusion; diabetic macular edema, diabetic retinopathy (including all stages of non-proliferative and proliferative diabetic retinopathy), myopic macular degeneration, branch retinal vein occlusion, hemiretinal vein occlusion, and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectasia; neovascular glaucoma; vitreous hemorrhage; retinal and choroidal neovascularization secondary to retinal diseases including, but not limited to, uveitis, trauma, retinal degenerative disorders, inherited retinal and / or choroidal diseases, ocular tumors, and corneal and iris neovascularization. In a preferred embodiment, the nucleic acid is delivered to the subject in a vector, preferably a recombinant AAV (rAAV) vector described herein, or a pharmaceutical composition comprising such a vector and a pharmaceutically acceptable excipient, preferably the rAAV vector comprises a capsid protein of SEQ ID NO: 48 or a sequence comprising at least 90% identity thereto.

[0167] In a related aspect, a nucleic acid described herein is provided for use in treating a VEGF-related ocular disease (e.g., a VEGF-A-related ocular disease) or for the manufacture of a medicament for treating a VEGF-related ocular disease. In another related aspect, an rAAV comprising a nucleic acid described herein is provided for use in treating a VEGF-related ocular disease or for the manufacture of a medicament for treating a VEGF-related ocular disease. In a preferred embodiment, the rAAV comprises a capsid sequence of SEQ ID NO: 48 or a sequence comprising at least 90% identity thereto, and is administered intravitreally to a subject, preferably by a single intravitreal injection, to treat a VEGF-related ocular disease.

[0168] In certain preferred embodiments, methods are provided for treating and / or preventing wet (neovascular, exudative) age-related macular degeneration in a subject (e.g., a human subject) by administering to the subject an effective amount of an rAAV comprising a nucleic acid described herein or a pharmaceutical composition comprising such an rAAV and a pharmaceutically acceptable excipient. Diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; or myopic choroidal neovascularization, as well as all other forms of abnormal ocular and retinal neovascularization, including but not limited to, idiopathic retinal neovascularization, neovascular glaucoma, retinopathy of prematurity, radiation retinopathy, central serous retinopathy, diabetic vitreous hemorrhage, pseudoxanthoma elasticum, Coats' disease, and other forms of peripheral retinal neovascularization. Preferably, the rAAV comprises a capsid protein of SEQ ID NO: 48 or a sequence comprising at least 90% identity thereto. In particularly preferred embodiments, methods are provided for treating wet age-related macular degeneration.

[0169] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding aflibercept and a nucleotide sequence encoding a second antiangiogenic polypeptide. In related embodiments, the second antiangiogenic polypeptide is selected from endostatin; tumstatin; angiostatin; and pigment epithelium-derived factor (PEDF). In some preferred embodiments, the second antiangiogenic polypeptide is PEDF. In particularly preferred embodiments, the rAAV vector comprises a capsid protein of SEQ ID NO: 48 and a nucleic acid comprising the following sequence (aflibercept + PEDF double construct) or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto: [ka] [ka] [ka] and uses thereof in treating wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; or myopic choroidal neovascularization, preferably wherein the vector is administered intraocularly to a human subject, preferably wherein intraocular administration comprises intravitreal injection (e.g., a single intravitreal injection), subretinal injection, or suprachoroidal injection.

[0170] In other aspects, the nucleic acid comprises a nucleotide sequence encoding a soluble fusion protein comprising aflibercept and one or more VEGF-binding moieties derived from the extracellular domain of VEGFR-3. In particularly preferred embodiments, an rAAV vector comprises the capsid protein of SEQ ID NO:48 and a nucleic acid comprising the following sequence (aflibercept + OPT-302 dual construct), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto: [ka] [ka] [ka] [ka] and uses thereof in treating wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; or myopic choroidal neovascularization, preferably wherein the vector is administered intraocularly to a human subject, preferably wherein intraocular administration comprises intravitreal injection (e.g., a single intravitreal injection), subretinal injection, or suprachoroidal injection.

[0171] In other aspects, the nucleic acid comprises a nucleotide sequence encoding aflibercept and a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that binds to and inhibits the activity of a pro-angiogenic protein. In preferred embodiments, the antibody or antigen-binding fragment thereof binds to human ang-1 or human ang-2. In particularly preferred embodiments, an rAAV vector comprising the capsid protein of SEQ ID NO: 48 and a nucleic acid comprising the following sequence (aflibercept + anti-Ang-2 HL construct) or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto: [ka] [ka] [ka] [ka] and an rAAV vector comprising the capsid protein of SEQ ID NO:48 and a nucleic acid comprising the following sequence (aflibercept + anti-Ang-2 LH construct) or a sequence at least 80%, at least 85%, at least 90%, at least 95%; at least 98% or at least 99% identical thereto: [ka] [ka] [ka] and their use in treating wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; or myopic choroidal neovascularization, preferably the vector is administered intraocularly to a human subject, preferably intraocular administration comprises intravitreal injection (e.g., a single intravitreal injection), subretinal injection, or suprachoroidal injection.

[0172] In some aspects, the nucleic acid comprises the nucleotide sequence encoding aflibercept and the nucleotide sequence encoding the interfering RNA that reduces the expression of pro-angiogenic protein.In a preferred embodiment, the nucleotide sequence encoding the interfering RNA encodes the natural or artificial miRNA that comprises the sense strand and the antisense strand that reduces the expression of pro-angiogenic protein.

[0173] In a related aspect, the interfering RNA reduces expression of human ang-1 and / or human ang-2. In a particularly preferred embodiment, an rAAV vector comprises the capsid protein of SEQ ID NO: 48 and a nucleic acid comprising the following sequence (aflibercept + human Ang-2 interfering RNA (SEQ ID NO: 13) construct), or a sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto: [ka] [ka] [ka] and their use in treating wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; or myopic choroidal neovascularization, preferably wherein the vector is administered intraocularly to a human subject, preferably wherein intraocular administration comprises intravitreal injection (e.g., a single intravitreal injection), subretinal injection, or suprachoroidal injection.

[0174] In a related aspect, the interfering RNA reduces expression of human VEGF-C. In a particularly preferred embodiment, an rAAV vector comprises the capsid protein of SEQ ID NO: 48 and a nucleic acid comprising the following sequence (aflibercept + human VEGF-C interfering RNA (SEQ ID NO: 19 / 20) construct), or a sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto: [ka] [ka] [ka] and their use in treating wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; or myopic choroidal neovascularization, preferably wherein the vector is administered intraocularly to a human subject, preferably wherein intraocular administration comprises intravitreal injection (e.g., a single intravitreal injection), subretinal injection, or suprachoroidal injection.

[0175] In a related aspect, the interfering RNA reduces expression of human VEGFR-3. In a particularly preferred embodiment, an rAAV vector comprises the capsid protein of SEQ ID NO: 48 and a nucleic acid comprising the following sequence (aflibercept + human VEGFR-3 interfering RNA (SEQ ID NO: 37) construct), or a sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto: [ka] [ka] [ka] and uses thereof in treating wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; or myopic choroidal neovascularization, preferably the vector is administered intraocularly to a human subject, preferably intraocular administration comprising intravitreal injection (e.g., a single intravitreal injection), subretinal injection, or suprachoroidal injection, and preferably administered to a subject by intravitreal injection (e.g., a single intravitreal injection) to treat wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; or myopic choroidal neovascularization.

[0176] Also provided herein are pharmaceutical compositions comprising: a) a nucleic acid described herein, preferably encapsidated within an rAAV (preferably an rAAV comprising the capsid protein of SEQ ID NO: 48); and b) a pharmaceutically acceptable carrier, diluent, excipient, or buffer. In some preferred embodiments, the nucleic acid comprises a nucleotide sequence selected from SEQ ID NOs: 64-70. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in human or non-human patients. Such excipients, carriers, diluents, and buffers include any pharmaceutical agent that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts, such as mineral acid salts, e.g., hydrochloride, hydrobromide, phosphate, sulfate, and the like; and salts of organic acids, e.g., acetate, propionate, malonate, benzoate, and the like, may be included therein. In addition, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances, etc., may be present in such vehicles.A wide variety of pharmaceutically acceptable excipients are known in the art and do not need to be discussed in detail here.Pharmaceutically acceptable excipients are fully described in various publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.

[0177] In some preferred embodiments, the pharmaceutical composition comprises Dulbecco's phosphate buffered saline (DPBS) and a non-ionic surfactant (eg, Pluronic® F68, preferably at about 0.005%).

[0178] In some embodiments, the pharmaceutical composition comprises 1×10 8 ~1×10 15 vector particles or vector genomes, 1 × 10 10 ~1×10 13 vector particles or vector genomes, or approximately 1 x 10 10 , about 2×10 10 , 3×10 10 , about 4×10 10 , about 5×10 10 , about 6×10 10 , about 7×10 10 , about 8×10 10 , about 9×10 10 , about 1×10 11 , about 2×10 11 , about 3×10 11 , about 4×10 11 , about 5×10 11 , about 6×10 11 , about 7×10 11 , about 8×10 11 , about 9×10 11 , about 1×10 12 , about 2×10 12 , about 3×10 12 , about 4×10 12 , about 5×10 12 , about 6×10 12 , about 7×10 12 , about 8×10 12 , about 9×10 12 Or about 1 x 10 13 In some embodiments, the pharmaceutical composition contains about 1 x 10 vector particles or vector genomes. 11 ~Approx. 1×10 12 Each vector particle or vector genome comprises a vector.

[0179] In some preferred embodiments, the pharmaceutical composition is administered intraocularly to a human having a VEGF-related ocular disorder, preferably, the pharmaceutical composition is administered via intravitreal, subretinal and / or suprachoroidal injection, more preferably, via a single intravitreal injection. [Example]

[0180] The following examples illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention in any way. Although the present invention has been described with respect to its preferred embodiments, various modifications thereof will become apparent to those skilled in the art upon reading the present application.

[0181] Example 1 The following examples describe a multi-mechanistic approach to antiangiogenic gene therapy using recombinant adeno-associated viruses (rAAVs) containing genetically modified capsid proteins that improve transduction of multiple retinal cells. The rAAV constructs described below provide sustained delivery of antiangiogenic agents from a single intravitreal dose, limiting the burden of repeated injections compared to the delivery of a single antiangiogenic agent, maintaining consistent levels of therapeutic gene products in relevant retinal cells, and improving therapeutic response. Each of the representative constructs described below contains nucleic acids encoding aflibercept (targeting VEGF-A, VEGF-B, and PIGF) and at least one other antiangiogenic agent, to enhance efficacy beyond the delivery of aflibercept alone.

[0182] Representative construct designs (AAV vector backbones) are illustrated in Figures 1A-B. In the first approach (Figure 1A), nucleotide sequences encoding aflibercept and a second antiangiogenic polypeptide, separated by an FT2A (ribosomal skipping peptide) sequence, are controlled by the ubiquitous CBA promoter (this is a bicistronic construct). The second antiangiogenic polypeptides exemplified herein include PEDF, a VEGFR-3-Fc fusion protein, and an anti-Ang-2 scFab fragment, selected based on their function in angiogenesis and / or reducing vascular permeability. In each case, the encoding gene was codon-optimized for human expression. In the second approach (Figure 1B), the nucleotide sequence encoding aflibercept is driven by a CAG promoter along with a nucleotide sequence encoding an interfering RNA targeting a pro-angiogenic protein. In each case, the RNAi sequence was embedded in a well-characterized miR-E backbone (e.g., as described in U.S. Patent Application Publication No. 2015-0018539, the contents of which are incorporated herein by reference, and Fellmann et al., Cell Reports, 5(6):1704-1713 (2013)) and placed within a hybrid chicken b-actin / rabbit b-globin intron (see FIG. 1B). The CBA promoter is a well-characterized ubiquitous promoter capable of driving sustained high-level expression. The CBA promoter is a hybrid of the human cytomegalovirus (CMV) upstream enhancer and the chicken β-actin (CBA) promoter, and also contains chicken b-actin exon 1, a hybrid chicken b-actin and rabbit b-globin intron, and a rabbit b-globin exon 3 fragment (creating an artificial splice site). The use of the miR-E backbone and its placement within an intron of the CAG promoter was validated using a model antigen (data not shown).

