Compositions and methods for selective gene expression in the treatment of ocular disease

Abstract

The present disclosure provides nucleic acids comprising one or more miRNA binding sequences to reduce off-target effects and maintain retinal cell-specific expression. Compositions comprising the nucleic acids and use of the compositions for the treatment of ophthalmic diseases are further provided.

Description

Compositions and Methods for Selective Gene Expression in the Treatment of Ocular DiseaseCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Provisional Patent Application Serial No.63 / 759,762, filed February 18, 2025, the entire contents of which are incorporated herein by reference.SEQUENCE LISTING SUBMISSION VIA EFS-WEB

[0002] A computer readable XML file, entitled “090400-5026 WO 01 Sequence Listing” created on February 16, 2026, with a file size of about 453,000 bytes contains the sequence listing for this application and is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0003] Cell specific gene delivery is an important aspect of gene therapy. Targeted gene delivery can be achieved by a variety of mechanisms, including route of administration, use of regulatory elements, and vector modification.

[0004] Selective delivery of genes to target cells (e.g., within the eye) has been achieved by exploiting these mechanisms; however, off-target expression may limit therapeutic potential of gene therapy approaches.

[0005] Thus, there remains a need for gene therapy for the treatment of diseases such as wet age-related macular degeneration (wetAMD) with improved selectivity of target cell gene expression.SUMMARY OF THE INVENTION

[0006] In one aspect, nucleic acids are provided encoding one or more genes for the treatment of an ocular disease, wherein the nucleic acid comprises one or more microRNA binding (ortarget) sites that decrease expression of the one or more genes in one or more off-target ocular cell types, while maintaining expression levels of the one or more genes in one or more target ocular cells. In some embodiments, decreased expression of the one or more genes in the off-target ocular cell(s) is measured relative to a nucleic acid that is otherwise identical but does not comprise the one or more microRNA binding sites. In other embodiments, decreased expression of the one or more genes in the off-target ocular cell(s) is measured relative to expression of the one or more genes in a target ocular cell (e.g., as a ratio of expression of the gene product in the off-target cell / target cell). In some embodiments, the one or more microRNA binding sites do not substantially decrease expression of the one or more genes in a target ocular cell. In some embodiments, a relative expression level is determined by comparing expression of a gene (comprised within a nucleic acid with the gene operably linked to the miRNA binding site and a promoter) in a cell relative to a control (in which the nucleic acid is without the miRNA binding site but otherwise identical). The relative expression should be high in a target ocular cell and low in an off-target ocular cell. In some embodiments, an expression preference for an miRNA binding (or target) site is calculated by averaging the expression levels in off-target cell types and dividing by the level in a target cell type. In some embodiments, an expression preference = (CE relative expression + IPE relative expression) / (2 X RPE relative expression), wherein CE refers to ciliary body epithelium (off-target cell), IPE refers to iris pigment epithelium (off-target cell) and RPE refers to retinal pigment epithelium (target cell).

[0007] In some embodiments, a target cell is a retinal pigment epithelium (RPE) cell. In some embodiments, an off-target cell is a ciliary body epithelium (CE) and / or iris pigment epithelium (IPE) cell. In some embodiments, the one or more microRNA binding sites are located within, or immediately 3’ or 5’ of, an untranslated region (UTR) of the one or more genes. In some embodiments, one or more microRNA binding sequences are located within or immediately 3’ or 5’ of a 5’ UTR. In other embodiments, one or more microRNA binding sequences are located within or immediately 3’ or 5’ of a 3’ UTR. In other embodiments, the one or more microRNA binding sites are located between a promoter sequence and the one or more genes, wherein the one or more genes are operably linked to the promoter sequence. In other embodiments, the one or more microRNA binding sites are located in any non-coding region of the nucleic acid that is transcribed to mRNA (e.g. between the 5’ UTR and the codingregion). The use of the term ‘immediately’ is understood to be synonymous with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 41, 42, 43, 44, 5, 46, 47, 48, 49 or 50 intervening nucleotide bases placed between the miRNA binding sequence and the adjacent UTR.

[0008] In some preferred embodiments, the one or more microRNA binding sites are complementary to microRNAs that are expressed at significantly higher levels in an off-target cell compared to a target cell. In some embodiments, the microRNAs are expressed at a relatively high level in a CE and / or IPE cell and at a relatively low level in an RPE cell. In some embodiments, the CE, IPE, and RPE cells are primate (e.g., human) ceils. In some preferred embodiments, an expression level of the one or more genes in a target cell is at least 70%, at least 80%, at least 90%, at least 95% or even greater than 100% of the expression level of the one or more genes from an otherwise identical nucleic acid not comprising the one or more microRNA binding sites. In other preferred embodiments, an expression level of the one or more genes in a non-target cell (e.g., IPE and / or CE cell) is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower compared to an expression level of the one or more genes an otherwise identical nucleic acid not comprising the one or more microRNA binding sites.

[0009] In some embodiments, the nucleic acid comprises one or more microRNA binding sites comprising a nucleotide sequence selected from those listed at Table 1 and / or Table 2 or a nucleotide sequence that has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some preferred embodiments, the nucleic acid comprises a microRNA binding site comprising a nucleotide sequence selected from SEQ ID Nos: 2, 4, 5, 8, 18, 25, 27, 28, 31, and 41. In a particularly preferred embodiment, the nucleic acid comprises one or multiple microRNA binding sites, each of which comprises the nucleotide sequence of SEQ ID NO: 18 or 41. In other preferred embodiments, the nucleic acid comprises a microRNA binding site comprising a nucleotide sequence selected from SEQ IDNos:47, 48, 49, 50, 71, 149, 160, 165, 172, 177, and 198. In some particularly preferred embodiments, the nucleic acid comprises at least one miRNA binding sequence of SEQ ID NO:50.

[0010] In some embodiments, there is provided a polynucleotide comprising at least one miR- 143-3p (SEQ ID NO:50) target sequence, and / or at least one miR-133a-3p (SEQ ID NO:49) target sequence, wherein the miRNA target sequences are operably linked to a transgene.

[0011] In one embodiment, the polynucleotide comprises at least one miR-143-3p target sequence, wherein the target sequence is operably linked to the transgene.

[0012] In one embodiment, the polynucleotide comprises at least one mir-133a-3p target sequence, wherein the target sequence is operably linked to the transgene.

[0013] In one embodiment, the polynucleotide comprises at least one miR-143-3p target sequence, and at least one miR-133a-3p target sequence, wherein the target sequences are operably linked to the transgene.

[0014] In one embodiment, the number of copies of each of the miRNA target sequences is independently selected from the group consisting of: one, two, three, and four.

[0015] In one embodiment, the polynucleotide comprises four miR-143-3p target sequence, and / or four tniR-I33a-3p target sequences, wherein the miRNA target sequences are operably linked to the transgene.

[0016] In one embodiment:

[0017] (a) the miR-143-3p target sequence comprises or consists of a nucleotide sequence that has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 50; and

[0018] (b) the miR-133a-3p target sequence comprises or consists of a nucleotide sequence that has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 49.

[0019] In one embodiment, the miRNA target sequences are located downstream, i.e., 3’, of the transgene. In other words, the miRNA target sequences may be located after the transgene in the 5’ to 3’ direction.

[0020] In one embodiment, the miRNA target sequences are located within the 3’-UTR of the transgene.

[0021] Both the individual target sequences and the clusters of target sequences may be contiguous with one another, separated by spacer sequences, or any combination of thereof.

[0022] Thus, in one embodiment, the miRNA target sequences are separated by spacer sequences.

[0023] In one embodiment, the polynucleotide further comprises a promoter. In one embodiment, the promoter is operably linked to the transgene.

[0024] In some aspects, a vector comprising a nucleic acid (or polynucleotide sequence) as herein disclosed is provided. In some embodiments, the vector is an expression vector. In some embodiments, the expression vector is a plasmid or virus. In some embodiments, the vector is a recombinant adeno-associated virus (rAAV).

[0025] In some aspects, a pharmaceutical composition is provided comprising a nucleic acid or vector as herein described and a pharmaceutically acceptable carrier.

[0026] In some aspects, treatment methods for a variety of ocular diseases is provided comprising administering a pharmaceutical composition as herein described to a mammal. In some embodiments, the mammal is a primate (e.g., a non-human primate or a human). In some embodiments, the ocular disease is associated with VEGF-A, including without limitation, wet age-related macular degeneration, diabetic macular edema; macular edema following retinal vein occlusion; diabetic retinopathy; and myopic choroidal neovascularization. In some embodiments, the method comprises ocularly administering the pharmaceutical composition to ahuman in need of treatment for an ocular disorder. In some embodiments, ocular administration comprises intravitreal, subretinal, periocular and / or suprachoroidal administration.

[0027] In other aspects, a method for selectively repressing expression of a gene product in a ciliary body epithelium cell is provided, the method comprising administering a heterologous nucleic acid (or vector comprising same) encoding the gene product and comprising one or more miRNA binding sequences to the eye of a mammal, whereby expression of the gene product in the ciliary body epithelium cell is repressed relative to expression of the gene product in one or more target retinal cells and / or is repressed in a ciliary body epithelium cell relative to expression of the gene product from an otherwise identical nucleic acid comprising the gene product and without the one or more miRNA binding sequences.

[0028] In other aspects, a method for selectively repressing expression of a gene product in both an iris pigment epithelium cell and a ciliary body epithelium cell is provided, the method comprising administering a heterologous nucleic acid (or vector comprising same) encoding the gene product and comprising one or more miRNA binding sequences to the eye of a mammal, whereby expression of the gene product in the ciliary body epithelium cell and in the iris pigment epithelium cell is repressed relative to expression of the gene product in one or more target retinal cells and / or is repressed in a ciliary body epithelium ceil and iris pigment epithelium cell relative to expression of the gene product from an otherwise identical nucleic acid comprising the gene product and without the one or more miRNA binding sequences.

[0029] In another embodiment, provided herein is a host cell comprising a nucleic acid as herein described. In some aspects, the host cell is a mammalian cell, including without limitation, a CHO cell, an HEK293 cell, a HeLa cell, a BHK21 cell, a Vero cell or a V27 cell. In other aspects, the host cell is a photoreceptor cell (e.g., rods; cones), a retinal ganglion cell (RGC), a glial cell (e.g., a Muller glial cell, a microglial cell), a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigmented epithelium (RPE) cell.DESCRIPTION OF THE DRAWINGS

[0030] FIGS. 1A-B Flowchart of Experimental Design. Fig. 1A: various miRNA binding sequences were placed within the 3’ untranslated region of a construct comprising a CAG promoter operably linked to nucleotide sequence encoding GFP. Figure IB: Expression of GFP was assessed in different cell types following transfection with the constructs.

[0031] FIGS.2A-B illustrate expression (measured by mean fluorescent intensity) of GFP from the various constructs (each with unique miRNA binding sequences) in ciliary body epithelium (CE) cells vs retinal pigment epithelium (RPE) cells (ARPE19, Figure 2A; hTerT, Figure 2B) normalized to a control construct containing random sequences with no match to the human genome. Constructs with a nucleotide sequence of SEQ ID NO:1 (hsa-miR-503-5p) are labeled.

[0032] FIGS.3A-C illustrate GFP expression (mean fluorescence intensity normalized to a construct with a CAG promoter driving transgene expression) in RPE cells (FIG. 3 A), CE cells (FIG. 3B) and Iris cells (FIG. 3C) following transduction at the specified multiplicities of infection (MOIs) with recombinant adeno-associated virus with a capsid protein of SEQ ID NO:361 and a heterologous nucleic acid comprising, in 5’-3’ direction: 5’ ITR, CAG promoter, nucleotide sequence encoding GFP, and 3’ ITR, and four copies of an miRNA binding sequence (Candidate 1 = SEQ ID NO:2; Candidate 2 = SEQ ID NO:4; Candidate 3 = SEQ ID NO: 5; Candidate 4 = SEQ ID NO:8; Candidate 5 = SEQ ID NO: 18) in the 3’ UTR region.

[0033] FIG. 4 illustrates relative transgene expression level across three cell types (retinal pigment epithelium (RPE); ciliary body epithelium (CE) and iris pigment epithelium (IPE) cells; top candidates are emphasized.

[0034] FIG. 5 illustrates relative expression levels in AAV format in RPE, CE and IPE cells with nucleic acid comprising a binding site (SEQ ID NO:50) for top candidate hsa-mir-143-3p.

[0035] FIG. 6 (1) illustrates transgene expression regulation by miRT (miR Target sequences). When the miRTs are incorporated in the transgene mRNA transcript of an AAVconstruct, the corresponding differentially expressed miRNAs can regulate the transgene expression in different tissues. In off-target tissues (right), abundant endogenous miRNA targets the miRT, leading to mRNA degradation and reduced transgene expression. In on-target tissues (left), the corresponding miRNA are present at low levels, allowing transgene expression without interference.