[0183] Construction of dual protein constructs: Codon-optimized genes for pigment epithelium-derived factor (PEDF), VEGF receptor 3 (VEGFR3)-Fc fusion protein, and anti-angiopoietin-2 (ANG2) single-chain Fab (scFab) fragment were excised from the shuttle vector and inserted into the AAV vector backbone on an NheI-MluI fragment between the CBA promoter and the SV40 late polyA (SV40pA) sequence. For coexpression with aflibercept, synthetic DNA included sequences encoding the C-terminal portion of aflibercept (AFLB), a furin cleavage site, and a T2A ribosomal skipping peptide upstream of the PEDF, VEGFR3-Fc, or anti-ANG2 scFab sequence. These synthetic DNAs were excised from the shuttle vector and inserted into pAAV-CAG-AFLB-SV40pA on an AvrII-MluI fragment. Plasmids were expanded in E. coli, and purified plasmid DNA was verified by restriction digestion and sequencing.

[0184] Construction details for protein + RNAi constructs

[0185] Construction of pAAV-CAG-miR-E-("target sequence")-AFLB-SV40

[0186] The pAAV-CAG-AFLB-SV40 construct expressing human codon-optimized aflibercept was synthesized as previously described. miR-E-(target) miRNA transgenes encoding hairpins targeting ANGPT2, VEGF-C, or VEGFR3, containing a region of the CAG beta-actin intron encoded between the SgrAI and NheI restriction cloning sites, were synthesized and cloned into pUC57 using Genscript (Genscript, Picataway, NJ). The pUC57 and pAAV-CAG-AFLB-SV40-Kan-Stuffer plasmids were cleaved with various restriction enzymes (New England Biolabs) as indicated, and the backbone DNA was also treated with recombinant shrimp alkaline phosphatase (rSAP, M0371L, New England Biolabs) during digestion to remove free phosphates on the cleaved DNA ends. The DNA fragments were added at a 7:1 molar ratio of insert to backbone and ligated using Quick Ligase (#M2200L, New England Biolabs) according to the manufacturer's instructions. The ligated plasmid was transformed into NEB Stable bacterial competent cells (#C3040H, New England Biolabs) according to the manufacturer's instructions, and the cells were spread on kanamycin 50 mg / ml plates (#L1025, Teknova, Hollister, CA) and grown at 30°C.

[0187] Preparation of pAAV-CAG-miR-E-AFLB-SV40

[0188] Miniprep cultures were grown from the resulting colonies, and DNA was prepared using the GeneJET Plasmid Miniprep Kit (Cat. No. 0503, ThermoFisher, Waltham, MA) and restriction digested to identify positive clones. 50 ml cultures in Terrific Broth were grown from one positive clone of each construct, and DNA was prepared using the Qiagen EndoFree Plasmid Maxi Kit (Cat. No. 12362, Qiagen, Hilden, Germany).

[0189] Restriction digestion and sequencing of pAAV-CAG-RFP657-miRNA plasmid variants

[0190] Maxiprep plasmid DNA (0.5 mg) was digested with various restriction enzymes (New England BioLabs) according to the manufacturer's instructions and analyzed by agarose gel electrophoresis. Sanger DNA sequencing was performed by ELIM using primers.

[0191] A summary of the constructs designed and tested in the examples herein is provided below: [Table 5]

[0192] Aflibercept is expressed at therapeutic levels in human RPE and RGC cells after delivery in rAAV virions containing the capsid protein of SEQ ID NO: 48, resulting in efficient blockade of VEGF-A, VEGF-B, and PIGF-mediated activity in these cells. See U.S. Patent Application Publication No. 2020 / 0282077 A1, the contents of which are incorporated herein by reference.

[0193] Studies were conducted to evaluate the effect of including a second transgene (encoding a protein or RNAi) in the AAV expression plasmid on the expression of aflibercept following transfection of HEK293T cells.

[0194] Briefly, HEK293T cells were cultured in 12-well plates at 2.0 × 10 in 1.0 ml of DMEM / 10% FBS medium. 5 Cells were seeded at 1000 x g / well. The following day, 1.0 mg of plasmid DNA (containing nucleotide sequences encoding aflibercept and a second transgene under the control of the same promoter) complexed with 3.0 ml of FuGeneHD (Cat. No. E2691, Promega, Madison, WI) was added to the cells in triplicate wells. 48 hours after transfection, the cell supernatant was collected and spun at 2000 g to remove cell debris. The medium was then assayed for the presence of aflibercept (ALFB) via ELISA.

[0195] For Western blot analysis, media from transduced HEK293T cells was pooled from three replicates, mixed with 4x LDS (B0007, Thermo), 10x reducing agent (B0009, Thermo), and denatured at 70°C for 10 min. Samples were loaded onto 10-well Bolt 4-12% Bis-Tris Plus polyacrylamide gels (Invitrogen, NW04120BOX) and run at 200 V for 32 min in 1x MOPS buffer (NP000102, Thermo). The separated proteins were transferred to nitrocellulose filters (1704158, BioRad) using a BioRad TransBlot Turbo device (BioRad) for 7 minutes and probed with anti-human IgG Fc cross-adsorbed secondary antibody, HRP (ThermoFisher, 31413) at 1:500 in iBind Flex solution (SLF2020, Thermo). Proteins were visualized using SuperSignal West Dura chemiluminescent substrate (ThermoFisher 34076) and imaged on a ChemiDoc MP (BioRad, Hercules, CA).

[0196] Cell lysates for ELISA (secreted free AFLB, ANGPT2, and VEGF-A levels) were prepared in M-PER lysis buffer (#78501, Thermo Scientific) supplemented with 1x Halt protease and phosphatase inhibitor cocktail (#78440, Thermo Scientific) according to the manufacturer's instructions. Cell media and lysates were diluted appropriately for each sample and used to measure free AFLB levels using an Aflibercept ELISA kit (to measure free AFLB levels) (Cat. No. IG-AA115, Eagle Biosciences, Nashua, NH), Quantikine Human VEGF-A, according to the supplier's instructions. Secreted analyte levels were assessed using an ELISA kit (DVE00, R&D Systems) and a Quantikine human ANGPT2 ELISA kit (DANG20, R&D Systems). Optical density (OD) was measured within 15 minutes of pipetting the stop solution using a Cytation 3 (BioTek, Winooski, VT) photometer at 450 nm (referenced to OD620 nm). Media concentrations were defined based on the generated standard curve.

[0197] Figure 3A demonstrates the results of Western blot analysis of AFLB from the medium of HEK293T cells after transfection with an AAV plasmid encoding AFLB and a second transgene (PEDF, VEGFR3, anti-Ang HL, or anti-AngLH) under the control of the CAG promoter. Results are normalized to AFLB levels in the medium of HEK293T cells transfected with an AAV plasmid encoding only AFLB under the control of the CBA promoter. Elyea (a commercially available preparation) serves as a positive control for aflibercept. The addition of a second transgene encoding a polypeptide product significantly reduces aflibercept expression by approximately 50-60%.

[0198] Figure 3B demonstrates the expression of aflibercept from various constructs encoding either a second polypeptide (PEDF, anti-Ang2 LH, anti-Ang2 HL) or an interfering RNA (targeting Ang2, FEGR3, or VEGF-C), normalized to AFLB levels in the medium of HEK293T cells transfected with an AAV plasmid encoding only AFLB under the control of the CBA promoter. Notably, plasmids encoding AFLB and an interfering RNA do not result in a significant reduction in aflibercept expression. Expression from the CAG promoter was observed to be 25% higher than that from the CBA promoter.

[0199] Next, studies were performed to evaluate the effect of including a second transgene (encoding a protein or RNAi) in the AAV expression plasmid on the expression of aflibercept following transduction of human retinal pigment epithelial (RPE) cells with recombinant AAV virus containing the capsid protein of SEQ ID NO:48.

[0200] For RPE transduction, human stem cell-derived retinal pigment epithelial cells (RPE) were differentiated from embryonic stem cells (ESI-017) according to published protocols (Buchholz D 2013, Leach L 2015). RPE cells were grown on Matrigel (Corning) in XVIVO-10 medium (Lonza) in a 96-well plate format for 30 days. Prior to transduction, three wells were harvested and counted for accurate calculation of the multiplicity of infection (MOI). Virus was added to cells in XVIVO-10 medium for 48 hours based on the respective viral titer in a total volume of 100 μL per well. Medium was collected on days 3, 7, 11, 15, and 19 and replaced with 200 μL of medium per well. Medium samples were stored at 4°C until processing.

[0201] Cell lysates for ELISA (to assess secreted free AFLB, ANGPT2, and VEGF-A levels) were prepared in M-PER lysis buffer (#78501, Thermo) supplemented with 1x Halt protease and phosphatase inhibitor cocktail (78440, Thermo) according to the manufacturer's instructions. Cell media and lysates were appropriately diluted for each sample and used to assess secreted analyte levels using an aflibercept ELISA kit (to measure free AFLB levels) (catalog number IG-AA115, Eagle Biosciences, Nashua, NH), a Quantikine human VEGF-A ELISA kit (DVE00, R&D Systems), and a Quantikine human ANGPT2 ELISA kit (DANG20, R&D Systems) according to the supplier's instructions. Optical density (OD) was measured within 15 minutes of pipetting the stop solution using a Cytation 3 (BioTek, Winooski, VT) photometer at 450 nm (OD 620 nm reference). Media concentrations were defined based on the generated standard curve.

[0202] As can be seen in Figure 4, a dose-dependent increase in aflibercept expression is seen with rAAV expressing aflibercept and an RNAi targeting VEGF-C at a high multiplicity of infection (MOI) close to that of the control (rAAV expressing aflibercept alone operably linked to a CAG promoter) (day 8 shown). All aflibercept-expressing constructs are able to completely neutralize endogenous VEGF-A at all multiplicities of infection (MOI) tested. The amounts shown in Figure 4 are free, active aflibercept in the medium of RPE cells after transduction.

[0203] Figures 5A and 5B illustrate the analysis of free aflibercept in RPE supernatants on days 7 (Figure 5A) and 11 (Figure 5B) after transduction with rAAV carrying the indicated expression cassette (and capsid protein of SEQ ID NO: 48). Similar to HEK293T cells, expression of a second protein dramatically reduces the level of aflibercept expression in transduced RPE cells. In contrast, some reduction in aflibercept expression was observed with coexpression of interfering RNA, although aflibercept expression remained robust at both MOIs. Surprisingly, the CBA promoter was determined to be much weaker than the CAG promoter in driving expression in RPE cells. See also Figures 6A-B, which illustrate some reduction in aflibercept expression at higher MOIs with coexpression of interfering RNA, although this is much less pronounced than with coexpression of a second polypeptide.

[0204] Importantly, aflibercept co-expressed with interfering RNA was functional and capable of binding to VEGF-A produced by RPE cells (see Figures 7A-B, which illustrate neutralization of VEGF-A at an MOI of greater than 40 for AFLB+VEGFC-RNAi and AFLB+ANG2 RNAi constructs).

[0205] conclusion

[0206] In HEK293T cells, aflibercept expression was reduced by approximately 20% when driven by the CBA promoter compared to the CAG promoter; in RPE cells, aflibercept expression was approximately 13-fold weaker when driven by the CBA promoter compared to the CAG promoter.

[0207] In HEK293T cells, RNAi had no significant effect on transgene expression compared to the control (aflibercept alone under the control of the same promoter), but all dual-protein constructs showed an approximately 50% reduction in aflibercept compared to the control (aflibercept alone under the control of the same promoter) in HEK293T cells. In RPE cells, under most conditions, a slight but non-significant reduction in free aflibercept levels compared to the control was observed in RPE cells for constructs containing RNAi, but all dual-protein constructs expressed approximately 5-10-fold less aflibercept than their control counterparts.