[0036] FIGS.7A-D (2) illustrates a method to identify miRTs regulating AAV therapeutic transgene expression in ocular tissues (7 A) Human donor ocular tissues were collected and isolated miRNA were sequenced on an Illumina platform. The resulting miRNA profiles from different ocular tissue types were combined to construct a miRNA profile database of human ocular tissues. (7B) By comparing expression levels in different tissues and examining their representation in different donors, miRNAs that met the pre-defined differential expression pattern across different ocular tissue types were identified from the database. (7C) The corresponding miRTs of differentially expressed miRNAs were incorporated into lentivirus screening constructs and evaluated in the relevant cell types. (7D) Lead miRT candidates with desired expression profiles in the lentiviral screening were incorporated into AAV constructs and further characterized in relevant cell types

[0037] FIG. 8A-C (3) Analysis and discovery criteria for miRT candidates. (8A) General analysis pipeline for miRNA database construction and miRT candidate discovery. The sequencing data’s quality was first evaluated by FastQC, followed by trimming of adapter sequences with Cutadapt. The pair-end reads were then merged with PEAR. The alignment to the reference sequences was performed by Bowtie2 and the alignment count was performed by Samtools. Finally, the differentially expressed miRNA candidates were identified by DESeq2. (8B) Normalized expression heatmap of top differentially expressed miRNAs across ciliary body (CB), iris, neural retina (NR), and RPE. Each row represents one miRNA, and each column represents one ocular tissue sample. (8C) Selection criteria for miRNA candidates. The ideal candidates meeting all four differential expression criteria are highlighted with a red outline. Candidates meeting only two selection criteria of the same off-target tissue are highlighted with ablue outline and shaded in grey, these candidates can be used to regulate expressions in a single off-target tissue.

[0038] FIGS. 9A-C (SI A-C) MA plots of miRNA expression level between each on / off-target tissue pair (9 A) MA plot comparing miRNA expression between ciliary body and neural retina tissues. (9B) MA plot comparing miRNA expression between ciliary body and RPE tissues. (9C) MA plot comparing miRNA expression between iris and neural retina tissues. (9D) MA plot comparing miRNA expression between iris and RPE tissues.

[0039] FIGS. 10A-F (4A-) Lentiviral miRTs screening design and protein expression results (10A) Lentiviral construct design for miRT screening. (10B) Lentiviral miRTs screening workflow and assays. (10C) Relative expression of CE vs ARPE19 in the lentiviral miRTs screen. Out of 155 candidates, 112 show lower relative expression in CE than ARPE19 (indicated as the dots below the green line). (10D) Relative expression of IPE vs ARPE19 in the lentiviral miRTs screen. Out of 155 candidates, 145 show lower relative expression in IPE than ARPE19 (indicated as the dots below the blue line). (10E) Expression preference of all candidates’ relative protein expression data in the lentiviral miRTs screen. Expression preference lower than a threshold of 1 (indicated by the red dashed line) suggests preferential expression in on-target tissues over off-target tissues. (1 OF) Relative protein expression of top miRTs candidates in lentiviral screen across all screening cell types, they were selected for AAV characterization. The relative expression across different cell types of the same miRT candidate are connected by black lines.

[0040] FIGS. 11 A-B (S3A) Protein and mRNA expression across three cell types in the lentiviral miRTs screen (11 A) Relative protein expression across three cell types in the lentiviral miRTs screen. Lines connect individual miRT candidates across different cell types. A total of 108 candidates achieved higher expression in on-target ARPE 19 cells compared to both off-target CE and IPE cells. (1 IB) Relative mRNA expression across three cell types in the lentiviral miRTs screen. Lines connect individual miRT candidates across different cell types. A total of133 candidates achieved higher expression in on-target ARPE19 cells compared to both off-target CE and IPE cells.

[0041] FIGS. 12A-D (S2) Lentiviral miRTs screening mRNA expression results (12A) Relative expression of CE vs ARPE19 in the lentiviral miRTs screen. Out of 155 candidates, 145 show lower relative expression in CE than ARPE19 (indicated as the dots below the green line). (12B) Relative expression of IPE vs ARPE1 in the lentiviral miRTs screen. Out of 155 candidates, 140 show lower relative expression in IPE than ARPE19 (indicated as the dots below the blue line). (12C) Expression preference of all candidates’ relative mRNA expression data in the lentiviral screen. Expression preference lower than a threshold of 1 (indicated by the red dashed line) suggests preferential expression in on-target tissues over off-target tissues. (12D) Relative mRNA expression of top miRTs candidates in lentiviral screen across all screening cell types, they were selected for AAV characterization. The relative expression across different cell types of the same miRT candidate are connected by black lines.

[0042] FIGS. 13A-D (5A-) AAV miRT characterization and protein expression (13 A) AAV construct design for miRT characterization. (13B) AAV miRT characterization workflow and assays. (13C) Expression preference of 11 AAV characterization candidates’ relative protein expression. (13D) Relative protein expression of AAV characterization candidates across 3 cell types. ** p-value < 0.01, *** p-value < 0.001 (Two-way ANOVA test)

[0043] FIGS. 14A- (S4A-) AAV miRTs characterization and mRNA expression (14A) Expression preference of 11 AAV characterization candidates’ relative mRNA expression. (14B) Relative mRNA expression of AAV characterization candidates across 3 cell types. ** p-value < 0.01, *** p-value < 0.001 (two-way ANOVA test)

[0044] FIG. 1SA-C (6A-) The miRNA and mRNA profile comparison between miRT-143-3p and control AAV constructs transduction across CE, IPE and iPSC-RPE (15A) Principal component analysis plot for miRNA profile comparisons. (1 B) Principal component analysis plot for mRNA profile comparison. (15C) Gene ontology over-representation analysis for IPE mRNA profile. Enriched biological processes were plotted on the graphs. The x-axis representsthe number of enriched genes found in each biological process and the color of the bar represents the enrichment score of each biological process.

[0045] FIG. 16A-F (S5) MA plots comparing the miRNA and mRNA profiles of miRT-143-3p and control AAV constructs transduced in multiple cell types. MA plots comparing the miRNA profiles of miRT-143-3p and control AAV constructs transduced in (16A) CE cells, (16B) IPE cells, (16C) iPSC-RPE cells, and comparing mRNA profiles of miRT-143-3p and control AAV constructs transduced in (16D) CE cells, (16E) IPE cells, and (16F) iPSC-RPE cells.

[0046] FIG. 17A-B (7A-) Unintended targeting miRNA identification for miRT-143-3p. (17A) Seed regions of miRNAs that were found in the miRT-143-3p sequence. (17B) Expression level of potential unintended targeting miRNAs and hsa-miR143-3p across four ocular tissue types.

[0047] FIG. 18 (S6) The miRNA profile comparison across cell models (18 ) Principal component analysis plot of miRNA profile across different RPE cell models. The distances between ARPE19, iPSC-RPE and primary RPE suggest that miRNA profile variations were observed between these cell models. (18B) Normalized hsa-miR-143-3p expression level across different cell models. The expression levels of hsa-miR-143-3p in three RPE cell models are over 20-folds lower than the expression level in CE and IPE.DETAILED DESCRIPTION OF THE INVENTION

[0048] Definitions

[0049] The term "isolated" designates a biological material (cell, nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present). For example, a polynucleotide present in the natural state in a plant or an animal is not isolated, however the same polynucleotide separated from the adjacent nucleic acids in which it is naturally present, is considered "isolated."

[0050] As used herein, a "coding region" or "coding sequence" is a portion of polynucleotide which consists of codons translatable into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding 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, then that a single vector can contain just a single coding region or can comprise two or more coding regions.

[0051] As used herein, the term "regulatory region" refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence 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 a coding region is intended for expression in a eukaryotic cell, a poly adenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[0052] As used herein, the term “nucleic acid” is interchangeable with “polynucleotide” or “nucleic acid molecule” and a polymer of nucleotides is intended. In some aspects, the nucleic acid is a DNA molecule. In other aspects, the nucleic acid is an RNA molecule, e.g., a messenger RNA (mRNA) molecule.

[0053] A polynucleotide which encodes a gene product, e.g., a polypeptide, can include a promoter and / or other transcription or translation control elements operably associated with one or more coding regions. In an operable association a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory regions in such a way 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 promoterfunction results in the transcription of 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 ability of the promoter to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.

[0054] " Transcriptional control sequences" or “expression control sequences” refer 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, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, 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 as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0055] A “CAG promoter” is composed of (C) the cytomegalovirus (CMV) early enhancer element, (A) the promoter, the first exon and the first intron of chicken beta-actin gene, (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 p-actin promoter directs efficient production of interleukin-5. Gene, 79(2), 269-277, the contents of which are incorporated herein by reference.

[0056] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picomaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).(0057] The term "expression" as used herein refers to a process by which a polynucleotide produces a gene product, for example, an RNA or a polypeptide. It includes without limitation transcription of the polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), primary miRNA, small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and the translation of an mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0058] The term “messenger RNA” (mRNA) refers to one type of RNA molecule. In vivo, transcription of DNA usually results in premature RNA, which has to be processed into so-called messenger RNA, usually abbreviated as mRNA. Typically, an mRNA comprises a 5’-cap, a 5-UTR of a gene, an open reading frame / coding sequence, a 3'-UTR of a gene and a poly(A). miRNA or microRNA as used herein, refers to a small non-coding RNA molecule which may function in post-transcriptional regulation of gene expression (e.g., by RNA silencing, such as by cleavage of the mRNA, destabilization of the mRNA by shortening its polyA tail, and / or by interfering with the efficiency of translation of the mRNA into a polypeptide by a ribosome). A mature miRNA is typically about 22-23 nucleotides long.

[0059] As used herein, the term "miRNA (or microRNA) binding site" or “miRNA (or microRNA) binding sequence” refers to a sequence within a polynucleotide (nucleic acid sequence), e.g., within a DNA or within an RNA or RNA transcript, that has sufficient complementarity to all or a region of a miRNA to interact with, associate with or bind to the miRNA. In exemplary embodiments, miRNA binding sites are included in RNA sequences, e.g. mRNAs, for example, in the 5' UTR and / or 3' UTR of an mRNA. A miRNA binding site having sufficient complementarity to the miRNA refers to a degree of complementarity sufficient to facilitate miRNA- mediated regulation of the mRNA, e.g., miRNA-mediated translational repression or degradation of the mRNA. In exemplary aspects of the disclosure, a miRNAbinding site having sufficient complementarity to the miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated degradation of the mRNA, e.g., miRNA-guided RISC-mediated cleavage of mRNA, The miRNA binding site can have complementarity to, for example, a 19-25 nucleotide long miRNA sequence, to a 1 -23 nucleotide long miRNA, most typically to a 22-nucleotide long miRNA sequence, A miRNA binding site may be complementary to only a portion of a miRNA, e,g., to a portion 1, 2, 3 or 4 nucleotides shorter that a naturally occurring miRNA. Full or complete complementarity (e.g., fully complementary or completely complementary over all or a significant portion of a naturally occurring miRNA) is preferred when the desired regulation is RNA or mRNA degradation. In some embodiments, a miRNA binding site includes a sequence that has complementarity (e.g,, partial or complete complementarity) with a miRNA seed sequence. In particular embodiments, the miRNA binding site includes a sequence that has complete complementarity with a miRNA seed sequence. In some embodiments, a miRNA binding site includes a sequence that has complementarity (e.g., partial or complete complementarity) with a miRNA sequence. In particular embodiments, the miRNA binding site includes a sequence that has complete complementarity with a miRNA sequence. In some embodiments, a miRNA binding site has complete complementarity with a miRNA sequence but for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotide substitutions, terminal additions, and / or truncations. In a preferred embodiment, a miRNA binding site has complete complementarity with a miRNA sequence but for 1, 2 or 3 nucleotide substitutions, terminal additions, and / or truncations. In a very preferred embodiment, a miRNA binding site has complete complementarity with a miRNA sequence.

[0060] By “operably linked”, it is to be understood that individual components are linked together in a manner which enables them to carry out their function substantially unhindered.

[0061] A "vector" refers to any vehicle for the cloning of and / or transfer of a nucleic acid into a host cell. A vector can be a replicon to which another nucleic acid segment can be attached so as to bring about the replication of the attached segment, The term "vector" includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors are known and used in the art including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion, of a polynucleotide into asuitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini.

[0062] Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and / or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ), -glucuronidase (Gus), and the like. Selectable markers can also be considered to be reporters.

[0063] Eukaryotic viral vectors that can be used include, but are not limited to, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, poxvirus, e.g., vaccinia virus vectors, baculovirus vectors, or herpesvirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

[0064] “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. In general, a coding sequence is located 3' to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters." Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as "cell-specific promoters" or "tissuespecific promoters." Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as "developmentally-specificpromoters" or "cell differentiation-specific promoters," Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as "inducible promoters" or "regulatable promoters." It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.