[0208] Example 2 Characterization of dual protein constructs Characterization of constructs expressing aflibercept plus anti-Ang-2 scFab

[0209] HEK293T cells were transfected with an AAV plasmid containing a nucleotide sequence encoding aflibercept and a nucleotide sequence expressing an anti-Ang-2 scFab in a bicistronic configuration driven by a CAG promoter (see Figure 1A). Briefly, HEK293T cells were transfected into 12-well plates at 2.0 x 10 in 1.0 ml of DMEM / 10% FBS medium. 5 Cells were seeded at 1000 x g / well. The following day, 1.0 mg of plasmid DNA complexed with 3.0 ml of FuGeneHD (Cat. No. E2691, Promega, Madison, WI) was added to the cells in triplicate wells. 48 hours after transfection, the cell supernatant was collected and spun at 2000 g to remove cell debris. The medium was then assayed for the presence of ALFB via ELISA.

[0210] Western Blot - Media (6.25 μl) from transfected HEK293T cells was mixed with 12.5 μl of 4x LDS, 5 μl of 10x reducing agent, and 26.25 μl of 1x PBS (final volume = 50 μl) and denatured at 70°C for 10 min. 40 μl of sample was loaded onto a 10-well Bolt 4-12% Bis-Tris Plus polyacrylamide gel (Invitrogen, NW04120BOX) and run at 200 V for 32 min in 1x MOPS buffer. Separated proteins were transferred to nitrocellulose filters using an iBlot 2 device (ThermoFisher) for 7 min and probed with anti-human IgG F(ab')2 secondary antibody (ThermoFisher 31482 1:1000) using an iBind Flex device (ThermoFisher). Proteins were visualized using SuperSignal West Dura chemiluminescent substrate (ThermoFisher 34076) and imaged on a ChemiDoc MP (BioRad, Hercules, CA).

[0211] Functional anti-ANG2 ELISA using ANG2-coated plates—Nunc MaxiSorp flat-bottom plates (Invitrogen, 44-2404-21) were coated with 100 μl of 1.0 μg / μl recombinant human angiopoietin-2 (R&D, 623-AN / CF) in PBS, sealed with adhesive tape, and placed at 4°C overnight. The following day, the coating solution was aspirated, and the plates were washed three times with 300 μl of PBST (PBS / 0.05% Tween® 20). The plates were blocked with 200 μl of PBS / 2.0% BSA for 2 hours at room temperature. After the 2-hour incubation, the blocking solution was aspirated, and the plates were washed three times with 300 μl of PBST. Media from transfected HEK293T cells was diluted in PBS / 0.2% BSA, and 100 μl of the diluted media was added to the plate and incubated at room temperature for 2 hours with gentle shaking. The plate was washed three times with 300 μl of PBST. 100 μl of anti-human IgG F(ab')2 secondary antibody (ThermoFisher 31482, 1:20,000) was added to the plate and incubated at room temperature for 1 hour with gentle shaking. The plate was washed three times with 300 μl of PBST. The plate was developed with 100 μl of TMB ELISA substrate (Abcam, ab171522) for 5–15 minutes at room temperature. The TMB reaction was stopped with 100 μl of 450 nm stop solution for TMB substrate (Abcam, ab171529). Plates were read at 450 nm and 540 nm (as a reference blank for the plate) using a Cytation 5 device (BioTek).

[0212] ANG2 competitive ELISA—Media from transfected HEK293T cells was diluted in PBS / 0.2% BSA and mixed with an equal volume of 2.0 ng / ml recombinant human angiopoietin-2 (R&D, 623-AN / CF) and incubated overnight at room temperature (final ANG2 concentration = 1,000 pg / ml). The following day, the concentration of free ANG2 was determined using an Angiopoietin-2 human ELISA kit (Invitrogen, KHC1641) interpolated from a freshly prepared recombinant human angiopoietin-2 (R&D, 623 / AN-CF) standard.

[0213] ANG2 receptor competition assay—nunc MaxiSorp flat-bottom plates (Invitrogen, 44-2404-21) were coated with 100 μl of 1.0 μg / ml recombinant human Tie-2 Fc chimeric protein (R&D, 313-TI / CF) in PBS, sealed with adhesive tape, and placed at 4°C overnight. The competition mix was prepared using a dilution of medium from transfected HEK293T cells with an equal volume of 80 ng / ml N-terminal FLAG-tagged human angiopoietin-2 (Adipogen, AG-40B-0114-C010) (final ANG2-Flag concentration = 80 ng / ml). The following day, the coating solution was aspirated, and the plates were washed three times with 300 μl of PBST. The plates were blocked with 200 μl of PBS / 2.0% BSA for 2 hours at room temperature. After 2 hours of incubation, the blocking solution was aspirated and the plate was washed three times with 300 μl of PBST. 100 μl of the competition mix sample was added to the plate along with freshly prepared ANG2-Flag standards and incubated at room temperature for 2 hours with gentle shaking. The plate was aspirated and washed three times with 300 μl of PBST. 100 μl of DYKDDDDK (SEQ ID NO: 71) epitope tag horseradish peroxidase-conjugated antibody (R&D HAM85291 1:10000) was added to the plate and incubated at room temperature for 1 hour with gentle shaking. The plate was washed three times with 300 μl of PBST and developed with 100 μl of TMB ELISA substrate (Abcam, ab171522) for 5-15 minutes at room temperature. The TMB reaction was stopped with 100 μl of 450 nm stop solution for TMB substrate (Abcam, ab171529). Plates were read at 450 nm and 540 nm (as a reference blank for the plate) using a Cytation 5 device (BioTek), and the concentration of ANG2-Flag was interpolated from the ANG2-Flag standard.

[0214] Figure 8A illustrates the expression of protein products of the expected size for anti-Ang-2 HL and HL Fab. Figure 8B illustrates that the expressed anti-Ang-2 Fab is functional (binding of the expressed protein to an Ang-2-coated plate is shown). Figure 8C illustrates that the expressed anti-Ang-2 Fab blocks Ang-2 binding to an antibody-coated plate (competitive ELISA). Figure 8D illustrates that the expressed anti-Ang-2 Fab blocks Ang-2 binding to Tie-2 (receptor competition assay).

[0215] Dose-dependent expression of anti-Ang-2 Fab (LH and HL formats) from the dual protein construct was demonstrated in transfected HEK cells. The expressed anti-Ang-2 Fab protein was of the correct size and was functional in binding to Ang2 and blocking Ang2 binding to its receptor.

[0216] Studies were then conducted to characterize the expression of aflibercept and anti-Ang-2 Fab following transduction of human retinal pigment epithelial (RPE) cells with recombinant AAV viruses containing the capsid protein of SEQ ID NO: 48 and nucleic acids encoding aflibercept and anti-Ang-2 Fab (LH or HL).

[0217] Figure 9 illustrates the expression of a single protein of the correct size (53 kD) for the anti-Ang-2 scFab (LH and HL), along with similar expression levels of the LH and HL conformations. Briefly, RPE cells were transduced in 96-well plates at MOIs of 5,000 and 1,000 with rAAV containing the capsid protein of SEQ ID NO: 48 and nucleic acids encoding aflibercept and the anti-Ang-2 scFab. The medium (0.2 ml) was replaced on day 3 and collected on day 7 after transduction. Twenty-five ml of medium from the 5,000 MOI sample was run on a 4-12% SDS-PAGE, transferred to nitrocellulose using iBlot, and probed on an iBind Flex to detect anti-Ang-2 scFab using an HRP-conjugated anti-Hu-Fab antibody and aflibercept using an anti-Hu-Fc antibody.

[0218] Figure 10A illustrates the dose-dependent expression of functional anti-Ang-2 scFab in RPE cells transduced at MOIs of 1,000 and 5,000. Briefly, dilutions of medium (day 11) from transduced RPE cells were incubated on Ang-2-coated plates. Ang-2-bound anti-Ang-2 scFab was detected using an HRP-conjugated anti-Hu-Fab antibody. The LH format was slightly less effective at binding to Ang2 than the HL format, especially at lower MOIs.

[0219] Figure 10B illustrates the results of a competition ELISA using day 11 samples of transduced RPE. Briefly, 1000 pg / ml of Ang2 protein was incubated overnight with serial dilutions of medium from transduced RPE cells. The competition mixtures were assayed by Ang-2 ELISA (Invitrogen KHC1641).

[0220] The binding affinities of anti-Ang-2 scFabs LH and HL were then compared via SPR by Biacore assay performed at Genscript.

[0221] Immobilization of Angiopoietin-2 onto a CM5 sensor chip. Angiopoietin-2 immobilization was performed at 25°C, with HBS-EP+ used as the running buffer. The sensor chip surface of flow cells 1 and 2 was activated with freshly mixed 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) for 420 seconds (10 μL / min). Angiopoietin-2 diluted in 10 mmol / L NaAC (pH 4.5) was then injected into flow cell 2, achieving conjugation of 243.1 response units. After the amine coupling reaction, remaining active coupling sites on the chip surface were blocked by a 420-second injection of 1 mol / L ethanolamine hydrochloride. For affinity measurements, the assay was performed at 25°C, with HBS-EP+ as the running buffer. Diluted V2.2 / V2.3 was injected over the surfaces of flow cells 1 and 2 as the association phase, followed by running buffer as the dissociation phase. The running configurations are listed below (sample concentrations (nM) = 1.5625, 3.125, 6.25, 12.5, 25, 50, 100). [Table 6]

[0222] The results are provided in Figure 11. The LH configuration (V2.3) had a 5.6-fold faster association (higher k value) than the HL configuration (V2.2), suggesting stronger binding. The LH configuration had a 91-fold faster dissociation (higher k value) than HL, suggesting weaker binding. The HL configuration had an overall binding affinity of 1.46 nM, and LH had a binding affinity of 23.8 nM. Thus, the LH configuration has approximately 16-fold higher Ang-2 binding affinity than the HL configuration. These values are consistent with the published affinity for the anti-Ang2 arm of faricimab.

[0223] Next, PEDF expression in HEK293T cells was assessed after transfection with an AAV plasmid dual protein construct encoding aflibercept and PEDF. Briefly, HEK293T cells were plated in 12-well plates at 2.0 × 10 in 1.0 ml of DMEM / 10% FBS medium. 5 Cells were seeded at 1000 ng / well. HEK293T cells were used due to their high transfectability and protein expression. The following day, 1.0 mg of plasmid DNA complexed with 3.0 ml of FuGene6 (catalog no. E2691, Promega, Madison, WI) was added to the cells in duplicate wells. Two days after transfection, the supernatant was collected. Cell debris was pelleted by centrifugation in a microcentrifuge at 12,000 g for 10 minutes at 4°C. The supernatant was collected and stored at 4°C. A condition without plasmid was included in the transfection as a negative control.

[0224] For SDS-PAGE and Western blot, PEDF samples were diluted 1:10 before performing the SDS-PAGE / Western blot assay. Aflibercept samples were diluted 1:50. The diluted media was combined with 4x sample buffer and 10x reducing buffer according to the ThermoFisher iBlot system. Samples were then boiled at 90°C for 5 minutes. A 4-12% Bis / Tris reducing gel was run in 1x MOPS buffer at 125V for 1.5 hours. Proteins were transferred to nitrocellulose using a BioRad Trans Blot Turbo System with the preset MIXED protocol for 7 minutes. The membrane was blocked in iBind Flex buffer for 1-2 minutes and then loaded into an iBind Flex Western Device according to the manufacturer's instructions. Primary antibodies (anti-PEDF EMD Millipore, 1:1000, anti-human FC HRP ThermoFisher 1:2000) were used together with species-specific secondary antibodies conjugated to HRP (1:10000), detected with Femto ECL substrate, and imaged on a BioRad ChemiDoc system.

[0225] ELISA (R&D, Human Serpin F1 / PEDF ELISA Cat. No. DY1177) was performed according to the manufacturer's instructions. Media was diluted 1:1000 or 1:10000 before running the assay.

[0226] Figures 12A-B illustrate the expression of a single protein product of the expected size of PEDF from transfected HEK293T cells, with production of approximately 17 mg / ml of PEDF.