[0065] The term "plasmid" refers to an extra-chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular doublestranded DNA molecules. Such elements can be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single-or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0066] A polynucleotide or polypeptide has a certain percent "sequence identity" to 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 a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FAST A, available in the Genetics Computing Group (GCG) package, from 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. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. 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).

[0067] The terminology “retinal cell” refers herein to any of the cell types that comprise the retina, such as, without limitation, retinal ganglion (RG) cells, amacrine cells, horizontal cells,bipolar cells, photoreceptor cells, Mtiller glial cells, microglial cells, and retinal pigmented epithelium (RPE). The terminology “photoreceptor cells” refers herein to, without limitation, rod cells or “rods” and cone cells or “cones”. The terminology “Mtiller cells” or “Mtiller glia” refers to glial cells that support neurons in the vertebrate retina.

[0068] The present disclosure is based, inter alia, on the surprising discovery that incorporating one or more miRNA binding sequences (such as those listed at Table 1 and Table 2) into a nucleic acid encoding a gene product can reduce of expression of the gene product in off-target cells such as ciliary body epithelium cells and iris pigment epithelium cells while maintaining adequate (e.g., therapeutic) expression levels of the gene product in target cells such as retinal pigment epithelium cells.

[0069] In various aspects, the present disclosure provides nucleic acids encoding gene(s) useful for the treatment of an ocular disease, wherein the nucleic acid comprises one or more microRNA binding sequences (that decrease the expression level of the encoded gene produces) in an off-target cell or tissue, while maintaining the expression level of the encoded gene produces) in a target cell or tissue. Also provided are vectors comprising the nucleic acids and pharmaceutical compositions comprising the nucleic acids or vectors and their use in the treatment of various ocular diseases.

[0070] In some embodiments, an expression level of a gene encoded by a nucleic acid comprising one or more microRNA binding sequences as described herein in a non-target cell or tissue is reduced by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to an expression level of the gene encoded by an otherwise identical nucleic acid that does not comprise the one or more microRNA binding sequences in the same non-target cell. In some preferred embodiments, the non-target cell is a ciliary body epithelium (CE) cell. In other preferred embodiments, the non-target cell is an iris pigment epithelium (IPE) cell.

[0071] In particularly preferred embodiments, an expression level of a gene encoded by a nucleic acid comprising one or more microRNA binding sequences as described herein isreduced by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% in a CE and in an IPE cell compared to an expression level of the gene encoded by an otherwise identical nucleic acid that does not comprise the one or more microRNA binding sequences in the same CE cell and IPE cell.

[0072] In related embodiments, an expression level of a gene encoded by a nucleic acid comprising one or more microRNA binding sequences as described herein in a target cell or tissue is reduced by less than 30%, less than 20%, less than 15%, less than 10%, or less than 5% compared to an expression level of the gene encoded by an otherwise identical nucleic acid that does not comprise the one or more microRNA binding sequences in the same target cell. In some preferred embodiments, the target cell is a retinal pigment epithelium (RPE) cell. In some embodiments, an expression of a gene encoded by a nucleic acid comprising one or more microRNA binding sequences as described herein in a target cell or tissue is substantially the same or is increased (e.g., by up to about 3%, by up to about 5%, by up to about 8%, by up to about 10%, by up to about 15%, by up to about 20% or more) compared to an expression level of the gene encoded by an otherwise identical nucleic acid that does not comprise the one or more microRNA binding sequences in the same target cell. In some embodiments, expression level of the gene is increased by about 5% to about 30% or by about 5% to about 25% or by about 5% to about 20% in the target cell.

[0073] In some embodiments, the one or more microRNA binding sequence(s) are selected from those set forth at Table 1Table 1miRNA Binding Sequence (DNA) SEQ ID NO:CTGCAGAACTGTTCCCGCTGCTA 1CTGGAAGTGCCCATACTACAGT 2CGAATCCACCACGAACAACTTC 3TCCAACACTGTACTGGAAGATG 4 CCTGGCAGCGGAAACAATACCCC 5 AGCAAAAATGTGCTAGTGCCAAA 6CGATGTAGTCCAAAGGCACATA 7CCCATGGCTTTTAGCCCTACAT 8CTGCAGGAGACAGAGTCGGTG 9 GCTGCAAACATCCGACTGAAAG 10 GAAGTAAACATCCACCTCCCAG 11 TCCATCATTACCCGGCAGTATTA 12 GGAAAGCTAGGAGGCCGTATAG 13 ACAAACCACAGTGTGCTGCTG 14 CACAAATTCGGTTCTACAGGGTA 15 AGCCAAGCTCAGACGGATCCGA 16 CCTGGCCGTGTGGTTAGTGATT 17 GAATTCACCAAGGGCAACCTCT 18 CTGTGTTTCAGCTCAGTAGGCA 19 CTAGTGGTCCTAAACATTTCAC 20 AAGTGTTGTCCGTGAATGATT 21 TGCAAGCCTTGGGTGTGGGAGG 22 TGCTCACAAGCAGCTAAGCCCT 23 miRNA Binding Sequence (RNA) SEQIDNO: CUGCAGAACUGUUCCCGCUGCUA 24 CUGGAAGUGCCCAUACUACAGU 25 CGAAUCCACCACGAACAACUUC 26 UCCAACACUGUACUGGAAGAUG 27 CCUGGCAGCGGAAACAAUACCCC 28 AGCAAAAAUGUGCUAGUGCCAAA 29 CGAUGUAGUCCAAAGGCACAUA 30 CCCAUGGCUUUUAGCCCUACAU 31 CUGCAGGAGACAGAGUCGGUG 32 GCUGCAAACAUCCGACUGAAAG 33 GAAGUAAACAUCCACCUCCCAG 34 UCCAUCAUUACCCGGCAGUAUUA 35 GGAAAGCUAGGAGGCCGUAUAG 36 ACAAACCACAGUGUGCUGCUG 37 CACAAAUUCGGUUCUACAGGGUA 38 AGCCAAGCUCAGACGGAUCCGA 39 CCUGGCCGUGUGGUUAGUGAUU 40 GAAUUCACCAAGGGCAACCUCU 41 CUGUGUUUCAGCUCAGUAGGCA 42 CUAGUGGUCCUAAACAUUUCAC 43 AAGUGUUGUCCGUGAAUGAUU 44 UGCAAGCCUUGGGUGUGGGAGG 45UGCUCACAAGCAGCUAAGCCCU 4610074] In some embodiments, a nucleic acid molecule or vector comprising same is provided comprising at least one, two, three, four or more (distinct, similar or identical) miRNA binding sequences comprising a nucleotide sequence selected from those set forth as SEQ ID Nos: 1-23. In some preferred embodiments, the nucleic acid comprises at least one, two, three, four or more (distinct, similar or identical) miRNA binding sequences comprising a nucleotide sequence selected from SEQ ID Nos: 2, 4, 5, 8 and 18. In some particularly preferred embodiments, the nucleic acid comprises multiple miRNA binding sites, at least two of which comprise the nucleotide sequence set forth as SEQ ID NO: 18. In some embodiments, the nucleic acid molecule comprises at least one, two, three, four or more (distinct, similar or identical) miRNA binding sequences, at least one or more (and preferably all) of which is selected from a nucleotide sequence comprising, consisting essentially of, or consisting 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 related embodiments, the nucleic acid molecule comprises at least one, two, three, four or more miRNA binding sequences selected from a nucleotide sequence comprising, consisting essentially of, or consisting 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 that is the reverse complement of a sequence selected from those listed in Table 1.