[0227] VEGFR3-Fc expression in HEK293T cells was assessed after transfection with an AAV plasmid dual protein construct encoding aflibercept and VEGFR3-Fc. Briefly, HEK293T cells were plated in 12-well plates at 2.0 x 10 ng / well in 1.0 ml of DMEM / 10% FBS medium. 5Cells were seeded at 1000 ng / well. HEK293T cells were used due to their high transfectability and protein expression. The following day, 1.0 mg of plasmid DNA complexed with 3.0 ml of FuGene6 (catalog no. E2691, Promega, Madison, WI) was added to the cells in duplicate wells. Two days after transfection, the supernatant was collected. Cell debris was pelleted by centrifugation in a microcentrifuge at 12,000 g for 10 minutes at 4°C. The supernatant was collected and stored at 4°C. A condition without plasmid was included in the transfection as a negative control.

[0228] For reductive Western blotting, cell broth (20 ml) was mixed with 10 ml of 4x LDS, 4 ml of 10x reducing agent, and 6 ml of water (final volume = 40 ml) and denatured at 70°C for 10 min. Samples were loaded onto a 12-well Bolt 4-12% Bis-Tris Plus polyacrylamide gel (Invitrogen, NW04122BOX) and run at 100 V for 75 min in 1x MOPS buffer. Separated proteins were transferred to nitrocellulose filters using an iBlot 2 device (ThermoFisher) for 10 min and probed with HRP-conjugated goat anti-human IgG Fc (ThermoFisher Scientific, catalog no. 31413, 1:2000) using an iBind Flex device (ThermoFisher). Proteins were visualized using SuperSignal West Dura chemiluminescent substrate (ThermoFisher 34076) and imaged on a ChemiDoc MP (BioRad, Hercules, CA).

[0229] For non-reducing Western blotting, cell supernatant (30 ml) was mixed with 10 ml of 4x LDS (final volume = 40 ml) and denatured at 70°C for 10 min. Samples were loaded onto 12-well Bolt 4-12% Bis-Tris Plus polyacrylamide gels (Invitrogen, NW04122BOX) and run at 100 V for 120 min in 1x MOPS buffer. Separated proteins were transferred to nitrocellulose filters using an iBlot 2 device (ThermoFisher) for 10 min and probed with HRP-conjugated goat anti-human IgG Fc (ThermoFisher Scientific, catalog no. 31413, 1:2000) using an iBind Flex device (ThermoFisher). Proteins were visualized using SuperSignal West Dura chemiluminescent substrate (ThermoFisher 34076) and imaged on a ChemiDoc MP (BioRad, Hercules, CA).

[0230] Figure 13A compares the expression of aflibercept after transfection of HEK293T cells with dual-protein AAV constructs also encoding PEDF, VEGFR3-Fc, anti-Ang-2 HL, or anti-Ang-2 LH. Figure 13B illustrates the dramatic reduction in aflibercept when co-expressed with VEGFR3-Fc due to the formation of VEGFR3-Fc / aflibercept heterodimers.

[0231] Example 3 Characterization of constructs encoding aflibercept+RNAi The general strategy used was to: (i) design shRNAs against human targets; (ii) select shRNA sequences based on (a) knockdown of endogenous expression of the target using lentivirus and (b) homology to NHP sequences; and (iii) embed miR-E containing sequences from the selected shRNAs within the intron of CAG in anti-VEGF expression plasmids or in combination with RFP to assess RNAi function alone.

[0232] Both Ang2 and VEGFR3 are expressed by endothelial cells - transduction of endothelial cells by rAAV containing the capsid protein of SEQ ID NO:48 was confirmed.

[0233] Characterization of constructs containing RNAi targeting Ang-2

[0234] Construction of pAAV-CAG-miR-E(Ang-2)-AFLB-SV40 (see Figure 14; constructs pP143.001 encode RFP675, and pP141.001 encodes aflibercept but are otherwise identical; pP143.001 is an miRNA-only efficacy control for in vitro studies). A pAAV-CAG-AFLB-SV40 construct expressing human codon-optimized aflibercept VEGF-A / B Trap was synthesized. A miR-E-(ANG-2) miRNA transgene containing a region of the CAG beta-actin intron encoded between the SgrAI and NheI restriction cloning sites was synthesized and cloned into pUC57 using Genscript (Genscript, Picataway, NJ). The pUC57 and pAAV-CAG-AFLB-SV40-Kan-Stuffer plasmids were digested with various restriction enzymes (New England Biolabs) as indicated. The backbone DNA was also treated with recombinant shrimp alkaline phosphatase (rSAP, M0371L, New England Biolabs) during digestion to remove free phosphates on the cleaved DNA ends. The DNA fragments were added at a 7:1 molar ratio of insert to backbone and ligated using Quick Ligase (#M2200L, New England Biolabs) according to the manufacturer's instructions. The ligated plasmids were transformed into NEB Stable bacterial competent cells (#C3040H, New England Biolabs) according to the manufacturer's instructions, and the cells were spread on 50 mg / ml kanamycin plates (#L1025, Teknova, Hollister, CA) and grown at 30°C.

[0235] Miniprep cultures were grown from the resulting colonies (7.1), and DNA was prepared using the GeneJET Plasmid Miniprep Kit (Cat. No. 0503, ThermoFisher, Waltham, MA) and restriction digested to identify positive clones. A 50 ml culture in Terrific Broth was grown from one positive clone, and DNA was prepared using the Qiagen EndoFree Plasmid Maxi Kit (Cat. No. 12362, Qiagen, Hilden, Germany). Maxiprep plasmid DNA (0.5 mg) was digested with various restriction enzymes (New England BioLabs) according to the manufacturer's instructions and analyzed by agarose gel electrophoresis. Sanger DNA sequencing was performed by ELIM using primers.

[0236] Several shRNA sequences targeting Ang-2 (five sequences listed in Table 1) were evaluated for their ability to reduce Ang-2 expression in HUVEC cells. Briefly, pooled human umbilical vein endothelial cells were procured from Lonza (catalog number C2519A) and cultured in Lonza endothelial cell growth medium (EGM-2, catalog number CC-3162) according to the manufacturer's instructions. HUVECs were passaged using PBS (without Ca and Mg), 0.05% trypsin, and defined trypsin inhibitors. HUVECs were placed in plastic (uncoated) cell cultureware, and placed on 1 cm 2 Cells were seeded at a density of 2,500 cells per well. Assays were typically performed in 24-well cell culture plates, with 0.5 mL of EGM-2 refreshed every other day. HUVECs were typically used only before passage 8, at which point new cultures were initiated.

[0237] HUVEC transduction - HUVECs were transduced into 1cm 2Cells were seeded at 2,500 cells per well. After two days, cells were confluent, and single wells were dissociated and counted. Multiplicity of infection was calculated using qPCR-derived viral titers and cell counts. An appropriate volume of AAV was applied to cells in 0.5 mL of EGM-2 medium. This was incubated for 48 hours, and final assay dissection was performed one week after transduction.

[0238] Generation of ANG2 shRNA lentiviral system - The pLKO.1-shANG2 plasmid was generated by ligation of annealed phosphorylated oligos corresponding to five unique target sequences in human ANG2 identified from the Broad RNAi Consortium (Table 1) into the pLKO.1 vector (Sigma Aldrich, catalog no. SCH001) via EcoRI and AgeI (New England Biolabs) restriction cloning. Plasmids were verified by sequencing, similar to AAV vectors. Maxi prep DNA was generated similar to AAV vectors. HEK293T cells were cultured in 6-well plates at 5.0 x 10 in 2.0 ml of DMEM / 10% FBS medium. 5Cells were seeded at 1000 x g / well. The following day, 0.5 μg of pSF-GFP plasmid DNA complexed with 2.7 μl of FuGene6 (catalog number E2691, Promega, Madison, WI) and 4.6 μL of MISSION lentiviral packaging mix (Sigma Aldrich, catalog number SHP001) were added to the cells. The following day, the medium was replaced with 2 mL of fresh medium. The day after that, the medium containing the lentivirus was collected and replaced with fresh medium on the cells. The supernatant was collected, filtered with a 0.45 μm syringe filter (Millipore Sigma, catalog number SLHVM33RS), aliquoted, and stored at -80°C. The lentivirus was titrated using the Lenti-X™ qRT-PCR Titration Kit (Takara, catalog number 631235) according to the qPCR manufacturer's instructions. Human umbilical vein endothelial cells (HUVECs) (#PCS-100-013, ATCC) were plated in 6-well plates at 2.0 × 10 in 2 ml of complete EGM-2 medium (CC-3162, Lonza). 5 Immediately after plating, cells were transduced with lentivirus at a multiplicity of infection (MOI) of 25 viral genomes per cell. After 48 hours, the medium containing lentivirus was removed and replaced with fresh medium containing 1.5 μg / mL puromycin (10 mg / mL stock solution, Sigma-Aldrich, Cat. No. P9620-10 ml). After 72 hours of puromycin selection, the medium containing dead, uninfected cells was removed. Cells expressing shRNA were continuously cultured in 1.0 μg / mL puromycin in all experiments to maintain shRNA expression. The medium and lysates of infected HUVECs were analyzed by ELISA for ANGPT2.

[0239] Figures 15A-B illustrate the percentage of Ang-2 (secreted and cellular) in HUVEC cells after transduction with lentivirus containing each of the candidate shRNAs. All constructs performed well (>50% KD for secreted Ang-2), with shRNA #5 showing the greatest reduction in Ang-2 protein from HUVEC. Notably, shRNAs #1, #4, and #5 are perfect matches in non-human primates. shRNA #5 was selected for inclusion in an AAV plasmid construct encoding aflibercept and an miRNA targeting Ang-2. Ang-2 shRNAs #1-5 contain the following sense and antisense strand sequences: [Table 7]

[0240] Next, human retinal microvascular endothelial cells were transduced with an rAAV containing the capsid protein of SEQ ID NO: 48 and a nucleic acid encoding a miRNA containing the sense and antisense strands of shRNA #5 (the sense and antisense strands of shRNA #5 were embedded within mir-E, and the miRNA was placed within a hybrid intron of the CAG promoter). An rAAV containing the capsid protein of SEQ ID NO: 48 and a nucleic acid encoding GFP under the control of the CAG promoter was used as a control.

[0241] Briefly, human retinal microvascular endothelial cells were purchased from Cell Systems (catalog number ACBRI 181) along with "The System" (catalog number CSS-A101) containing medium, coating matrix, and passaging reagents. All passaging, cryopreservation, and cell thawing were performed according to the manufacturer's instructions. Cultures were vialed by Cell Systems at passage 3, and upon receipt, the vials were expanded and cryopreserved as banks. Experiments were only performed on cultures prior to passage 9. Experiments were typically performed in 24-well plates and passaged at a 1:3 ratio (1 cm 2The medium volume was 1 mL per well and the medium was replenished every other day until passaging.

[0242] For RMVEC transduction, RMVECs were plated at 1E+4 cells / cm in 24-well cell culture plates coated with attachment factors. 2 Cells were seeded at a density of 1000 x g / ml, sufficient to reach confluence 3 days after seeding. AAV carrying a CAG-GFP payload was added to the cells at an MOI calculated by cell counting and qPCR-derived viral titer at the time of transduction for 48 hours. The transduction volume was the same as the standard culture volume (1 ml per well of a 24-well plate). After transduction, medium was replenished every other day thereafter until the final readout 7 days after transduction.

[0243] ELISA for Secreted Free AFLB, ANGPT2, and VEGF-A Levels—Cell lysates for ELISA were prepared in M-PER lysis buffer (#78501, Thermo) supplemented with 1x Halt protease and phosphatase inhibitor cocktail (78440, Thermo) according to the manufacturer's instructions. Cell media and lysates were diluted appropriately for each sample and used to assess secreted analyte levels using an aflibercept ELISA kit (to measure free AFLB levels) (catalog number IG-AA115, Eagle Biosciences, Nashua, NH), a Quantikine human VEGF-A ELISA kit (DVE00, R&D Systems), and a Quantikine human ANGPT2 ELISA kit (DANG20, R&D Systems) according to the supplier's instructions. Optical density (OD) was measured within 15 minutes of pipetting the stop solution using a Cytation 3 (BioTek, Winooski, VT) photometer at 450 nm (OD 620 nm reference). Media concentrations were defined based on the generated standard curve.