[0075] Preferably, one or more microRNA binding sequence(s) selected from those set forth at Table 1, when present in a nucleic acid encoding a gene product, act to suppress expression of the gene in a ciliary body epithelium cell, without substantially affecting expression of the gene in a target cell.(0076] In some embodiments, the one or more microRNA binding sequence(s) are selected from those set forth at Table 2 (Sequence Identifiers are provided after each sequence in parentheses):Table 2microRNA microRNA sequence microRNA Binding Sequence hsa-miR-184 TGGACGG AGAACTGAT AAGGGT (2021 ACCCTTATCAGTTCTCCGTCCA (47) hsa-miR-145-5p GTCC AGTTTTCCC AGGAAI CCCT (203) AGGGATTCCTGGGAAAACTGGAC (48) hsa-tniFM33a-3p TTTGGTCCCCTTCAACCAGCTG (204) CAGC JGGTTGAAGGGGAC C AAA (49) hsa-miR-143-3p TGAGATGAAGCACTGTAGCTC (205) GAGCI ACAGTGCnCATCTCA (bO) hsa-miR-l-Sp TGGAATGTAAAGAAGTATGTAT (206) ATACATACTTCTTTAC ATTCCA (51) hsa miR 13!:a 5p TATGGCnTTTATTCCTATGTGA (207} TCAC ATAGGAATAAAAAGC C ATA (52) hsa-tniR-135a-2'3p ATGTAGGGATGGAAGCCATGAA 1208) TTCATGGCTTCCATCCCTAC AT (53) hsa-miR-423-3p AGCTCGGTCTGAGGCCCCTCAGT (209) ACTGAGGGGCCTCAGACCGAGCT (54) hsa-miR-99b-3p CAAGCICGTGTCTGTGGGTCCG (210) CGGACCCACAGACACGAGCTTG (5-5) hsa-m(R-1247-5p ACCCGICCCG1 ICGICCCCGGA (211) TCCGGGGACGAACGGGACGGGT (56) hsa-miR-3909 TGTC CTCTAGGGC CTGCAGTCT (212) AGACTGCAGGCCCIAGAGGACA (57) iisa-i:iiR-145'3p GGATTC CTGGAAATACTGTTCT (213) AGAACAGTATTTCGAGGAATCC (58) i •isa iniR-671-.3p TCCGGTTCTCAGGGCTCCACC (214) GGTGGAGCCCTGAGAACCGGA (59) isa-miR 2113 ATTTGTGCTTGGCTCTGTCAC (215) GTGACAGAGCCAAGCACAAAT (60) h5a«mjR.-1251-5p ACTCTAGCTGCCAAAGGCGCT (216) AGCGCCTTTGGCAGCTAGAGT (61) hsa-let-7er5p TGAGGTAGGAGGTTGTATAGTT (217) AACTATACAACCTCCTACCTCA (62) hsa-raiR-3605-3p CCTCCGTGT7AC CTGTCCTCTAG (218) CTAGAGGACAGGTAACACGGAGG (63) Tisa-miR-421 AICAACAGACAHAAIIGGGCGC (219) GCGCCCAATTAATGTCTGTTGAT (64) tisa-m(R-887-5p CTTGGGAGCCCTGTTAGACTC (22.0 ) GAGTCT. AACAGGGCTCGCA. AG (65) Hsa-m(R-365b-5p AGGGACTTTC AGGGGC AGCTGT (221 ) ACAGCTGCCCCTGAAAGTCCCT (66) tisa-riiR-6516-5p TTTGCAGTAACAGGTGTGAGCA (222) TGC 1CACACC 1 GTTACTGCAAA (67) hsa-i'iiR-477O TGAGATGACACTGTAGCT (223) AGCTACAGTGTCATCTCA (68) hsa-miR-5O6-3p TAAGGCACCCTTCTGAGTAGA (224) TCTACTCAGAAGGGTGCC IT A (69) hsa-miR-224-5p TCAAGTCACTAGTGGTTCCGTTTAG (225) CTAAACGGAACCACTAGTGACTTGA (70) hsa-miR*892a CACTGTGTCCTTTCTGCGTAG (226) CTACGCAGAAAGGACACAGTG (71) hsa-miR-1298-5p TTCATTCGGCTGTCCAGATGTA (227) TACATCTGGACAGCCGAATGAA (72) hsa-mi. R-143-5p GGTGCAGTGCTGGATCTGTGGT (228) ACCAGAGATGCAGCACTGCACC (73) hsa-miR-1911-5p TGAG1 ACCGCCA1GTCTGTTGGG (229) CCCAACAGACATGGCGGTACTCA (74) hsa-miR-708-3p CAACTAGACTGTGAGCT (CT AG (230) CTAGAAGCTCACAGTCTAGTTG (75) lisa-mi R-148a-3p TCAGTGCACTACAGAACTTTGT (231) ACAAAGTTCTGTAGTGCACTGA (76) hsa-miR-204-5p TTCCCTTTGTCATCCTATGCCT (232) AGGCATAGGATGACAAAGGGAA (77) hsa-rniR-452-5p AACTGTTTGCAGAGGAAACTGA (233) TCAGTTTCCTCTGCAAACAGTT (78) Jisa-miR-30a-3p CTTTCAGTCGGATGTTTGCAGC (234) GCTGCAAACATCCGACTGAAAG (79) hsa-miR-:133b TTTGG7CCCCTTCAACCAGCTA (235) TAGCTGGTTGAAGGGGACCAAA(8O) hsa-miR-148a-5p AAAGTTCTGAGACACTCCGACT (236) AGTCGGAGTGTCTCAGAACTTT (81) hsa-miR-27b-5p AGAGCI TAGCTGATIGGTGAAC (237) GTTCACCAATCAGCTAAGCTCT (82) hsa-miR-652-3p AATGGCGCCACTAGGGTTGTG (238) CACAACCCTAGTGGCGCCATT (S3) lisa-rniR-1298-3p CATCIGGGCAACTGACTGAAC (239) GTTCAGTCAGTTGCCCAGATG (84) lisa-miR-30e-3p CTTTCAGTCGGATGTrTACAGC (240) GCTGTAAAC AICCGACTGAAAG (85) hsa-miR-133a-5p AGCTGGTAAAATGGAACCAAAT (241) ATTTGGTTCCATTTTACCAGCT (86)l»sa-tniR’1247’3p CCCCGGGAACGTCGAGACTGGAGC 242 GCTCCAGTCTCGACGTTCCCGGGG (87)hsa-miR-1261 ATGGATAAGGCTTTGGCTT (243} AAGCCAAAGCCTTATCCAT (88) hsa rniR 4443 TTGGAGGCGTGGGTTTT (244) AAAAC C CACGCCTCCAA (89) hsa-miR-12135 T A AAG GTTTGTTTGTA AA (245) TTTACAAAC AAACCTTTA (90 ) li5a-miR-27b-3p TTCACAGTGGCTAAGTTCTGC (246) GCAGAAC TTAGCCAC TGI GAA (91) hsa-miR-224-3p AAAAT GGTGCCCT AG IGACTACA (247) TGTAGTCACTAGGGCACCATTTT (92) hsa-miR-1264 CAAGTCTTATTTGAGCACCTGTT (248) AACAGGTGCTCAAATAAGACTTG (93) hsa-miR-581 TCTTGTGTTCTCTAGATCAGT (249) ACTGATCTAGAGAACACAAGA (94) lisa- nR-24-3p TGGCTCAGTTCAGCAGGAACAG (250) CTGTTCCTGCTGAACTGAGCCA (95) h sa miR-3074 3p GATATCAGCTCAGTAGGCACCG (251) CGGTGCCTACTGAGCTGATATC (96) h sa- T iR 548uti-3p CAAAAACTGCAGTTACTTTTGC (252) GCAAAAGTAACTGCAGTTTTTG (97) hsa-TiR 1250-bp ACGGTGCTGGATGTGGCCi n (253) AAAGGCCACATCCAGCACCGT (98) hsa-miR-1912-5p CTCATTGCATGGGCTGTGTATA (254) TATACACAGCCCAf GCAAIGAG (99) 0sa-mR-6866-5p TTAGAGGCTGGAATAGAGATTCT (255) AGAATCTCTATTCCAGCCTCTAA (100) lisa-miR-5694 CAGATCATGGGACTGTCTCAG (256) CTGAGACAGTCCCATGATCTG (101) hsa'JWiR'1911«3p CACCAGGCATTGTGGTCTCC (257) GGAGACCACAATGCCTGGTG |102) hs;i-riR-6873-3p TTCTCTCTGTCTTTCTCTCTCAG (258) CTGAGAGAGAAAGACAGAGAGAA (103) lisa-ri'iR-1912-3p TACCCAGAG CATGCAGTGTGAA (259) TTCACACTGCATGCTCTGGGTA ( 104) hs;i:riR 24 1 op TGCCTACTGAGCTGATATCAGT (260 ) ACTGATATCAGCTC AGTAGGCA ( 105) hsa TiR 892c-3p CACTGTTTCCTTTCTGAGTGGA (261) TCCACTCAGAAAGGAAACAGTG (106) hsa-miR-30c-5p I GI AAAC A I CC I ACACTC 1CAGC (262) GC IGAGAGI GI AGGA I G I T I ACA (107) hsa-miR-891a-5p TGCAACGAACCTGAGCCACTGA (263) TCAGTGGCTCAGGTTCGTTGCA ( 108) hsa-miR-338-3p TCCAGC ATC AGTGATTTTGTTG (264) CAACAAAATCACTCATGCTGGA ( 109) hsa-miR-335-3p TTTTTCATTATTGCTCCTGACC (265) GGTCAGGAGCAATAATGAAAAA (110) hsa-miR-448 TTGCATATGTAGGATGTCCCAT (266) ATGGGACATCCTACATATGCAA (111) lisa-riR-88S-5p TACTCAAAAAGCTGTCAGTCA (267) TGACTGACAGCTTTTTGAGTA (112) hsa*miR’892b CACTGGCTCCTTTCTGGGTAGA (268) TCTACCCAGAAAGGAGCCAGTG (113) h sa - mi R-548am - 3 p CAAAAACTGCAGTTACTTTTGT (269) ACAAAAGTAACTGQAGTTTTTG (114) hsa -niR-211-5p TTCCCTTTGTC ATC CTTCGCCT (270) AGGCGAAGGATGACAAAGGGAA ( 115) h sa-mi R 509 3 5p TACTG C AGAOGTGGC AATCATG (271 ) CATGATTGCCACGTCTGCAGTA (116) hsa-miR-514a-3p ATTGAC AC 0 CTGIGAG I AGA (272) TCTAC1CACAGAAGIGICAAT (117) h sa-mi R-455-5p TATGTGCCTTTGGACTACATCG (273) CGATCTAGICCAAAGGCACAIA (118) hsa-miR-363-3p AATTGCACGGTATCCATCTGTA (274) TACAGATGGATACCGTGCAATT (119) hsa-miR-5O8-3p TGATTGTAGCGTTTTGGAGTAGA (275 ) TCTACTCCAAAAGGCTACAATCA (120) hsa-miR-221’3p AGCTACATTGTCTGCTGGGTTTC (276) GAAACCCAGCAGACAATGTAGCT (121) h sa- mi R- 187-3 p TCGTGTCTTGTGTTGCAGCCGG (277) CCGGCTGCAACACAAGACACGA (122) hsa-miR-195-3p CCAATATTGGCTGTGCTGCTCC (278) GGAGCAGCACAGCCAATATTGG (123) hsa miR-509-3:> TGATTGGTACGTCTGTGGGTAG (279) CTACC C ACAG ACGTAC C AATC A ( 124) hsa-miR.532-3p CCTCCCACACCCAAGGCn GCA (280) TGCAAGCCTTGGGTGTGGGAGG (125) hsa-miR-5100 TTCAGATCCCAGCGGTGCCTCT (281) AGAGGCACCGCTGGGATCTGAA (126) hsa-miR-455-3p GCAGTCCATGGGCATATACAC (282) GTGTATATGCCCATGGACTGC (127) hsa-miR-5O9-5p TACTGCAGACAGTGGCAATCA (283) TGATTGCCACTGTCTGC AGTA ( 128) hsa-miR-505-3p CGTCAACACTTGCTGGTTTCCT (284) AGGAAACCAGCAAGTGTTGACG (129) hsa-miR-146a-3p CCTCTGAAATTCAGTTCTTCAG (285) CTGAAGAACTGAATTTCAGAGG (130)hsa-miR-7704 CCGGGTCGGCGGCGACGTG (286) CACGTCGCCGCCGACCCCG (131)hsa-mifir508-5p TACTCCAGAGGGCGTCACTCATG {287) CATGAGTGACGCCCTCTGGAGTA (132) hsa-let-7d-5p AGAGGTAGTAGGTTGCATAGTT (288) AACTATGCAACCTACTACCTCT (133) h$a miR 125a-5p TCCCTGAGACCCTTTAACCTGTGA (289) TCACAGGTTAAAGGGTCTCAGGG A (134) l:s;i-i'iiR 3283p CTGGCCCTCTCTGCCCTTCCGT (290) ACGGAAGGGCAGAGAGGGCCAG (135) hsa-miR-504-5p AGACCCTGGTCTGCACTCTATC (291) GATAGAGTGCAGACCAGGGTCT (136) h sa >niR-4662;; -5p TTAGCCAATTGTCCATCTTTAG (292) CTAAAG ATGGACAATTGGCTAA (137) hsa-miR~423'5p TGAGGGGCAGAGAGCGAGACTTT (293) AAAGICICGClCICrGCCCCTCA (138) hsa-miR-3166 C GCAGACAATGCCTACTGGC CTA (294) TAGGCCAGTAGGCATTGTCTGCG ( 139) hsa-miR-549a-3p TGACAACTATGGATGAGCTCT(295) AGAGCTCATCCATAGTTGTCA (140) hsa-;r!iR-549a-5p AGCTCATCCATAGTTGTCACTG (296) CAGIGACAACTATGGATGAGCT (141) l)sa-n'iR-132-5p ACCGTGGCTTTCGATTGTTACT (297 ) AGTAAC AATCGAAAGC C ACGGT ( 142) hsa-rr.iR 211 3p GCAGGGACAGGAAAGGGGTGC (298) GCACCCCTTTGCTGTCCCTGC (143) hsa rriR 193a 5:> TGGGTCTTTGCGGGCGAGATGA (299) TCATCTCGCCCGCAAAGACCCA (144) hsa-miR-675Q-3p GAAC ICACCCTC1GCICCCAG (300) CTGGGAGCAGAGGGTGAGTTC (145) hsa-miR-128-3p 1 C AC AG1 GAAC CGG1 CT CTTT (301) AAAGAGACCGGTTCACTGTGA (146) hsa-mlR-7977 TTCCCAGCCAACGCACCA (302) TGGTGCGTTGGCTGGGAA ( 147) hsa-miR-10394-3p TGGGCGCGCCGGGACTGTGAGAC (303) GTCTCACAGTCCCGGC GC GCC CA (148) hsa-miR-3184-5p TGAGGGGCCTCAGACCGAGCTTTT (304) AAAAGCTCGGTCTGAGGCCCCTCA (149 hsa-ntiR-4530 CCCAGCAGGACGGGAGCG (305) CGCTCCCGTCCTGCTGGG (150) hsa-iriR-513t;-5p TTCTCAAGGAGGTGTCGTTTAT ( 306 ) ATAAACGACACCTCGnGAGAA (151) hsa-miR-365a-3p TAATGC C C CTAA AAATCCTTAT (307) ATAAGGATTTTTAGGGGCATTA (152) h$aj.miR’212"5p ACCTTGGCTCTAGACTGCTTACT(3O8) AGTAAGCAGTCTAGAGCCAAGGT (153) hsa- mjR-6 / 16-3[: TCCGAACTCTCCATTCCTCTGC (309) GC AG AGGAATGG AG AGTTC GGA ( 154) hsa-miR’676-3p CTGTCCTAAGGTTGTTGAGTT I 310 j AACTC AAC AACCTTAGGAC AG ( 155) hsa-miR-365b-3p TAATGCCCCTAAAAATCCTTAT (311) ATAAGGATTTTTAGGGGCATTA (156) hsa-miR-1249-3p ACGCCCTTCCCCCCCTTCTTCA (312) TGAAGAAGGGGGGGAAGGGCGT (157) hsa-miR-125b-5p TCCCTGAGAC C CTAACTTGTGA (313) TCACAAGTTAGGGTCTCAGGGA (158) hsa-ffiiR-363-5p CGGGTGGATCACGATGCAATTT (314) AAATTGCATCGTGATCCACCCG (159) hsa-miR-514a-5p TACTCTGGAGAGTGACAATCATG (315) CATGATTGTCACTCTCCAGAGTA (160) hsa-miR-1303 TTTAGAGACGGGGTCTTGCTCT (316) AGAGCAAGACCCCGTCTCTAAA (161) hsa-let-7a-5p TGAGGTAGTAGGTTGTATAGTT (317) AACTATACAACCTACTACCTCA (162) hsa miR 1468 bp CTCCGTTTGCCTGTTTCGCTG (318) CAGCGAAACAGGCAAACGGAG (163) hsa-miR-6809-3p CTTCTCTrCTCTCCTTCCCAG (319) CTGGGAAGGAGAGAAGAGAAG (164) hsa-let-7e-3p CTATACGGCCTCCTAGCTTTCC (320) GGAAAGCTAGGAGGCCGTATAG ( 165) hsa-miR-26b-3p GC TGTTCTCC ATTACTTGGCT (321) AGCC AAGTAATG GAGAAC AGG ( 166 ) hsa-miR-4636 AACTCGTGTTC AAAGCCTTTAG (322) CTAAAGGCTTTGAACACGAGTT (1B7) hsa-miR-513b-5p TTCACAAGGAGGTGTCATTTAT (323) ATAAATGACACCTCCTTGTGAA (168) hsa-rniR^205-5p TCCTTCATTCCACCGGAGTCTG (324) CAGACTCCGGTGGAATGAAGGA (169) ■isii-rnjR-506 5p TATTCAGGAAGGTGTTACTTAA (325) TTAAGTAACACCTTCCTGAATA (170) hsa-miR-3928-3p GGAGGAACCTTGGAGCTTCGGC (326) GCCGAAGCTCCAAGGTTCCTCC (171) isa miR-296-5p AGGGCCCCCCCTCAATCCTGT (327) ACAGGATTGAGGGGGGGCCCT ( 172) hsa-miR-6747-3p TCCTGCCHCC TCTGCACCAG (328) CTGGTGCAGAGGAAGGCAGGA (173) bsa-miR-187-5p GGC I ACAACACAGGACCCGGGC (329) GCCCGGGTCCTGTGTTGTAGCC (174)hsa-miR-23b-5p TGGGnCCTGGCATGCTGATTT (330) AAATCAGCATGCCAGGAACCCA (175)lisa-miR-4431 GCGACTCTGAAAACTAGAAGGT (331) ACCnCTAGTTTTCAGAGTCGC (176) hsa-miR-744-3p CTGTTGCCACTAACCTCAACCT (332) AGGTTGAGGTTAGTGGCAACAG (177) hsa-miR-3184-3p AAAGTCTCGCTCTCTGCCCCTCA (333) TGAGGGGCAGAGAGCGAGACTTT (178) hsa-miR-3652 CGGCTGGAGGTGTGAGGA (334) TCCTCACACCTCCAGCCG (179) hsa-miR-7153-3p CACCATGGACGGTTTACC (335) GGTAAACCGTCCATGGTG (180) hsa-miR-6775-3p AGGCCCTGTCC7CTGCCCCAG (336) CTGGGGCAGAGGACAGGGCCT (181) hsa-miR-3612 AGGAGGCATCTTGAGAAATGGA (337) TCCATTTCTCAAGATGCCTCCT (182) hsa-mlR-516b-5p ATCTGGAGGTAAGAAGCACTTT (338) AAAGTGGTTCTTACCTCCAGAT (183) hsa-miR- 1295a TTAGGCCGCAGATCTGGGTGA (339) TC ACCCAGATCTGCGGCCTAA ( 184) hsa-miR-6514-3p CTGCCTGTTCTTCCACTCCAG (340) CTGGAGTGGAAGAACAGGCAG (185) hsa-tniR-296-3p GAGGGTTGGGTGGAGGCTCTCC (341) GGAGAGCCTCCACCCAACCCTC (186) hsa-miR-4649-5p TGGGCGAGGGGTGGGCTCTCAGAG (342 CTCTGAGAGCCCACCCCTCGCCCA 187 hsa-miR-4741 CGGGCTGTCCGGAGGGGTCGGCT (343) AGCCGACCCCTCCGGACAGCCCG (188 hsa-miR-1306-3p ACGTTGGCTCTGGTGGTG (344) CACCACCAGAGCCAACGT (189) hsa-mlR-99b-5p CACCCGTAGAACCGACCTTGCG (345) CGCAAGGTCGGTTCTACGGGTG (190) hsa-miR- 193b-5p CGGGGTTTTGAGGGCGAGATGA (346) TCATCTCGCCCTCAAAACCCCG (191) hsa-mlR-25-5p AGGCGGAGACTTGGGCAATTG (347) CAATTGCCCAAGTCTCCGCCT ( 192) hsa-miR-6511b-3p CCTCACCACCCCTTCTGCCTGCA (348) TGCAGGCAGAAGGGGTGGTGAGG (193) hsa-miR-550a-5p AGTGCCTGAGGGAGTAAGAGCCC (349) GGGCTCTTACTCCCTCAGGCACT (194) hsa-miR-30b-3p CTGGGAGGTGGATGTTTACTTC (350) GAAGTAAACATCCACCTCCCAG (195} hsa-miR-128-l-5p CGGGGCCGTAGCACTGTCTGAGA (351) TCTCAGACAGTGCTACGGCCCCG (196) hsa-miR-222-3p AGCTACATCTGGCTACTGGGT (352) ACCCAGTAGCCAGATGTAGCT (197) hsa-miR-338-5p AACAATATCCTGGTGCTGAGTG (353) CACTCAGCACCAGGATATTGTT (198) hsa-miR-92a-3p TAnGCACTTGTCCCGGCCTGT (354) ACAGGCCGGGACAAGTGCAATA (199} hsa-mlR-744-5p TGCGGGGCTAGGGCTAACAGCA (355) TGCTGTTAGCCCTAGCCCCGCA (200)hsa-mlR-664a-3p TATTCATTTATCCCCAGCCTACA (356) TGTAGGCTGGGGATAAATGAATA (201)