[0244] RT-qPCR of mature miRNAs and targets from transduced cells. Cells were lysed in RLT on the plate, and total RNA containing miRNAs was purified using the Qiagen RNeasy kit (#74104, Qiagen) according to the manufacturer's instructions, with modifications for miRNA isolation suggested in the manufacturer's supplemental protocol. Briefly, the RLT lysate was filtered through a gDNA removal column, after which 1.5 volumes of 100% ethanol was added to the lysate. After loading onto the RNeasy mini column, the RW1 wash step was skipped and the lysate proceeded directly to the wash with buffer RPE. Total cDNA was generated from 100 ng of total RNA using the Maxima RT with dsDNA kit (#M1681, Thermo-Fisher) according to the manufacturer's instructions. qPCR was performed using TaqMan Fast Advanced Mastermix (#4444963, Thermo-Fisher) and a predesigned TaqMan probe set targeting ANGPT2 (Hs00169867_m1, Thermo), our human-optimized AFLB (AR7DTHZ, Thermo), and custom TaqMan assays for RPL32 (Hs07291819_s1, Thermo) as a housekeeping control for normalization. Measured levels of VEGF-C and AFLB were normalized to RPL32 expression and expressed as a function of percent reduction from untreated or vehicle-treated controls. miRNA-specific cDNA was generated using 10 ng of total RNA and a custom RT primer (CTU6249, Thermo) provided with the ANG2 custom miRNA Taqman assay, targeting the FL mature miRNA guide sequence: 5'-AAUGUUCAUACAAUGAGUAAGC-3' (SEQ ID NO: 72), using the TaqMan miRNA RT Kit (#4366596, Thermo) according to the manufacturer's instructions. qPCR was performed using TaqMan Fast Advanced Mastermix (#4444963, Thermo-Fisher) and a custom TaqMan probe set targeting ANG2 (CTU6249, Thermo).A standard curve was generated from a custom miRvana miRNA mimic for ANG2 (AKS063L, Thermo) with inputs of miRNA mimic RT product ranging from 1e9 to 1e2 copies per reaction. MiRNA concentrations were calculated from the generated standard curve.

[0245] Figure 16A illustrates the results of Ang-2 ELISA, showing a reduction in secreted and cellular Ang-2 protein (endogenously produced by RMVEC cells) by expression of miRNA targeting Ang-2 in transduced RMVEC cells. A significant reduction in Ang-2 secretion is observed at an MOI of 100K; a trend toward reduced secretion is also observed at an MOI of 10K. A marked reduction in Ang-2 was observed in cellular samples. Figure 16B illustrates a significant reduction in Ang-2 mRNA expression at an MOI of 100K in transduced RMVEC cells by Ang-2 qPCR.

[0246] Next, Ang-2 levels were evaluated in human RPE cells after transduction with rAAV (same as above for RMVEC cells). Figures 17A-B illustrate Ang-2 levels in RPE cells at day 8 (post-transduction) by RT-qPCR (Figure 17A - mRNA levels) and ELISA (Figure 17B - protein levels). A strong decrease in Ang-2 RNA levels is observed, and the KD increases with increasing MOI. Notably, Ang-2 protein levels in RPE are extremely low; however, a trend toward increased KD of Ang-2 protein is observed at all MOIs compared to cells transduced with the GFP control, with a trend toward higher KD with increasing MOI. Significance is observed at matched MOIs.

[0247] Next, the effect of including the RNAi of targeting Ang-2 or VEGF-C in the double construct on aflibercept expression was evaluated in RMVEC cells.Briefly, RMVEC cells were transduced with the rAAV that comprises the capsid protein of SEQ ID NO: 48 and (i) the nucleic acid that codes AFLB only (CAG-AFLB), (ii) the nucleic acid that codes AFLB and the miRNA that targets Ang-2 (CAG-AFLB-ANG2-RNAi), or (iii) the nucleic acid that codes AFLB and the miRNA that targets VEGF-C (CAG-AFLB-VEGFC-RNAi).

[0248] RMVECs were plated at 1E+4 cells / cm in attachment factor-coated 24-well cell culture plates. 2 Cells were seeded at a density of 1000 x g / ml, sufficient to reach confluence 3 days after seeding. AAV carrying a CAG-GFP payload was added to the cells at an MOI calculated by cell counting and qPCR-derived viral titer at the time of transduction for 48 hours. The transduction volume was the same as the standard culture volume (1 ml per well of a 24-well plate). After transduction, medium was replenished every other day thereafter until the final readout 7 days after transduction.

[0249] RT-qPCR of mature miRNAs and targets from transduced cells. Cells were lysed in RLT on the plate, and total RNA containing miRNAs was purified using the Qiagen RNeasy kit (#74104, Qiagen) according to the manufacturer's instructions, with modifications for miRNA isolation suggested in the manufacturer's supplemental protocol. Briefly, the RLT lysate was filtered through a gDNA removal column, after which 1.5 volumes of 100% ethanol was added to the lysate. After loading onto the RNeasy mini column, the RW1 wash step was skipped and the lysate proceeded directly to the wash with buffer RPE. Total cDNA was generated from 100 ng of total RNA using the Maxima RT with dsDNA kit (#M1681, Thermo-Fisher) according to the manufacturer's instructions. qPCR was performed using TaqMan Fast Advanced Mastermix (#4444963, Thermo-Fisher) and a predesigned TaqMan probe set targeting ANGPT2 (Hs00169867_m1, Thermo), our human-optimized AFLB (AR7DTHZ, Thermo), and custom TaqMan assays for RPL32 (Hs07291819_s1, Thermo) as a housekeeping control for normalization. Measured levels of VEGF-C and AFLB were normalized to RPL32 expression and expressed as a function of percent reduction from untreated or vehicle-treated controls. miRNA-specific cDNA was generated using 10 ng of total RNA and a custom RT primer (CTU6249, Thermo) provided with the ANG2 custom miRNA Taqman assay, targeting the FL mature miRNA guide sequence: 5'-AAUGUUCAUACAAUGAGUAAGC-3' (SEQ ID NO: 72), using the TaqMan miRNA RT Kit (#4366596, Thermo) according to the manufacturer's instructions. qPCR was performed using TaqMan Fast Advanced Mastermix (#4444963, Thermo-Fisher) and a custom TaqMan probe set targeting ANG2 (CTU6249, Thermo).A standard curve was generated from a custom miRvana miRNA mimic for ANG2 (AKS063L, Thermo) with inputs of miRNA mimic RT product ranging from 1e9 to 1e2 copies per reaction. MiRNA concentrations were calculated from the generated standard curve.

[0250] ELISA for secreted free AFLB, ANGPT2, and VEGF-A levels Cell lysates for ELISA were prepared in M-PER lysis buffer (#78501, Thermo) supplemented with 1x Halt protease and phosphatase inhibitor cocktail (#78440, Thermo) according to the manufacturer's instructions. Cell media and lysates were diluted appropriately for each sample and used to assess secreted analyte levels using an aflibercept ELISA kit (to measure free AFLB levels) (catalog number IG-AA115, Eagle Biosciences, Nashua, NH), a Quantikine human VEGF-A ELISA kit (DVE00, R&D Systems), and a Quantikine human ANGPT2 ELISA kit (DANG20, R&D Systems) according to the supplier's instructions. Optical density (OD) was measured within 15 minutes of pipetting the stop solution using a Cytation 3 (BioTek, Winooski, VT) photometer at 450 nm (OD 620 nm is the reference). Media concentrations were defined based on the generated standard curve.

[0251] Aflibercept expression was evaluated on day 8. Figure 18A illustrates the increase in free aflibercept levels after transduction with miRNA constructs in both ANG2 and VEGF-C RNAi compared to transduction with CAG-AFLB control. Figure 19B illustrates the corresponding dose-dependent increase in aflibercept miRNA.

[0252] Ang-2 expression was assessed on day 8. Figure 19A illustrates a significant reduction in Ang-2 protein levels with all aflibercept-encoding constructs, and at 100k MOI, a further reduction in Ang-2 was observed with miRNAs targeting Ang-2 compared to other constructs. Figure 19B illustrates the dose-response relationship for the reduction in Ang-2 transcripts by RNAi targeting Ang-2. Aflibercept alone does not appear to affect Ang-2 transcriptionally, but may prevent VEGF-A-stimulated secretion, resulting in the observed reduction in the supernatant (Figure 19A). A dose-dependent increase in anti-Ang-2 miRNA (as a function of MOI) is observed (Figure 19C).

[0253] Characterization of VEGF miRNA-containing vectors The AAV plasmid pP145.001 (pAAV-CAG-miR-E-(VEGFC)-AFLB-SV40) (see Figure 2) was constructed as follows.

[0254] A pAAV-CAG-AFLB-SV40 construct expressing human codon-optimized aflibercept VEGF-A / B Trap was synthesized. The miR-E-(VEGF-C) miRNA transgene, containing a region of the CAG beta-actin intron encoded between the SgrAI and NheI restriction cloning sites, was synthesized and cloned into pUC57 using Genscript (Genscript, Picataway, NJ). The pUC57 and pAAV-CAG-AFLB-SV40-Kan-Stuffer plasmids were digested with various restriction enzymes (New England Biolabs) as indicated, and the backbone DNA was also treated with recombinant shrimp alkaline phosphatase (rSAP, M0371L, New England Biolabs) during digestion to remove free phosphates on the cleaved DNA ends. The DNA fragments were added at a 7:1 molar ratio of insert to backbone and ligated using Quick Ligase (#M2200L, New England Biolabs) according to the manufacturer's instructions. The ligated plasmid was transformed into NEB Stable bacterial competent cells (#C3040H, New England Biolabs) according to the manufacturer's instructions, and the cells were spread on kanamycin 50 mg / ml plates (#L1025, Teknova, Hollister, CA) and grown at 30°C.

[0255] Miniprep cultures were grown from the resulting colonies (7.1), and DNA was prepared using the GeneJET Plasmid Miniprep Kit (Cat. No. 0503, ThermoFisher, Waltham, MA) and restriction digested to identify positive clones. A 50 ml culture in Terrific Broth was grown from one positive clone, and DNA was prepared using the Qiagen EndoFree Plasmid Maxi Kit (Cat. No. 12362, Qiagen, Hilden, Germany).

[0256] Maxiprep plasmid DNA (0.5 mg) was digested with various restriction enzymes (New England BioLabs) according to the manufacturer's instructions and analyzed by agarose gel electrophoresis. Sanger DNA sequencing was performed by ELIM using primers.

[0257] Several shRNA sequences targeting VEGF-C were evaluated for their ability to reduce the expression of VEGF-C in HEK293T cells.

[0258] Generation of VEGF-C shRNA lentiviral system - The pLKO.1-shVEGFC plasmid was generated by ligation of annealed phosphorylated oligos corresponding to five unique target sequences in human VEGF-C identified from the Broad RNAi Consortium (Table 1) into the pLKO.1 vector (Sigma Aldrich, catalog no. SCH001) via EcoRI and AgeI (New England Biolabs) restriction cloning. Plasmids were verified by sequencing, similar to AAV vectors. Maxi prep DNA was generated similar to AAV vectors. HEK293T cells were seeded in 6-well plates at 5.0 x 10^5 cells / well in 2.0 ml of DMEM / 10% FBS medium. The following day, 0.5 μg of pSF-GFP plasmid DNA complexed with 2.7 μl of FuGene6 (catalog number E2691, Promega, Madison, WI) and 4.6 μL of MISSION lentiviral packaging mix (Sigma Aldrich, catalog number SHP001) were added to the cells. The following day, the medium was replaced with 2 mL of fresh medium. The day after that, the medium containing the lentivirus was collected and replaced with fresh medium on the cells. The supernatant was collected, filtered with a 0.45 μm syringe filter (Millipore Sigma, catalog number SLHVM33RS), aliquoted, and stored at -80°C. The lentivirus was titrated using a Lenti-X™ qRT-PCR titration kit (Takara, catalog number 631235) according to the qPCR manufacturer's instructions. MCF7 cells (#HTB-22, ATCC) were plated in 6-well plates at 5.0 × 10 cells / well in 2 ml of EMEM / 10% FBS medium supplemented with 0.01 mg / mL human recombinant insulin (#I9278-5ML, Sigma Aldrich). 5Immediately after plating, cells were transduced with lentivirus at a multiplicity of infection (MOI) of 25 viral genomes per cell. After 48 hours, the medium containing lentivirus was removed and replaced with fresh medium containing 0.75 μg / mL puromycin (10 mg / mL stock solution, Sigma-Aldrich, Cat. No. P9620-10 ml). After 72 hours of puromycin selection, the medium containing dead, uninfected cells was removed. Cells expressing shRNA were continuously cultured in 0.5 μg / mL puromycin in all experiments to maintain shRNA expression. Cells cultured with puromycin were lysed in RLT, and total RNA was purified using the Qiagen RNEasy kit according to the manufacturer's instructions.

[0259] qPCR analysis of MCF7 VEGF-C knockdown. Cells cultured with puromycin were lysed in RLT on the plate, and total RNA was purified using the Qiagen RNeasy kit (#74104, Qiagen) according to the manufacturer's instructions. Total cDNA was generated from 5 μg of total RNA using the Maxima RT with dsDNA kit (#M1681, Thermo-Fisher) according to the manufacturer's instructions. qPCR was performed using TaqMan Fast Advanced Mastermix (#4444963, Thermo-Fisher) and a predesigned TaqMan probe set targeting VEGF-C (Hs01099203_m1, Thermo) with RPL32 (Hs07291819_s1, Thermo) as a housekeeping control for normalization. Measured levels of VEGF-C were normalized to RPL32 expression and expressed as a function of percent reduction from non-targeting shRNA.

[0260] RT-qPCR of mature miRNAs and targets from transduced cells. Cells were lysed in RLT on the plate, and total RNA containing miRNAs was purified using the Qiagen RNeasy kit (#74104, Qiagen) according to the manufacturer's instructions, with modifications for miRNA isolation suggested in the manufacturer's supplemental protocol. Briefly, the RLT lysate was filtered through a gDNA removal column, after which 1.5 volumes of 100% ethanol was added to the lysate. After loading onto the RNeasy mini column, the RW1 wash step was skipped and the lysate proceeded directly to the wash with buffer RPE. Total cDNA was generated from 100 ng of total RNA using the Maxima RT with dsDNA kit (#M1681, Thermo-Fisher) according to the manufacturer's instructions. qPCR was performed using TaqMan Fast Advanced Mastermix (#4444963, Thermo-Fisher) and a predesigned TaqMan probe set targeting VEGF-C (Hs01099203_m1, Thermo), our custom TaqMan assay for human-optimized AFLB (AR7DTHZ, Thermo), and RPL32 (Hs07291819_s1, Thermo) as a housekeeping control for normalization. Measured levels of VEGF-C and AFLB were normalized to RPL32 expression and expressed as a function of percent reduction from untreated or vehicle-treated controls. MiRNA-specific cDNA was produced using a TaqMan miRNA RT kit (#4366596, Thermo) according to the manufacturer's instructions, using 10 ng of total RNA and a custom RT primer (CTTZ9KC, Thermo) provided with the VEGF-C custom miRNA Taqman assay, targeting the FL mature miRNA guide sequence: 5'-AAUAACGUCUUGCUGAGGUAGC-3' (SEQ ID NO: 73).qPCR was performed using TaqMan Fast Advanced Mastermix (#4444963, Thermo-Fisher) and a custom TaqMan probe set targeting VEGF-C (CTTZ9KC, Thermo). A standard curve was generated from a custom miRvana miRNA mimic of VEGF-C (AKT949T, Thermo) with inputs of miRNA mimic RT product ranging from 1e9 to 1e2 copies per reaction. miRNA concentrations were calculated from the generated standard curve.

[0261] Figure 20 illustrates the percentage of VEGF-C RNA levels (normalized to RPL32) in HEK293T cells after transduction with shRNA. Potent knockdown of VEGF-C was observed in all constructs, with shRNA #2 resulting in >90% knockdown of VEGF-C. shRNA #2 was selected for inclusion in an AAV plasmid construct encoding aflibercept and an miRNA targeting VEGF-C. VEGF-C shRNAs #1-5 contain the following sense and antisense strand sequences: [Table 8]

[0262] Next, human RPE cells were transduced with an rAAV containing the capsid protein of SEQ ID NO: 48 and a nucleic acid encoding a miRNA containing the sense and antisense strands of shRNA #2 (the sense and antisense strands of shRNA #2 were embedded within mir-E, and the miRNA was placed within a hybrid intron of the CAG promoter.) An rAAV containing the capsid protein of SEQ ID NO: 48 and a nucleic acid encoding GFP under the control of the CAG promoter was used as a control.

[0263] RPE transduction - Human stem cell-derived retinal pigment epithelial cells (RPE) were differentiated from embryonic stem cells (ESI-017) according to published protocols (Buchholz D 2013, Leach L 2015). RPE cells were grown on Matrigel (Corning) in XVIVO-10 medium (Lonza) in a 96-well plate format for 30 days. Prior to transduction, three wells were harvested and counted for accurate calculation of the multiplicity of infection (MOI). Virus was added to cells in XVIVO10 medium for 48 hours based on the respective viral titer in a total volume of 100 μL per well. Medium was collected on days 3, 7, 11, 15, and 19 and replaced with 200 μL of medium per well. Medium samples were stored at 4°C until processing.

[0264] ELISA for Secreted Free AFLB, ANGPT2, and VEGF-A Levels—Cell lysates for ELISA were prepared in M-PER lysis buffer (#78501, Thermo) supplemented with 1x Halt protease and phosphatase inhibitor cocktail (78440, Thermo) according to the manufacturer's instructions. Cell media and lysates were diluted appropriately for each sample and used to assess secreted analyte levels using an aflibercept ELISA kit (to measure free AFLB levels) (catalog number IG-AA115, Eagle Biosciences, Nashua, NH), a Quantikine human VEGF-A ELISA kit (DVE00, R&D Systems), and a Quantikine human ANGPT2 ELISA kit (DANG20, R&D Systems) according to the supplier's instructions. Optical density (OD) was measured within 15 minutes of pipetting the stop solution using a Cytation 3 (BioTek, Winooski, VT) photometer at 450 nm (OD 620 nm reference). Media concentrations were defined based on the generated standard curve.

[0265] Figure 21A illustrates VEGF-C levels (by ELISA) in RPE cells after transduction with rAAV carrying the indicated constructs. A dose-dependent decrease in VEGF-C in the supernatant of RPE cells (which make VEGF-C endogenously) is observed, specific to the VEGF-C miRNA construct. A slight decrease in VEGF-C with the other constructs is observed at the highest MOI. The miRNAs targeting VEGF-C are functional.

[0266] FIG. 21B illustrates the corresponding dose-dependent decrease in VEGF-C mRNA specific to the VEGF-C construct (MOIs of 1.6, 1000, and 5000).

[0267] Figure 21C illustrates a dose-dependent increase in the expression of miRNAs targeting VEGF-C in RPE cells.

[0268] The ability of dual constructs (aflibercept plus miRNAs encoding VEGF-C or Ang-2) to neutralize VEGF-A in RPE cells was assessed 8 days after transduction with rAAV vectors containing the capsid protein of SEQ ID NO: 48 and nucleic acids comprising the dual constructs ("VEGF-C RNAi-AFLB," "Ang-2 RNAi-AFLB," "AFLB"). Figure 22A illustrates that all constructs expressing AFLB can completely neutralize endogenous VEGF-A at all MOIs tested. Notably, constructs not expressing AFLB ("GFP," "Ang2 RNAi") have no significant effect on VEGF-A levels in the culture medium. Figures 21B-C illustrate that VEGF-C protein (Figure 22B; ELISA) and mRNA levels (Figure 22C; RT-qPCR) were reduced in a dose-dependent manner in RPE cells after transduction with rAAV encoding miRNAs targeting VEGF-C.

[0269] Example 4 Dual RNAi vector A dual RNAi approach (targeting VEGF-C and Ang-2) was investigated. Representative embodiments included pP151.001, pP152.001, and pP153.001 (see Figure 2).

[0270] pP151 contains an Ang-2-targeting miRNA located within an artificial intron in the 3'UTR of the aflibercept coding region and an VEGF-C-targeting miRNA located within a hybrid intron of the CAG promoter. pP152 contains an Ang-2-targeting miRNA located within an artificial intron in the aflibercept coding region and an VEGF-C-targeting miRNA located within a hybrid intron of the CAG promoter. pP153 contains an Ang-2-targeting miRNA and an VEGF-C-targeting miRNA, each located at a different position within the hybrid intron of the CAG promoter.

[0271] rAAV containing a capsid containing SEQ ID NO: 48 transduced 25% or less of total CD31+ cells after intravitreal administration (1 x 10 12 vg / eye) (see Figure 23A - the rAAV contained nucleic acid encoding the GFP reporter gene).

[0272] Example 5 NHP model of angiogenesis A pilot pharmacology study was conducted in non-human primates (NHPs) to (i) evaluate acute ocular safety, (ii) measure expression of aflibercept and intracellular miRs targeting VEGF-C, and (iii) confirm the predominant miRNA species in vivo after intravitreal administration of an rAAV comprising the capsid protein of SEQ ID NO:48 and a nucleic acid comprising nucleotide sequences encoding aflibercept and an miR targeting VEGF-C. The nucleic acid includes sense and antisense strands corresponding to SEQ ID NOs:19 and 20, and the complete construct corresponding to SEQ ID NO:69.

[0273] As shown in Figure 24, ocular chamber and vitreous aflibercept (AFLB) levels were well within / above the range reported for efficacy. Robust retinal AFLB levels were detected. There was no evidence of uveitis or retinal abnormalities during the study.

[0274] As shown in Figure 25, high miRNA copy numbers were detected throughout the entire retina of all eyes of NHPs in the study. Interanimal expression levels are comparable to those observed by AFLB ELISA. MiSeq data confirm the full-length 22-bp targeting VEGF-C as the predominant miRNA species, as shown in transfected HEK293T and transduced RPE cells.

[0275] Next, a proof-of-concept study was initiated to investigate the efficacy of rAAV in an NHP model of angiogenesis. See, e.g., Goody et al., Experimental Eye Research, 92(6):464-472 (2011). Briefly, African green monkey NHPs, n=7 per group, were inoculated with rAAV (comprising a capsid protein of SEQ ID NO: 48 and a nucleic acid comprising nucleotide sequences encoding aflibercept and a miR targeting VEGF-C) or vehicle at three doses (1x10 11 vg / eye, 3×10 11 vg / eye or 1 × 10 12 Intravitreal steroids (40 mg methylprednisolone IM weekly starting on Day 1 and 2 mg triamcinolone acetonide sub-Tenon's capsule injection) were discontinued 4 weeks after administration. Laser was administered 42 days after administration to induce choroidal neovascularization (CNV), and lesions were scored 2 and 4 weeks after CNV laser.

[0276] As illustrated in Figure 26, assessment of grade IV lesion incidence revealed that treatment with all doses of rAAV significantly blocked CNV, as demonstrated by the complete absence of clinically relevant grade IV lesions in all treatment groups (i.e., at all tested doses of rAAV). Compared to vehicle controls, see Figure 26. No dose response was observed, suggesting complete efficacy of rAAV at all administered doses.

[0277] Aqueous humor samples collected from NHPs 21 days after intravitreal administration of rAAV containing the capsid protein of SEQ ID NO: 48 and nucleic acids encoding aflibercept and a miR targeting VEGF-C (including the sense and antisense strands corresponding to SEQ ID NO: 19 and 20, and the complete construct corresponding to SEQ ID NO: 69), hereinafter referred to as rAAV SEQ ID NO: 48 CAG-AFLB-VEGFC-RNAi, were analyzed for aflibercept protein expression. As shown in Figure 28, the ocular chamber levels of aflibercept are dose-dependent. Furthermore, 1x10 12 At a dose of vg / eye, aflibercept expression from eyes administered rAAV SEQ ID NO:48 CAG-AFLB-VEGFC-RNAi was not inferior to an rAAV comprising the capsid protein of SEQ ID NO:48 and nucleic acid encoding only aflibercept, hereinafter referred to as rAAV SEQ ID NO:48 CAG-AFLB.