[0077] In some embodiments, a nucleic acid molecule or vector comprising a nucleic acid molecule is provided comprising at least one, two, three, four or more (distinct, similar or identical) miRNA binding sequences comprising a nucleotide sequence selected from those set forth as SEQ ID Nos:47-201. In some preferred embodiments, the nucleic acid comprises at least one, two, three, four or more (distinct, similar or identical) miRNA binding sequences comprising a nucleotide sequence selected from SEQ ID Nos: 47, 48, 49, 50, 71, 149, 160, 165, 172, 177, and 198. In some particularly preferred embodiments, the nucleic acid comprises at least one miRNA binding sequence of SEQ ID NO:50- In related embodiments, the nucleic acid comprises multiple miRNA binding sites, at least one of which comprise the nucleotide sequence set forth as SEQ ID NO:50. In some embodiments, the nucleic acid molecule comprises at least one, two, three, four or more (distinct, similar or identical) miRNA binding sequences, at leastone or more (and preferably all) of which is selected from a nucleotide sequence comprising, consisting essentially of, or consisting 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. In related embodiments, the nucleic acid molecule comprises at least one, two, three, four or more miRNA binding sequences selected from a nucleotide sequence comprising, consisting essentially of, or consisting 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 that is the reverse complement of a sequence selected from those listed in Table 2.

[0078] Preferably, one or more microRNA binding sequence(s) selected from those set forth at Table 2, when present in a nucleic acid encoding a gene product, act to suppress expression of the gene in a ciliary body epithelium cell and in an iris pigment epithelium cell, while substantially maintaining expression of the gene in a target cell. In some preferred embodiments, an expression level of a gene of interest on a nucleic acid comprising the gene of interest under the control of a promoter and further comprising one or more microRNA binding sequence(s) selected from those set forth at Table 2 (e.g., SEQ ID NO:50) is expressed is less than about 40%, preferably less than about 30% compared to a control expression level and is least about 80%, preferably at least 90% and even at least 100% in a target cell or tissue (e.g., an RPE cell) relative to a control. In some aspects, a control is an expression level of the gene of interest in the same cell wherein the gene of interest is on an otherwise identical nucleic acid that does not comprise a microRNA binding sequence. In some aspects, an expression level is measured as an miRNA and / or protein level.

[0079] In some embodiments, a nucleic acid as herein described comprises one or more miRNA binding sequences located within an untranslated region (UTR) of one or more encoded genes. In some embodiments, one or more miRNA binding sequences are located within a 5’ UTR of one or more encoded genes. In other embodiments, one or more miRNA binding sequences are located within a 3’ UTR of one or more encoded genes. In other embodiments, one or more miRNA binding sequences are located within a 5’ UTR and one or more miRNA binding sequences are located within a 3’ UTR of one or more encoded genes. In related embodiments, a nucleic acid as herein described comprises one or more miRNA binding sequences located between a promoter sequence and one or more encoded genes operably linked to the promoter sequence.

[0080] In some embodiments, a nucleic acid as herein described comprises one or more microRNA binding sequences and further comprises a nucleotide sequence encoding one or more gene products, wherein the one or more gene products comprise polypeptides and / or interfering RNA molecules.

[0081] The uses of the encoded gene product include, but are not limited to, enhancing the level of a factor in a cell, enhancing the level of a factor in a neighboring cell through secretion of a factor, decreasing the level of a factor in a cell, or decreasing the level of a factor in a neighboring cell through secretion of a factor. The gene product can be designed to supplement the level of a defective of missing gene product, decrease the level of a defective of missing gene product, introduce a new supporting gene product, supplement the level of a supporting gene product, decrease the level of a hindering gene product, or both decrease the level of a hindering gene product and introduce or supplement the level of a supporting gene product.

[0082] Gene products encoded by a nucleic acid as herein described can be used to alter the level of gene products or gene product activity directly or indirectly linked to retinal diseases and trauma. Genes whose gene products are directly or indirectly linked to genetic diseases include, e.g., ADP-ribosylation factor-like 6 (ARL6); BBSome interacting protein 1 (BBIP1); BBSome protein 1 (BBS1); BBSome protein 2 (BBS2); BBSome protein 4 (BBS4); BBSome protein 5 (BBS5); BBSome protein 7 (BBS7); BBSome protein 9 (BBS9); BBSome protein 10 (BBS 10); BBSome protein 12 (BBS 12); centrosomal protein 290 kDa (CEP290); intraflagellar transportprotein 172 (IFT172); intraflagellar transport protein 27 (IFT27); inositol polyphosphate-5- phosphatase E (INPP5E); inwardly-rectifying potassium channel subfamily J member 13 (KCNJ13); leucine zipper transcription factor like-1 (LZTFL1); McKusick-Kaufinan syndrome protein (MICKS); Meckel syndrome type 1 protein (MKS1); nephronophthisis 3 protein (NPHP1); serologically-defined colon cancer antigen 8 (SDCCAG8); tripartite motif-containing protein 32 (TRIM32); tetratricopeptide repeat domain 8 (TTC8); Batten disease protein (CLN3); Rab escort protein 1 (CHM); (PRDM13); (RGR; (TEAD1); arylhydrocarbon-interacting receptor protein-like 1 (AIPL1); cone-rod otx-like photoreceptor homeobox transcription factor (CRX); guanylate cyclase activating protein 1A (GUCA1A); retinal-specific guanylate cyclase (GUCY2D); phosphatidylinositol transfer membrane-associated family member 3 (PITPNM3); prominin 1 (PROMI); peripherin (PRPH); peripherin 2 (PRPH2); regulating synaptic membrane exocytosis protein 1 (RIMS1); semaphorin 4A (SEMA4A); human homolog of C.elegans uncl 19 protein (UNCI 19); ATP-binding cassette transporter - retinal (ABCA4); ADAM metallopeptidase domain 9 (ADAM9); activating transcription factor 6 (ATF6); chromosome 21 open reading frame 2 (C21orf2); chromosome 8 open reading frame 37 (C8orf37); calcium channel; voltage-dependent; alpha 2 / delta subunit 4 (CACNA2D4); cadherin-related family member 1 (protocadherin 21) (CDHR1); ceramide kinase-like protein (CERKL); cone photoreceptor cGMP-gated cation channel alpha subunit (CNGA3); cone cyclic nucleotide-gated cation channel beta 3 subunit (CNGB3); cyclin M4 (CNNM4); guanine nucleotide binding protein (G protein); alpha transducing activity polypeptide 2 (GNAT2); potassium channel subfamily V member 2 (KCNV2); Phosphodiesterase 6C (PDE6C); Phosphodiesterase 6H (PDE6H); proteome of centriole 1 centriolar protein B (POC1B); RAB28 member of RAS oncogene family (RAB28); retina and anterior neural fold homeobox 2 transcription factor (RAX2); 11 -cis retinol dehydrogenase 5 (RDH5); RP GTPase regulator-interacting protein 1 (RPGRIP1); tubulin tyrosine ligase-like family member 5 (TTLL5); L-type voltage-gated calcium channel alpha-1 subunit (CACNA1F); retinitis pigmentosa GTPase regulator (RPGR); (GNAT1); (PDE6B); (RHO); CABP4); GPR179); (GRK1); GRM6); LRIT3); SLC24A1); TRPM1); NYX); 0PN1LW); 0PN1MW); blue cone opsin (OPN1SW); frataxin (FXN);(IMPDH1); (0TX2); CRB1); DTHD1GDF6); IFT140); IQCB1); LCA5); LRAT); NMNAT1); RD3); RDH12); RPE65); SPATA7); TULP1); mitochondiral genes (KSS, LHON, MT-ATP6, MT-TH, MT-TL1, MT-TP, MT-TS2, mitochondrially encoded NADH dehydrogenases [MT-ND]); (BEST1); C1QTNF5EFEMP1); ELOVL4); FSCN2); GUCA1B); HMCN1); IMPG1); RPIL1); TIMP3); DRAM2); MFN2); NR2F1); optic atrophy 1 (OPA1); TMEM126A);TIMM8A); CA4); HK1); KLHL7); NR2E3); NRL); OR2W3); PRPF3); PRPF4); PRPF6); PRPF8); PRPF31); ROM1); retinitis pigmentosa protein 1 (RP1); RP9); SNRNP200); SPP2); TOPORS); ARL2BP); C2orf71); CLRN1); CNGA1); CNGB1); CYP4V2); DHDDS); DHX38); EMC1); EYS); F AMI 61 A); GPR125); HGSNAT); IDH3B); IMPG2); KIAA1549); KIZ); MAK); MERTK); MVK); NEK2); NEURODI); PDE6A); PDE6G); PRCD); RBP3); RLBP1); SLC7A14); USH2A); ZNF408); ZNF513); OFD1); RP2); retinoschisin (RSI); ABHD12); CDH23); CEP250); CIB2); DFNB31); GPR98); HARS); MYO7A); PCDH15); USH1C);USH1G); NDP); PGK1); CAPN5); FZD4); ITM2B); LRP5); MIR204); RBI); TSPAN12); C12orf65); CDH3); MFRP); OAT); PLA2G5); RBP4); RGS9); RGS9BP); ARMS2; ERCC6); FBLN5); HTRA1); TLR3); and TLR4).

[0083] Other gene products (and encoding sequences) that can be encoded by a nucleic acid as herein described include those disclosed in U. S, Patent Nos. 11,357,870 and 11,345,390, the entire contents of each of which is incorporated herein by reference.

[0084] Yet other gene products that can be encoded by a nucleic acid as herein described include gene products that function as immune modulators, e.g., complement factors (e.g., complement factor H), toll-like receptors, are called “immunomodulatory genes”. Exemplary immunomodulatory genes include cytokines, chemokines, and the fusion proteins or antibodies that are specific for them and / or their receptors, e.g. the anti-IL-6 fusion protein Rilonacept™, the Complement Factor H-specific antibody lampamizumab, etc. Other gene product that can be encoded by a nucleic acid as herein described include gene products that function as neuroprotective factors, e.g., platelet derived growth factor receptor (PDGFR); glial derived neurotrophic factor (GDNF); rod-derived con viability factor (RdCVF); fibroblast growth factor (FGF); neurturin (NTN); ciliary neurotrophic factor (CNTF); nerve growth factor (NGF); neurotrophin-4 (NT4); brain derived neurotrophic factor (BDNF); epidermal growth factor. Other gene products that can be encoded by a nucleic acid as herein described include gene products that function as light responsive opsins, e.g., opsin; rhodopsin; channel rhodopsin; halo rhodopsin.

[0085] Genes whose gene products induce or promote apoptosis are referred to herein as “pro-apoptotic genes” and the products of those genes (mRNA; protein) are referred to as “pro-apoptotic gene products.” Pro-apoptotic targets include, e.g., Bax gene products; Bid gene products; Bak gene products; Bad gene products; Bcl-2; Bcl-Xl. Anti-apoptotic gene products include X-linked inhibitor of apoptosis.

[0086] Genes whose gene products induce or promote angiogenesis are referred to herein as “pro-angiogenic genes” and the products of those genes (mRNA; protein) are referred to as “pro-angiogenic gene products.” Pro-angiogenic targets include, e.g., vascular endothelial growth factor (VEGFa, VEGFb, VEGFc, VEGFd); vascular endothelial growth factor receptor 1 (VEGFR1); vascular endothelial growth factor receptor 2 (VEGFR2); vascular endothelial growth factor receptor 3 (VEGFR3); Fms-Related Tyrosine Kinase 1 (Fltl); placenta growth factor (PGF); Platelet-derived growth factor (PDGF); angiopoietins; sonic hedgehog. Genes whose gene products inhibit angiogenesis are referred to herein as “anti-angiogenic genes” and the products of those genes (mRNA; protein) are referred to as “anti-angiogenic gene products.” Anti-angiogenic gene products include endostatin; tumstatin; angiostatin; pigment epithelium-derived factor (PEDF), and fusion proteins or antibodies that are specific for pro-angiogenic targets and / or their receptors, e.g. the anti-VEGF fusion proteins sFLTl or Eylea, the VEGF- specific antibodies Lucentis™ and Avastin™, etc.

[0087] In some preferred embodiments, the gene product(s) delivered by the subject AAV variants act to inhibit angiogenesis. In certain preferred embodiments, the gene product(s) delivered by the subject AAV variants act to inhibit the activity of one or more mammalian VEGF proteins selected from the group consisting of VEGFa, VEGFb, VEGFc, VEGFd and PGF. In particularly preferred embodiments, the gene product(s) delivered by the subject AAV variants inhibit the activity of VEGFa. VEGFa has 9 isoforms generated by alternative splicing, the most physiologically relevant of which is VEGF165. VEGFa levels have been found to be elevated in the vitreous of patients with wet age-related macular degeneration, diabetic macular edema and retinal vein occlusion. Gene product(s) which inhibit the activity of VEGFa in the eye and which are therefore effective to treat patients with elevated vitreous VEGFa include, but are not limited to, Aflibercept, Ranibizumab, Brolucizumab, Bevacizumab, and soluble fms-liketyrosine kinase 1 (sFLTl) (GenBank Acc. No. U01134). Other gene products that can be encoded by a nucleic acid as herein describe include, without limitation, aflibercept, ranibizumab, a single-chain version of ranibizumab (sc-ranibizumab LH or HL), brolucizumab, sc-ranibizumab fused to the IgG Fc domain (sc-Ranibizumab-Fc), or brolucizumab fused to the IgG Fc domain (brolucizumab-Fc), as described in U. S. Patent No. 11,766,489, the entire contents of which are incorporated herein by reference.

[0088] In some embodiments, the nucleic acid comprises (i) a nucleotide sequence encoding a first anti-angiogenic polypeptide (e.g., aflibercept) and (ii) a nucleotide sequence encoding one or more interfering RNA molecule(s) that reduce expression of one or more pro-angiogenic target genes. In some embodiments, the RNA molecule is a short hairpin RNA (shRNA). In other embodiments, the RNA molecule is a primary miRNA molecule. In some embodiments, the nucleic acid comprises an expression cassette comprising (i) a nucleotide sequence encoding a first anti-angiogenic polypeptide, operably linked to an expression control sequence and (ii) a nucleotide sequence encoding an interfering RNA molecule that reduces expression of one or more pro-angiogenic target genes, operably linked to an expression control sequence. In some embodiments, the nucleotide sequence encoding the anti-angiogenic polypeptide and the nucleotide sequence encoding the interfering RNA molecule are operably linked to distinct expression control sequences. In preferred embodiments, expression of the anti-angiogenic polypeptide and the interfering RNA molecule are driven by a common (i.e., the same) expression control sequence. In some embodiments, the expression control sequence(s) comprise(s) a constitutive promoter such as a CAG or CB A promoter. In other embodiments, the expression control sequence(s) comprise(s) a cell-specific promoter. In other embodiments, the nucleic acid encodes one or more gene products as disclosed in WIPO Publication No.WO2022 / 232178 (e.g., an interfering RNA as set forth in any of Tables 1-3), the entire contents of which are incorporated herein by reference.

[0089] In some particularly preferred embodiments, the anti-angiogenic polypeptide encoded by the nucleic acid is aflibercept. A preferred nucleotide sequence encoding aflibercept, codon-optimized for expression in humans, is provided below:ATGGTTTCTTACTGGGACACCGGCGTGCTGCTGTGTGCCCTGCTTTCTTGTCTGCTGCTGACC GGCTCTAGCAGCGGCTCTGATACCGGCAGACCCTTCGTGGAAATGTACAGCGAGATCCCCGA GATCATCCACATGACCGAGGGCAGAGAGCTGGTCATCCCTTGCAGAGTGACAAGCCCCAAC ATCACCGTGACTCTGAAGAAGTTCCCTCTGGACACACTGATCCCCGACGGCAAGAGAATCAT CTGGGACAGCCGGAAGGGCTTCATCATCAGCAACGCCACCTACAAAGAGATCGGCCTGCTG ACCTGTGAAGCCACCGTGAATGGCCACCTGTACAAGACCAACTACCTGACACACAGACAGA CCAACACCATCATCGACGTGGTGCTGAGCCCTAGCCACGGCATTGAACTGTCTGTGGGCGAG AAGCTGGTGCTGAACTGTACCGCCAGAACCGAGCTGAACGTGGGCATCGACTTCAACTGGG AGTACCCCAGCAGCAAGCACCAGCACAAGAAACTGGTCAACCGGGACCTGAAAACCCAGAG CGGCAGCGAGATGAAGAAATTCCTGAGCACCCTGACCATCGACGGCGTGACCAGAAGTGAC CAGGGCCTGTACACATGTGCCGCCAGCTCTGGCCTGATGACCAAGAAAAACAGCACCTTCGT GCGGGTGCACGAGAAGGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCG GCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACC CCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTG GTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAAT AGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAG AGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCTCCAAG GCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGA CAAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTG GAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACA GCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGCAGATGGCAGCAGGG CAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCC TGAGCCTGTCTCCTGGCAAA (SEQ ID NO:357)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:357 and / or comprises a stop codon (e.g. TGA) at the end of the sequence. In some embodiments, the aflibercept gene product comprises the following amino acid sequence or a sequence at least 90%, 95%, 97%, 98%, or at least 99% identical thereto and optionally does not comprise the terminal lysine:

[0090] MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVT SPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNT IIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKK FLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO:358)

[0091] In some preferred embodiments, a nucleic acid as herein described comprises nucleotide sequence encoding aflibercept and further comprises nucleotide sequence encoding an interfering RNA molecule that targets VEGF-C, VEGF-D, VEGFR1, VEGFR2 and / or VEGFR3.

[0092] In particularly preferred aspects, the interfering RNA molecule targets human VEGF-C and / or human VEGF-D. Representative human VEGF-C sequences can be found at e.g., GenBank Accession numbers NM_005429 and X94216. Representative human VEGF-D sequences can be found, e.g., at GenBank Accession number AJ000185.1. Suitable target sequences within the human VEGF-C gene as well as representative interfering RNA molecules targeting human VEGF-C include those in US Patent No. 7,517,864 (e.g., Table II) and US Patent Application Publication No. US2011 / 0293625 (e.g., SEQ ID Nos: 1-3 and 7-12), the contents of each of which are incorporated herein by reference. In particularly preferred embodiments, the interfering RNA molecule is an miRNA that targets human VEGF-C and comprises a sense strand sequence of CUACCUCAGCAAGACGUUAUU (SEQ ID NO:359) and an antisense strand sequence of AAUAACGUCUUGCUGAGGUAG (SEQ ID NO:360) or sequences at least 80% identical thereto.

[0093] In embodiments where a nucleic acid as herein describes encodes an anti-angiogenic polypeptide and one or more interfering RNAs, the sequence encoding the interfering RNA(s) may be placed within a natural or artificial intron (e.g., an artificial intron, within a transcription control sequence, within a 5' UTR region of a gene, within the coding sequence of a gene or within the 3' UTR region of a gene). In some aspects, the interfering RNA is placed within a synthetic U2 or U12 based intron or within an interferon regulating factor 7 intron 4 (IRF7int4; 93 bp).

[0094] In some preferred aspects, the interfering RNA is placed within an artificial intron in a transcription control sequence. In some preferred aspects, the intron is located within the hybridchicken p-actin and rabbit p-globin intron of the CAG promoter, whereby the intron is cotranscribed within a pre-mRNA by Pol-II and cleaved out of the pre-mRNA by RNA splicing. The spliced intron containing the pre-miRNA structure is further processed into mature miRNA capable of silencing a pro-angiogenic target gene.(0095] In other aspects, the interfering RNA is placed within an artificial intron that is located within the coding sequence of a gene (e.g., encoding aflibercept), whereby the intron is cotranscribed within a pre-mRNA by Pol-II and cleaved out of the pre-mRNA by RNA splicing. The spliced intron containing the pre-miRNA structure is further processed into mature miRNA capable of silencing a pro-angiogenic target gene.{0096] In other aspects, the sequence encoding the interfering RNA is placed within the 5' UTR or 3' UTR region of a gene but is not within an intron, in which case some portion (e.g., 50%) of the transcribed pre-mRNA is translated into the encoded protein and some portion (e.g., 50%) of the transcribed pre-mRNA is processed into active shRNA or miRNA. In some preferred aspects, the interfering RNA is placed within a 3’ UTR region of a gene.