[0278] Intraocular inflammation in NHPs was examined using a slit lamp biomicroscope at designated time points.Scoring was applied to qualitative clinical ophthalmological findings using the Non-Human Primate Ophthalmology Test Scoring System, with summary scores derived from test components.At designated time points, intraocular pressure (IOP) measurements were collected using a TonoVet (iCare, Finland) tonometer set to canine (d) calibration settings.Three measurements were taken from each eye at each time point, and the mean IOP was determined.

[0279] Compared to vehicle-treated eyes, which consistently demonstrated lower integrated clinical scores,12 vg / eye of rAAV SEQ ID NO: 48 CAG-AFLB or 1 × 10 12 Eyes that received rAAV SEQ ID NO: 48 CAG-AFLB-VEGFC-RNAi at 100 vg / eye exhibited mild to moderate intraocular inflammation, peaking approximately 28 days after IVT injection (see Figure 29).

[0280] At week 22, vehicle, 1 × 10 11 vg / eye or 3 × 10 11 There was no or only mild intraocular inflammation in eyes treated with rAAV SEQ ID NO: 48 CAG-AFLB-VEGFC-RNAi at 1 × 10 vg / eye, whereas 1 × 10 12 vg / eye of rAAV SEQ ID NO: 48 CAG-AFLB or 1 × 10 12 Half or more of the eyes treated with rAAV SEQ ID NO: 48 CAG-AFLB-VEGFC-RNAi (vg / eye) showed mild to moderate intraocular inflammation. See Figure 29. The inflammatory response mainly included mild chamber cells, posterior corneal deposits, vitreous cell findings, and deposits on the anterior capsular membrane of the lens. Intraocular pressure (IOP) remained normal in all groups.

[0281] Retinal volume and central retinal thickness were evaluated in NHPs. Briefly, optical coherence tomography (OCT) was performed at designated time points using a Heidelberg Spectralis OCT Plus with eye tracking and HEYEX image capture and analysis software. A total volume scan encompassing the posterior retina was performed. Retinal thickness maps and cross-sectional images were obtained at the time of pre-laser testing.

[0282] OCT-derived retinal volume and thickness showed stable global retinal thickness from baseline to week 22, indicating that no retinal edema or degeneration-related thinning occurred during any post-treatment observation period. The mean total retinal volume and mean central retinal thickness within the applied ETDRS grid remained stable throughout the study (see Figure 30).

[0283] Full-field electroretinography (ffERG) was performed at 84 days and 22 weeks to compare changes in retinal function in NHPs. Briefly, a minimum 25-minute period of dark adaptation preceded scotopic ffERG recording. Dark adaptation was achieved by holding sedated monkeys in a travel cage located in a darkened room with access to dim red light. Pupils were dilated with phenylephrine (10%) and augmented with cyclopentolate (1%) at the onset of dark adaptation and potentially again prior to stimulus exposure to ensure animals had maximal pupil dilation at the time of stimulus induction.

[0284] A minimum 10-minute period of light adaptation preceded photopic ffERG recording, with the eyes held open and the DTL electrodes maintained in place. Pupils were dilated with phenylephrine (10%) and augmented with cyclopentolate (1%) at the onset of light adaptation and potentially again prior to stimulus exposure to ensure that monkeys had maximal pupillary dilation at the time of stimulus induction.

[0285] The following procedures were performed using the Veris platform to ISCEV standards for toxicology studies and included the following irritant exposures:

[0286] Dark 0.16 cd-s m² stimulation (measured rod-driven responses of ON-type bipolar cells, b-wave)

[0287] Dark 2.51 cd-s m² stimulus (rod- and cone-driven responses of both photoreceptors, a-wave, and ON-type bipolar cells, b-wave)

[0288] Photopic 2.51 cd-s m² stimulation (cone-driven responses of both photoreceptors, a-wave, and on- and off-bipolar cells, b-wave)

[0289] Photopic 30Hz flicker stimulus at 2.51cd-s m² (cone-driven response)

[0290] NHPs were tested in the dark before testing in the light, and always received a sequence of stimulus exposures of increasing intensity for a given adaptation. A single stimulus exposure always preceded a flicker stimulus exposure to avoid bleaching and affecting retinal adaptation.

[0291] To verify consistency and establish the range of inherent variability in ffERGs, each stimulus at each time point was captured by two independent trials, each trial being a composite of three separate, sequential stimulus inductions.

[0292] Data recording followed a format guided by ISCEV standards.

[0293] Stimulation guidance was indicated by a marker

[0294] The time-integrated luminance of the stimulus as well as the background was recorded as an absolute value.

[0295] Time and date of stimulation induction

[0296] · Pupil diameter

[0297] Corneal electrode type and position

[0298] No statistically significant differences in scotopic A-wave, scotopic B-wave, or photopic flicker were observed between treatment groups at the same time point or between different time points within the same treatment group (all p>0.05, two-way ANOVA followed by Tukey-Krammer HSD). The mean amplitudes of scotopic A-wave, scotopic B-wave, and photopic flicker are shown in Figure 31.

[0299] Conclusions—rAAV comprising the capsid protein of SEQ ID NO: 48 and aflibercept plus a nucleic acid encoding a miR targeting VEGF-C completely abolished grade IV lesion development and significantly attenuated CNV development compared to vehicle-treated controls, supporting the safety and efficacy of rAAV in treating various diseases associated with ocular neovascularization, such as wet AMD.

[0300] Example 6 HUVEC proliferation and migration assays An in vitro angiogenesis assay was performed to evaluate the effects of (i) aflibercept plus a VEGF-C-targeting miRNA (CAG-AFLB-VEGFC-RNAi), (ii) aflibercept alone (CAG-AFLB), or (iii) a plasmid encoding GFP (CAG-GFP) on the proliferation and migration of human umbilical vein endothelial cells (HUVECs) after electroporation. Briefly, HUVECs were lifted using 0.05% trypsin-EDTA and electroporated according to the Thermo Fisher Neon Electroporator kit instructions. Two million cells per condition were resuspended in R buffer with the appropriate amount of plasmid. One microgram of total DNA was transfected per condition (CAG-AFLB-VEGFC-RNAi, CAG-AFLB, or CAG-GFP). Equimolar concentrations of the CAG-AFLB-VEGFC-RNAi and CAG-AFLB plasmids were transfected. The length of the CAG-AFLB plasmid (6,660 bp; 1.4 × 10 11 copies / μg) of the CAG-AFLB-VEGFC-RNAi plasmid (10,711 bp: 8.6 × 10 10 Because the total DNA content was shorter than 100 copies / μg, extra CAG-GFP plasmid was added to the CAG-AFLB condition to equalize the total DNA. A mock transfection "shock" was also performed as a control. Cells were electroporated with a single pulse of 1350V for 30 ms. The medium was changed 4 hours after electroporation to remove residual R buffer. After electroporation, cells were plated for proliferation or migration assays.

[0301] HUVEC cell counting for proliferation assay

[0302] Four days after electroporation, cells were lifted with 0.05% trypsin-EDTA. Trypsin was quenched with an equal volume of complete medium. Cells were centrifuged at 400×g for 5 minutes and resuspended in 50 μl of complete medium. The cell suspension was counted using a BD countess cell counter. Six replicates were counted per condition. The entire experiment was performed three separate times.

[0303] HUVEC cell counting for migration assay

[0304] Four days after electroporation, cells were lifted with 0.05% trypsin-EDTA. Trypsin was quenched with an equal volume of complete medium. Cells were centrifuged at 400 × g for 5 minutes and counted. 25,000 cells were seeded in starvation EGM-2 medium (without VEGF) into the upper compartment of 8-μm-pore transwell inserts coated with 0.1% gelatin according to Nareshkumar et al. Scientific Reports 8.1 (2018): 1-16. The lower compartment contained complete EGM-2 medium to create a growth factor gradient. Four hours after seeding, cultures were fixed and washed with PBS. Nuclei were counterstained with DAPI for 5 minutes at room temperature. The upper compartment was then completely scraped using a rubber scraper. Images were acquired at 50x magnification using a Zeiss AxioVert.A1 fluorescence microscope. Four images per insert were acquired in an unbiased grid pattern with three replicates per transfection condition. The entire experiment was performed three separate times. DAPI quantification was performed using FIJI software. Schindelin, et al. Nature methods. 9.7 (2012): 676-682. Briefly, a threshold was applied to each image and converted to a binary mask. DAPI points were then quantified using the "Analyze Particles" function. The threshold was the same within each experiment but varied between experimental replicates due to variability in DAPI staining intensity.

[0305] As shown in Figure 27, HUVEC cells transfected with plasmid DNA containing nucleic acids encoding aflibercept and a miR targeting VEGF-C resulted in a reduction in the number of cells present in the culture system compared to a plasmid containing GFP under the control of the CAG promoter or the shock condition control (Figure 27A). The nucleic acids encoding aflibercept and a miR targeting VEGF-C included the sense and antisense strands corresponding to SEQ ID NOs: 19 and 20, as well as the complete construct corresponding to SEQ ID NO: 69. Furthermore, compared to the shock condition, fewer cells migrated through the transwell membrane after transfection with plasmid DNA containing nucleic acids encoding aflibercept and a miR targeting VEGF-C (Figure 27B). Importantly, in both assays, the plasmid containing nucleic acids encoding aflibercept and a miR targeting VEGF-C was not inferior to cells transfected with a plasmid containing only aflibercept under the control of the CAG promoter. These data demonstrate robust inhibition of endothelial cell proliferation and migration by nucleic acids encoding aflibercept and a miR targeting VEGF-C in HUVEC cells.