[0097] In some embodiments of the nucleic acids disclosed herein comprising one or more miRNA binding sites, a nucleotide sequence encoding a gene product of interest is operably linked to a constitutive promoter. Suitable constitutive promoters include e.g. cytomegalovirus promoter (CMV) (Stinski et al. (1985) Journal of Virology 55(2): 431-441), CMV early enhancer / chicken p-actin (CBA) promoter / rabbit p-globin intron (CAG) (Miyazaki et al. (1989) Gene 79(2): 269-277, CBSB(Jacobson et al. (2006) Molecular Therapy 13(6): 1074-1084), human elongation factor la promoter (EFla) (Kim et al. (1990) Gene 91(2): 217-223), human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al. (1984) Gene 32(3): 409-417, mitochondrial heavy-strand promoter (Loderio et al. (2012) PNAS 109(17): 6513-6518), ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261: 101-105). In other embodiments, a nucleotide sequence encoding a gene product of interest is operably linked to an inducible promoter. In some instances, a nucleotide sequence encoding a gene product of interest is operably linked to a tissue-specific or cell type-specific regulatory element. For example, in some instances, a nucleotide sequence encoding a gene product of interest is operably linked to aphotoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), e.g., a regulatory element that confers selective expression of the operably linked gene in a photoreceptor cell. Suitable photoreceptor-specific regulatory elements include, e.g., a rhodopsin promoter; a rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); a beta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015); a retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra); an interphotoreceptor retinoid-binding protein (IRBP) gene enhancer (Nicoud et al. (2007) supra); an IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225), an opsin gene promoter (Tucker et al. (1994) PNAS 91:2611-2615), a retinoschisin gene promoter (Park et al. (2009) Gene Therapy 16(7): 916-926), a CRX homeodomain protein gene promoter (Furukawa et al. (2002) The Journal of Neuroscience 22(5): 1640-1647), a guanine nucleotide binding protein alpha transducing activity polypeptide 1 (GNAT1) gene promoter (Lee et al. (2010) Gene Therapy 17:1390-1399), a neural retina-specific leucine zipper protein (NRL) gene promoter (Akimoto et al. (2006) PNAS 103(10): 3890-3895), human cone arrestin (hCAR) promoter (Li et al. (2002) Biochemistry and Molecular Biology 43: 1375-1383), and the PR2.1, PR1.7, PR1.5, and PR1.1 promoters (Ye et al. (2016) Human Gene Therapy 27(1): 72-82)). In some instances, a nucleotide sequence encoding a gene product of interest is operably linked to a retinal pigment epithelia (RPE) cell-specific regulatory element (e.g., a RPE-specific promoter), e.g., a regulatory element that confers selective expression of the operably linked gene in a RPE cell. Suitable RPE-specific regulatory elements include, e.g., an RPE65 gene promoter (Meur et al. (2007) Gene Therapy 14: 292-303), a cellular retinaldehyde-binding protein (CRALBP) gene promoter (Kennedy et al. (1998) Journal of Biological Chemistry 273: 5591-5598), a pigment epithelium-derived factor (PEDF aka serpin Fl) gene promoter (Kojima et al. (2006) Molecular and Cellular Biochemistry 293(1-2): 63-69), and a vitelliform macular dystrophy (VMD2) promoter (Esumi et al. (2004) The Journal of Biological Chemistry 279(18): 19064-19073). In some instances, a nucleotide sequence encoding a gene product of interest is operably linked to a Muller glia cell-specific regulatory element (e.g., a glial-specific promoter), e.g., a regulatory element that confers selective expression of the operably linked gene in a retinal glial cell. Suitable glial-specific regulatory elements include, e.g., a glial fibrillary acidic protein (GFAP) promoter (Besnard et al. (1991) Journal of Biological Chemistry 266(28): 18877-18883). In some instances, a nucleotide sequence encoding a gene product of interest is operably linked to a bipolar cell-specific regulatory element (e.g., abipolar-specific promoter), e.g., a regulatory element that confers selective expression of the operably linked gene in a bipolar cell. Suitable bipolar-specific regulatory elements include, e.g., a GRM6 promoter (Cronin et al. (2014) EMBO Molecular Medicine 6(9): 1175-1190).

[0098] The expression control sequence may also comprise one or more elements downstream of the gene coding sequence such as a Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), which has been shown to enhance AAV transgene expression in the retina.

[0099] Non-Viral Vectors

[0100] In some embodiments, a non- viral vector (e.g., an expression plasmid or synthetic vectors such as a lipid nanoparticle) is provided comprising a nucleic acid as herein described. In some embodiments, the non-viral vector comprises a nucleotide sequence encoding an anti-angiogenic polypeptide (e.g., aflibercept) and / or a nucleotide sequence encoding one or more interfering RNA(s), and one or more miRNA binding sequences (preferably selected from those listed at Table 1 and Table 2) that reduce expression of the anti-angiogenic polypeptide and RNAi molecule in a non-target cell (e.g., a CE cell). Preferably, the non-viral vector is a plasmid comprising an expression cassette comprising a nucleic acid as herein described.

[0101] Viral Vectors

[0102] In some embodiments, a viral vector comprising a nucleic acid as herein described is provided. In preferred embodiments, the viral vector comprises a nucleic acid comprising a nucleotide sequence encoding an anti-angiogenic polypeptide (e.g., aflibercept) and / or a nucleotide sequence encoding one or more interfering RNA(s), and one or more miRNA binding sequences that decrease expression of the anti-angiogenic polypeptide and / or interfering RNA in a non-target cell. Examples of suitable viral vectors include but are not limited to adenoviral, retroviral, lentiviral, herpesvirus and adeno-associated virus (AAV) vectors.

[0103] In a preferred embodiment, the viral vector includes a portion of a parvovirus genome, such as an AAV genome with the rep and cap genes deleted and / or replaced by an expressioncassette comprising sequence encoding a first anti-angiogenic polypeptide (e.g., aflibercept) and nucleotide sequence encoding one or more interfering RNA(s) as herein described and / or comprising nucleotide sequence encoding a first and second anti-angiogenic polypeptide and their associated expression control sequences. The expression cassette is typically inserted adjacent to one or two (i.e., is flanked by) AAV TRs or TR elements adequate for viral replication (Xiao et al., 1997, J. Virol. 71(2): 941-948), in place of the nucleic acid encoding viral rep and cap proteins. Other regulatory sequences suitable for use in facilitating tissuespecific expression in the target cell may also be included.

[0104] In some embodiments, the AAV viral vector comprises a nucleic acid comprising: (a) an AAV2 terminal repeat (b) a transcription control sequence (c) nucleotide sequence encoding an anti-angiogenic polypeptide (d) nucleotide sequence(s) encoding an RNAi molecule as herein described (d) a polyadenylation sequence and (e) an AAV2 terminal repeat, wherein the nucleic acid comprises one or more rniRNA binding sites (preferably selected from those listed at Table 1 and Table 2) that reduce expression of the anti-angiogenic polypeptide and RNAi molecule in a non-target cell (e.g., a CE and / or IPE cell).

[0105] Those skilled in the art will appreciate that an AAV vector comprising a transgene and lacking virus proteins needed for viral replication (e.g., cap and rep), cannot replicate since such proteins are necessary for virus replication and packaging. Helper viruses include, typically, adenovirus or herpes simplex virus. Alternatively, as discussed below, the helper functions (El a, Elb, E2a, E4, and VA RNA) can be provided to a packaging cell including by transfecting the cell with one or more nucleic acids encoding the various helper elements and / or the cell can comprise the nucleic acid encoding the helper protein. For instance, HEK 293 were generated by transforming human cells with adenovirus 5 DNA and now express a number of adenoviral genes, including, but not limited to El and E3 (see, e.g., Graham et al., 1977, J. Gen. Virol. 36:59-72). Thus, those helper functions can be provided by the HEK 293 packaging cell without the need of supplying them to the cell by, e.g., a plasmid encoding them.

[0106] The viral vector may be any suitable nucleic acid construct, such as a DNA or RNA construct and may be single stranded, double stranded, or duplexed (i.e., self-complementaiy as described in WO 2001 / 92551).

[0107] The viral capsid component of the packaged viral vectors may be a parvovirus capsid. AAV Cap and chimeric capsids are preferred. For example, the viral capsid may be an AAV capsid (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 AAV8, AAV9, AAV10, AAV11, AAV 12, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAVrhlO, AAVrh74, RHM4-1, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAV-LK03, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat 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).

[0108] In some embodiments, the viral capsid component of the packaged viral vector is a variant of a native AAV capsid (i.e., comprises one or more modifications relative 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 those described in U. S. Patent Application Publication Number 2019 / 0255192A1 (e.g., comprising the amino acid sequence of any of SEQ ID NOs: 42-59 of 2019 / 0255192A1), the entire contents of which are incorporated herein by reference.

[0109] In a particularly preferred embodiment, the capsid comprises a VP1 capsid protein comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:361. In preferred embodiments, the rAAV vector comprises a capsid with a variant capsid protein comprising the following amino acid sequence or a sequence at least 80%, 90%, 95% or 99% identical thereto:MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKAAERHKDDSRGLVLPGYKYLGPFN GLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNL GRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNF GQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHC DSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFH CHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSE YQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRT GNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQA GASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTN LQRGNLAISDQTKHARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFH PSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKEN SKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ IDNO:361)

[0110] The variant AAV capsid protein of SEQ ID NO:361 contains the following modifications relative to native AAV2 capsid: (i) a proline (P) to alanine (A) mutation at amino acid position 34, which is located inside the assembled capsid (VP1 protein only), and (ii) an insertion of 10 amino acids (leucine-alanine-iso leucine-serine-aspartic acid-glutamine-threonine-lysine-histidine-alanine / LAISDQTKHA (SEQ ID NO:362)) at amino acid position 588, which is 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:361 and comprising a P34A substitution and an LAISDQTKHA (SEQ ID NO:362) peptide insertion in the GH loop of the capsid, e.g., between two adjacent amino acids at a position between amino acids 570 and 611 of VP1, preferably between amino acids 588 and 589 of VP1 (numbering is relative to native AAV2 VP1 capsid).

[0111] In certain embodiments, lhe capsid protein comprises a peptide insertion in the GH-loop of the capsid protein relative to a corresponding parental AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence ISDQTKH (SEQ ID NO:363), preferably wherein the insertion peptide has from 1 to 3 spacer amino acids (Y1-Y3) at the amino and / or carboxyl terminus of amino acid sequence ISDQTKH (SEQ ID NO: 363), wherein each of Y1-Y3 is independently selected from Ala, Leu, Gly, Ser, Thr, and Pro. In specific embodiments, the peptide insertion comprises the amino acid sequence LAISDQTKHA (SEQ ID NO:362), preferably wherein the insertion site 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 relative to VP1 capsid of AAV2 or one or more corresponding substitutions in another AAV serotype, preferably wherein the capsid protein comprises a P34A amino acid substitution relative to VP1 capsid of AAV2 or the corresponding substitution in another AAV serotype.

[0112] In some embodiments, the capsid protein, when present in an AAV virion, confers increased infectivity of a retinal cell compared to the infectivity of a retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein. For example, in some embodiments, the variant capsid proteins disclosed herein, when present in an AAV virion, confer more efficient transduction of primate retinal cells than AAV virions comprising the corresponding parental AAV capsid protein or wild-type AAV capsid protein, e.g. the retinal cells take up more AAV virions comprising the subject variant AAV capsid protein than AAV virions comprising the parental AAV capsid protein or wild-type AAV. In some such embodiments, the AAV variant virion or variant rAAV exhibits at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold, increased transduction of a retinal cell, compared to the transduction of the retinal cell by a wild-type AAV virion or rAAV comprising the corresponding parental AAV capsid protein.

[0113] A full complement of AAV Cap proteins includes VP1, VP2, and VP3. The ORF comprising nucleotide sequences encoding AAV VP capsid proteins may comprise less than a full complement AAV Cap proteins or the full complement of AAV Cap proteins may be provided.

[0114] The invention includes packaging cells, which are encompassed by "host cells," which may be cultured to produce packaged viral vectors of the invention. The packaging cells of the invention generally include cells with heterologous (1) viral vector function(s), (2) packaging function(s), and (3) helper function(s). Each of these component functions is discussed in the ensuing sections.

[0115] Initially, the vectors can be made by several methods known to skilled artisans (see, e.g., WO 2013 / 063379). A preferred method is described in Grieger, et al. 2015, Molecular Therapy 24(2):287-297, the contents of which are incorporated by reference herein for all purposes. Briefly, efficient transfection of HEK293 cells is used as a starting point, wherein an adherent HEK293 cell line from a qualified clinical master cell bank is used to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allow for rapid and scalable rAAV production. Using the triple transfection method (e.g., WO 96 / 40240), the suspension HEK293 cell line generates greater than 105vector genome containing particles(vg) / cell or greater than 1014vg / L of cell culture when harvested 48 hours post-transfection. More specifically, triple transfection refers to the fact that the packaging cell is transfected with three plasmids: one plasmid encodes the AAV rep and cap genes, another plasmid encodes various helper functions (e.g., adenovirus or HSV proteins such as El a, Elb, E2a, E4, and VA RNA, and another plasmid encodes the transgene and its various control elements.

[0116] To achieve the desired yields, a number of variables are optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that resulted in high purity vector preps of AAV serotypes 1-6, 8, 9 and various chimeric capsids. This user-friendly process can be completed within one week, results in high full to empty particle ratios (>90% full particles), provides post-purification yields (>1 x 1013vg / L) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. This scalable manufacturing technology has been utilized to manufacture 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 into patients. In addition, a minimum of a 5-fold increase in overall vector production by implementing a perfusion method that entails harvesting rAAV from the culture media at numerous time-points post-transfection.

[0117] The packaging cells include viral vector functions, along with packaging and vector functions. The viral vector functions typically include a portion of a parvovirus genome, such as an AAV genome, with rep and cap deleted and replaced by the first anti-angiogenic polypeptide sequence that inhibits the activity of VEGF-A and at least one synthetic RNA molecule or a second anti-angiogenic polypeptide sequence and its associated expression control sequences. The viral vector functions include sufficient expression control sequences to result in replication of the viral vector for packaging. Typically, the viral vector includes a portion of a parvovirus genome, such as an AAV genome with rep and cap deleted and replaced by the 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. Appropriate expression control sequences areincluded, such as a tissue-specific promoter and other regulatory sequences suitable for use in facilitating tissue-specific expression of the transgene in the target cell. The transgene is typically a nucleic acid sequence that can be expressed to produce a therapeutic polypeptide or a marker polypeptide.