[0306] While the materials and methods of this invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention. In certain embodiments, for example, the following items are provided: (Item 1) A synthetic ribonucleic acid (RNA) molecule comprising a sense strand that comprises at least 90% sequence identity to a sequence selected from SEQ ID NOs: 19, 16, 22, 25, 28, 1, 4, 7, 10, 13, 31, 34, 37, 40 and 43, and an antisense strand that comprises at least 90% sequence identity to a sequence selected from SEQ ID NOs: 20, 17, 23, 26, 29, 2, 5, 8, 11, 14, 32, 35, 38, 41 and 44, respectively, preferably a synthetic RNA molecule comprising a sense strand that comprises at least 90% sequence identity to SEQ ID NO: 19 and an antisense strand that comprises at least 90% sequence identity to SEQ ID NO: 20. (Item 2) 2. The synthetic RNA molecule of item 1, wherein the RNA is a small interfering RNA (siRNA), a small hairpin RNA (shRNA) or an artificial microRNA (miRNA). (Item 3) 2. The synthetic RNA molecule of item 1, wherein the RNA is an shRNA comprising a loop containing the sequence CTCGAG or a sequence at least 90% identical thereto. (Item 4) The RNA is an artificial miRNA comprising an RNA having at least 90% sequence identity with the sequence shown in SEQ ID NO: 46 or 47, respectively, (X) n comprises a sense sequence comprising at least 90% sequence identity to a sequence selected from SEQ ID NOs: 19, 16, 22, 25, 28, 1, 4, 7, 10, 13, 31, 34, 37, 40, and 43; and (Y) n 20, 17, 23, 26, 29, 2, 5, 8, 11, 14, 32, 35, 38, 41, and 44. (Item 5) 5. The synthetic RNA of any one of items 1 to 4, further comprising an unpaired overhang sequence at the 5' and / or 3' end, the overhang sequence optionally comprising a sequence of repeated bases, the repeated bases optionally comprising repeated uracil (U) bases. (Item 6) (i) A nucleic acid comprising a nucleotide sequence encoding at least one synthetic RNA molecule according to any one of items 1 to 4. (Item 7) 7. The nucleic acid of item 6, further comprising (ii) a nucleotide sequence encoding a first antiangiogenic polypeptide that inhibits the activity of VEGF-A, preferably wherein the nucleotide sequences of (i) and (ii) are operably linked to an expression control sequence. (Item 8) 8. The nucleic acid of item 7, wherein the first antiangiogenic polypeptide is aflibercept, and preferably, the nucleotide sequence encoding the aflibercept comprises the nucleotide sequence of SEQ ID NO: 50 or a sequence at least 90% identical thereto. (Item 9) 9. The nucleic acid of item 7 or 8, wherein the nucleotide sequences of (i) and (ii) are operably linked to an expression control sequence. (Item 10) 10. The nucleic acid of item 9, wherein the nucleotide sequence encoding the first antiangiogenic polypeptide and the nucleotide sequence encoding the one or more synthetic RNA molecules are under the control of the same expression control sequence. (Item 11) 11. The nucleic acid of item 10, wherein the expression control sequence comprises a ubiquitous promoter. (Item 12) 12. The nucleic acid of item 11, wherein the promoter is a CAG or CBA promoter. (Item 13) 9. The nucleic acid of item 8, comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 69, 68, and 70, or a sequence at least 95% identical thereto. (Item 14) A composition comprising the synthetic RNA of any one of items 1 to 4 and / or the nucleic acid of any one of items 6 to 13. (Item 15) 15. The composition according to item 14, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. (Item 16) 14. A vector comprising the synthetic RNA molecule of any one of items 1 to 4 and / or the nucleic acid of any one of items 6 to 13. (Item 17) 17. The vector according to item 16, which is an expression plasmid. (Item 18) 18. The vector according to item 16 or 17, which is a viral vector. (Item 19) 19. The vector of item 18, wherein the viral vector is a recombinant adeno-associated virus (rAAV) vector, preferably wherein the rAAV vector comprises an AAV capsid of serotype 2, 5, or 8, or a variant thereof. (Item 20) 20. The vector of claim 19, wherein the rAAV has a capsid comprising a capsid protein comprising a heterologous peptide insertion having a length of 7, 8, 9, 10, or 11 amino acids covalently inserted into the GH loop of the capsid protein compared to the corresponding parental AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence ISDQTKH (SEQ ID NO: 74). (Item 21) 21. The vector according to item 20, wherein the inserted peptide has 1 to 3 spacer amino acids (Y1 to Y3) at the amino and / or carboxyl terminus of the amino acid sequence ISDQTKH (SEQ ID NO: 74). (Item 22) 22. The vector of item 20 or 21, wherein the inserted peptide is LAISDQTKHA (SEQ ID NO: 49). (Item 23) 23. The vector of any one of paragraphs 20 to 22, wherein the site of insertion is located between two adjacent amino acids at a position between amino acid 570 and amino acid 611 of VP1 of AAV2, or at a corresponding position in the capsid protein of another AAV serotype. (Item 24) 24. The vector of any one of paragraphs 20 to 23, wherein the site of insertion is located between the amino acid corresponding to amino acid 587 and amino acid 588 of VP1 of AAV2, or between the amino acid corresponding to amino acid 588 and amino acid 589 of VP1 of AAV2, or at a corresponding position in the capsid protein of another AAV serotype. (Item 25) 25. The vector of any one of items 20 to 24, wherein the capsid protein comprises one or more amino acid substitutions compared to VP1 of AAV2, or one or more corresponding substitutions in a capsid protein of another AAV serotype. (Item 26) The capsid protein has the following amino acids compared to VP1 of AAV2: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S 26. The vector of any one of paragraphs 20 to 25, comprising one or more of the following amino acid substitutions: 463Y, D472N, R484C, A524T, P535S, N551S, A593E, 1698V, V708I, V719M, S721L, and L735Q, or one or more corresponding substitutions in a capsid protein of another AAV serotype. (Item 27) 27. The vector of any one of items 20 to 26, wherein the variant capsid protein comprises a V708I amino acid substitution compared to VP1 of AAV2, or an amino acid substitution at the corresponding position in another capsid protein. (Item 28) 28. The vector of any one of items 20 to 27, wherein the rAAV has a capsid comprising a capsid protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 48, preferably at least 95% identical to SEQ ID NO: 48, more preferably comprising the amino acid sequence set forth in SEQ ID NO: 48. (Item 29) 29. The vector of item 28, wherein the rAAV comprises a heterologous nucleic acid comprising a sequence set forth in any one of SEQ ID NOs: 69, 64, 65, 66, 67, 68, and 70. (Item 30) 30. A host cell comprising the vector of any one of items 16 to 29. (Item 31) 30. A composition comprising the vector of any one of items 16 to 29. (Item 32) 32. The composition according to item 31, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. (Item 33) 32. A method for treating an eye disease associated with ocular neovascularization, preferably an eye disease associated with VEGF-A, in a subject, comprising administering to the subject the nucleic acid of any one of items 6 to 13, the pharmaceutical composition of item 15, the vector of any one of items 16 to 29, or the pharmaceutical composition of item 32. (Item 34) 34. The method of claim 33, wherein the subject is administered the pharmaceutical composition of claim 32, preferably wherein the pharmaceutical composition comprises the rAAV virion of claim 28 or 29. (Item 35) 35. The method of claim 34, wherein the rAAV virion or pharmaceutical composition is administered to the subject intraocularly, preferably by intravitreal, suprachoroidal, or subretinal injection, preferably by a single intravitreal injection, and no further doses of the rAAV virion or pharmaceutical composition are administered to the subject. (Item 36) The pharmaceutical composition is 1×10 8 ~1×10 15 vector particles, preferably 1 x 10 19 ~1×10 14 vector particles or approximately 1 x 10 10 ~Approx. 5×10 13 36. The method of claim 34 or 35, comprising vector particles. (Item 37) 37. The method of any one of items 23 to 36, wherein the disease associated with ocular angiogenesis is selected from the group consisting of wet (neovascular, exudative) age-related macular degeneration; macular edema after retinal vein occlusion; retinal neovascularization resulting from retinal vein occlusion; diabetic macular edema, diabetic retinopathy (including all stages of non-proliferative and proliferative diabetic retinopathy), myopic macular degeneration, branch retinal vein occlusion, hemiretinal vein occlusion, and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectasia; neovascular glaucoma; vitreous hemorrhage; retinal and choroidal neovascularization secondary to retinal diseases including, but not limited to, uveitis, trauma, retinal degenerative disorders, inherited retinal and / or choroidal diseases, ocular tumors, and corneal and iris neovascularization. (Item 38) Item 38. The method according to Item 37, wherein the disease associated with ocular neovascularization is selected from wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; and myopic choroidal neovascularization, preferably, the disease associated with ocular neovascularization is wet age-related macular degeneration. (Item 39) 32. The nucleic acid of any one of items 6 to 13, the pharmaceutical composition of item 15, the vector of any one of items 16 to 29 or the pharmaceutical composition of item 32 for use in the manufacture of a medicament for the treatment of a disease associated with ocular neovascularization. (Item 40) 40. The vector for use according to item 39, wherein the vector is an rAAV virion and is administered intraocularly, preferably by subretinal, suprachoroidal or intravitreal injection. (Item 41) 32。 The nucleic acid of any one of items 6 to 13, the pharmaceutical composition of item 14, the vector of any one of items 16 to 29 or the pharmaceutical composition of item 32 for use in treating a disease associated with ocular neovascularization. (Item 42) 6. A method for delivering synthetic RNA to a retinal cell, the method comprising contacting the retinal cell with the synthetic RNA of any one of items 1 to 5. (Item 43) 14. A method for delivering a heterologous nucleic acid to a retinal cell, the method comprising contacting the retinal cell with a nucleic acid according to any one of items 6 to 13.

Claims

1. A nucleic acid comprising a nucleotide sequence encoding an anti-angiogenic polypeptide operably linked to a CAG promoter and a nucleotide sequence to be transcribed into RNA, wherein the RNA comprises an interfering RNA targeting an RNA transcript of a pro-angiogenic protein, the interfering RNA being located within a natural or artificial intron, the interfering RNA being located within a hybrid chicken β-actin and rabbit β-globin intron of the CAG promoter, and / or the natural or artificial intron being located within the 5' or 3' untranslated region of the nucleotide sequence encoding the anti-angiogenic polypeptide and / or within the coding sequence of the nucleotide sequence encoding the anti-angiogenic polypeptide.

2. The nucleic acid described in claim 1, wherein the interfering RNA is a small interfering RNA (siRNA), a small hairpin RNA (shRNA) or an artificial microRNA (miRNA).

3. The nucleic acid described in claim 2, wherein the interfering RNA is a miRNA comprising a sense strand and an antisense strand embedded in a pri-miRNA scaffold derived from miR-30, miR-22, miR-15, miR-16, miR-103 or miR-107.

4. The nucleic acid described in claim 3, wherein the pri-miRNA scaffold has at least 90% sequence identity with the sequence shown in SEQ ID NO: 46 or 47.

5. The nucleic acid of claim 1, wherein the interfering RNA is located within the hybrid chicken β-actin and rabbit β-globin intron of the CAG promoter.

6. The nucleic acid of claim 1, wherein the anti-angiogenic polypeptide, when introduced into a host cell, is expressed at a higher level compared to the expression of a gene product in an otherwise identical nucleic acid that does not have a nucleotide sequence that is transcribed into RNA, the RNA comprising an interfering RNA, the interfering RNA being an artificial miRNA, and optionally the host cell being a HEK293 cell or a retinal pigment epithelial cell.

7. The nucleic acid of claim 1, wherein the anti-angiogenic polypeptide is selected from endostatin; tumstatin; angiostatin; pigment epithelium-derived factor (PEDF), a soluble receptor fusion protein that binds to and inhibits the activity of VEGF-A, VEGF-B, VEGF-C, VEGF-D and / or PIGF, and an antibody against VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, angiopoietin-1 or angiopoietin-2.

8. The nucleic acid of claim 1, wherein the pro-angiogenic protein is selected from VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, angiopoietin-1, and angiopoietin-2.

9. A vector comprising the nucleic acid described in claim 1.

10. The vector described in claim 9, which is an expression plasmid.

11. The vector described in claim 9, which is a viral vector.

12. The vector described in claim 11, wherein the viral vector is a recombinant adeno-associated virus (rAAV) vector, preferably wherein the rAAV vector comprises an AAV capsid of serotype 2, 5 or 8 or a variant thereof.

13. The vector of claim 12, wherein the rAAV has a capsid comprising a capsid protein that includes a heterologous peptide insertion having a length of 7, 8, 9, 10 or 11 amino acids covalently inserted into the GH loop of the capsid protein compared to the corresponding parent AAV capsid protein, and the peptide insertion comprises the amino acid sequence ISDQTKH (SEQ ID NO: 74).

14. The vector described in claim 13, wherein the inserted peptide is LAISDQTKHA (sequence number 49).

15. The vector of claim 13, wherein the site of insertion is located between two adjacent amino acids at a position between amino acid 570 and amino acid 611 of VP1 of AAV2, or at a corresponding position in the capsid protein of another AAV serotype, and optionally the site of insertion is located between the amino acid corresponding to amino acid 587 and the amino acid corresponding to amino acid 588 of VP1 of AAV2, or between the amino acid corresponding to amino acid 588 and the amino acid corresponding to amino acid 589 of VP1 of AAV2, or at a corresponding position in the capsid protein of another AAV serotype.

16. The vector of claim 13, wherein the capsid protein comprises a P34A amino acid substitution compared to VP1 of AAV2 and comprises an amino acid sequence at least 90% identical to sequence number 48.

17. A composition comprising the nucleic acid described in claim 1.

18. A composition comprising the nucleic acid of claim 1, or a pharmaceutical composition comprising the nucleic acid of claim 1 and a pharmaceutically acceptable carrier, for use in treating an ocular disease associated with ocular neovascularization in a subject.

19. A composition comprising the vector of claim 9, or a pharmaceutical composition comprising the vector of claim 9 and a pharmaceutically acceptable carrier, for use in treating an eye disease associated with ocular neovascularization in a subject.

20. The composition of claim 19, wherein the disease associated with ocular neovascularization is selected from the group consisting of wet (neovascular, exudative) age-related macular degeneration; macular edema following retinal vein occlusion; retinal neovascularization resulting from retinal vein occlusion; diabetic macular edema, diabetic retinopathy (including all stages of non-proliferative diabetic retinopathy and proliferative diabetic retinopathy), myopic macular degeneration, branch retinal vein occlusion, hemiretinal vein occlusion and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectasia; neovascular glaucoma; vitreous hemorrhage; retinal and choroidal neovascularization secondary to retinal diseases including, but not limited to, uveitis, trauma, retinal degenerative disorders, hereditary retinal and / or choroidal diseases, ocular tumors, and corneal and iris neovascularization.

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