[0118] The terminal repeats (TR(s)) (resolvable and non-resolvable) selected for use in the viral vectors are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5 and 6 being preferred. Resolvable AAV TRs need not have a wild-type TR sequence (e.g., a wild-type sequence may be altered by insertion, deletion, truncation or missense mutations), as long as the TR mediates the desired functions, e.g., virus packaging, integration, and / or provirus rescue, and the like. The TRs may be synthetic sequences that function as AAV inverted terminal repeats, such as the "double-D sequence" as described in U. S. Pat. No. 5,478,745 to Samulski et al., the entire disclosure of which is incorporated in its entirety herein by reference. Typically, but not necessarily, the TRs are from the same parvovirus, e.g., both TR sequences are from AAV2.

[0119] The packaging functions include capsid components. The capsid components are preferably from a parvoviral capsid, such as an AAV capsid or a chimeric AAV capsid function. Examples of suitable parvovirus viral capsid components are capsid components from the family Parvoviridae, such as an autonomous parvovirus or a Dependovirus. For example, the capsid components may be selected from AAV capsids, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrhlO, AAVrh74, RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAV Hu.26, AAV 1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9, AAV2i8G9, AAV2-TT, AAV2- TT-S312N, AAV3B-S312N, and AAV-LK03, and other novel capsids as yet unidentified or from non-human primate sources. Capsid components may include components from two or more AAV capsids.

[0120] The packaged viral vector generally includes sequence encoding one or more anti-angiogenic polypeptides and / or interfering RNAs as herein described and corresponding expression control sequence(s) flanked by TR elements, referred to herein as the "transgene" or "transgene expression cassette," sufficient to result in packaging of the vector DNA and subsequent expression of the interfering RNA and / or gene sequence in the transduced cell (e.g., aphotoreceptor). The viral vector functions may, for example, be supplied to the cell as a component of a plasmid or an amplicon. The viral vector functions may exist extrachromosomally within the cell line and / or may be integrated into the cell's chromosomal DNA.

[0121] Any method of introducing the nucleotide sequence carrying the viral vector functions into a cellular host for replication and packaging may be employed, 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 wherein the viral vector functions are provided by transfection using a virus vector; standard methods for producing viral infection may be used.

[0122] The packaging functions include genes for viral vector replication and packaging. Thus, for example, the packaging functions may include, as needed, functions necessary for viral gene expression, viral vector replication, rescue of the viral vector from the integrated state, viral gene expression, and packaging of the viral vector into a viral particle. The packaging functions may be supplied together or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon, a Baculovirus, or HSV helper construct. The packaging functions may exist extrachromosomally within the packaging cell, but are preferably integrated into the cell's chromosomal DNA. Examples include genes encoding AAV Rep and Cap proteins.

[0123] The helper functions include helper virus elements needed for establishing active infection of the packaging cell, which is required to initiate packaging of the viral vector.Examples include functions derived from adenovirus, baculovirus and / or herpes virus sufficient to result in packaging of the viral vector. For example, adenovirus helper functions will typically include adenovirus components Ela, Elb, E2a, E4, and VA RNA. The packaging functions may be supplied by infection of the packaging cell with the required virus. The packaging functions may be supplied together or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon. See, e.g., pXR helper plasmids as described in Rabinowitz et al., 2002, J. Virol. 76:791, and pDG plasmids described in Grimm et al., 1998, Human Gene Therapy 9:2745-2760. The packaging functions may exist extrachromosomally within the packaging cell, but are preferably integrated into the cell's chromosomal DNA (e.g., El or E3 in HEK 293 cells).

[0124] Any suitable helper virus functions may be employed. For example, where the packaging cells are insect cells, baculovirus may serve as a helper virus. Herpes virus may also be used as a helper virus in AAV packaging methods. Hybrid herpes viruses encoding the AAV Rep protein(s) may advantageously facilitate for more scalable AAV vector production schemes.

[0125] Any method of introducing the nucleotide sequence carrying the helper functions into a cellular host for replication and packaging may be employed, including but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal, embodiments wherein the helper functions are provided by transfection using a virus vector or infection using a helper virus; standard methods for producing viral infection may be used.

[0126] Any suitable permissive or packaging cell known in the art may be employed in the production of the packaged viral vector. Mammalian cells or insect cells are preferred. Examples of cells useful for the production of packaging cells in the practice of the invention include, for example, human cell lines, such as VERO, WI38, MRC5, A549, HEK 293 cells (which express functional adenoviral El under the control of a constitutive promoter), B-50 or any other HeLa cells, HepG2, Saos-2, HuH7, and HT1080 cell lines. In one aspect, the packaging cell is capable of growing in suspension culture, more preferably, the cell is capable of growing in serum-free culture. In one embodiment, the packaging cell is a HEK293 that grows in suspension in serum free medium. In another embodiment, the packaging cell is the HEK293 cell described in U. S. Pat. No. 9,441,206 and deposited as ATCC No. PTA 1 274. Numerous rAAV packaging cell lines are known in the art, including, but not limited to, those disclosed in WO 2002 / 46359. In another aspect, the packaging cell is cultured in the form of a cell stack (e.g., 10-layer cell stack seeded with HEK293 cells).

[0127] Cell lines for use as packaging cells include insect cell lines. Any insect cell which allows for replication of AAV and which can be maintained in culture can be used in accordance with the present invention. Examples include Spodoptera frugiperda, such as the Sf9 or Sf21 cell lines, Drosophila spp. cell lines, or mosquito cell lines, e.g., Aedes albopictus derived cell lines. A preferred cell line is the Spodoptera frugiperda Sf9 cell line. The following references are incorporated herein for their teachings concerning use of insect cells for expression ofheterologous polypeptides, methods of introducing nucleic acids into such cells, and methods of maintaining such cells in culture: Methods in Molecular Biology, ed. Richard, Humana Press, N J (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: 646-4650; 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. Pat. No.6,204,059.00128] Virus capsids according to the invention can be produced using any method known in the art, e.g., by expression from a baculovirus (Brown et al., (1994) Virology 198:477-488). As a further alternative, the virus vectors of the invention can be produced in insect cells using baculovirus vectors to deliver the rep / cap genes and rAAV template as described, for example, by Urabe et al., 2002, Human Gene Therapy 13:1935-1943.

[0129] In another aspect, the present invention provides for a method of rAAV production in insect cells wherein a baculovirus packaging system or vectors may be constructed to carry the AAV Rep and Cap coding region by engineering these genes into the polyhedrin coding region of a baculovirus vector and producing viral recombinants by transfection into a host cell. Notably when using Baculovirus production for AAV, preferably the AAV DNA vector product is a self- complementary AAV like molecule without using mutation to the AAV ITR. This appears to be a by-product of inefficient AAV rep nicking in insect cells which results in a self-complementary DNA molecule by virtue of lack of functional Rep enzyme activity. The host cell is a baculovirus-infected cell or has introduced therein additional nucleic acid encoding baculovirus helper functions or includes these baculovirus helper functions therein. These baculovirus viruses can express the AAV components and subsequently facilitate the production of the capsids.

[0130] During production, the packaging cells generally include one or more viral vector functions along with helper functions and packaging functions sufficient to result in replication and packaging of the viral vector. These various functions may be supplied together or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon, and they may exist extrachromosomally within the cell line or integrated into the cell's chromosomes.

[0131] The cells may be supplied with any one or more of the stated functions already incorporated, e.g., a cell line with one or more vector functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA, a cell line with one or more packaging functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA, or a cell line with helper functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA

[0132] The rAAV vector may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors are known in the art and include methods 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. Pat. No. 6,566,118 and WO 98 / 09657.

[0133] Pharmaceutical Compositions and Treatment Methods

[0134] In some embodiments, a pharmaceutical composition comprising a nucleic acid or vector as herein described and a pharmaceutically acceptable excipient is provided.

[0135] In preferred embodiments, the pharmaceutical composition comprises a vector, preferably a recombinant AAV (rAAV) vector as herein described, more preferably an rAAV vector comprising a capsid protein of SEQ ID NO:361 or sequence at least 90% identical thereto and a nucleic acid encoding one or more gene products and comprising one or more miRNA binding sequences selected from those listed at Table 1 and Table 2.

[0136] In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in a human or non-human patient. 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 can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pHbuffering substances, and the like, may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of 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) H. C. Ansel et al., eds., 7thed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rded. Amer.Pharmaceutical Assoc.

[0137] In some embodiments, the pharmaceutical composition comprises 1 x 108to 1 x 1015vector particles or vector genomes, 1 x 1010to 1 x 1013vector particles or vector genomes, or about 1 x 1010, about 2 x 1010, 3x 1010, about 4 x 1010, about 5 x 1010, about 6 x 1010, about 7 x 1010, about 8 x 1010, about 9 x 1010, about 1 x 1011, about 2 x 1011, about 3 x 10’1, about 4 x 1011, about 5 x 1011, about 6 x 1011, about 7 x 1011, about 8 x IO”, about 9 x 1011, about 1 x 1012, about 2 x 1012, about 3 x 1012, about 4 x 1012, about 5 x 1012, about 6 x 1012, about 7 x 1012, about 8 x 1012, about 9 x 1012or about 1 x 1013vector particles or vector genomes. In some aspects, the pharmaceutical composition comprises about 1 x 1011to about 1 x 1012vector particles or vector genomes.

[0138] In some embodiments, the pharmaceutical composition is administered to a subject (e.g., a human) intraocularly, preferably by periocular, subretinal, suprachoroidal, or intravitreal injection. In some preferred embodiments, the pharmaceutical composition is administered via intravitreal and / or subretinal injection, more preferably by a single intravitreal injection.

[0139] In some embodiments, the pharmaceutical composition is administered to a subject in order to treat an ocular disease.

[0140] Ocular diseases that can be treated using a variant rAAV vector or virion and / or method disclosed herein include, but are not limited to, monogenic diseases, complex genetic diseases, acquired diseases, and traumatic injuries. Examples of monogenic diseases include, but are not limited to, Bardet-Biedl syndrome; Batten’s Disease; Bietti’s Crystalline Dystrophy; choroideremia; chorioretinal atrophy; chorioretinal degeneration; cone or cone-rod dystrophies(autosomal dominant, autosomal recessive, and X-linked); congenital stationary night blindness (autosomal dominant, autosomal recessive, and X-linked); disorders of color vision, including achromatopsia (including ACHM2, ACHM3, ACHM4, and ACHM5), protanopia, deuteranopia, and tritanopia; Friedreich’s ataxia; Leber’s congenital amaurosis (autosomal dominant and autosomal recessive), including, but not limited to, LCA1, LCA2, LCA3, LCA4, LCA6, LCA7, LCA8, LCA12, and LCA15; Leber’s Hereditary Optic Neuropathy; macular dystrophy (autosomal dominant and autosomal recessive), including, but not limited to, acute macular degeneration, Best vitelliform macular dystrophy, pattern dystrophy, North Carolina Macular Dystrophy, inherited drusen, Sorsby’s fundus dystrophy, malattia levantanese, and genetically-determined retinopathy of prematurity; ocular-retinal developmental disease; ocular albinism; optic atrophies (autosomal dominant, autosomal recessive, and X-linked); retinitis pigmentosa (autosomal dominant, autosomal recessive, X-linked, and mitochondrially-inherited traits), examples of which include RP1, RP2, RP3, RP10, RP20, RP38, RP40, and RP43; X-linked retinoschisis; Stargardt disease; and Usher syndrome, including, but not limited to, USH1B, USH1C, USH1D, USH1F, USH1G, USH2A, USH2C, USH2D, AND USH3. Examples of complex genetic diseases include, but are not limited to, glaucoma (open angle, angle-closure, low-tension, normal-tension, congenital, neovascular, pigmentary, pseudoexfoliation); age-related and other forms of macular degeneration, both exudative and non-exudative forms (autosomal dominant and autosomal recessive), such as acute macular degeneration, vitelliform macular degeneration; retinopathy of prematurity; and Vogt Koyanagi-Harada (VKH) syndrome. Examples of acquired diseases include, but are not limited to, acute macular neuroretinopathy; anterior ischemic optic neuropathy and posterior ischemic optic neuropathy; Behcet's disease; branch retinal vein occlusion; choroidal neovascularization; diabetic retinopathy, including proliferative diabetic retinopathy and associated complications; diabetic uveitis; edema, such as macular edema, cystoid macular edema and diabetic macular edema; epiretinal membrane disorders; macular telangiectasia; multifocal choroiditis; non-retinopathy diabetic retinal dysfunction; ocular tumors; optic atrophies; retinal detachment; retinal disorders, such as central retinal vein occlusion, proliferative vitreoretinopathy (PVR), retinal arterial and venous occlusive disease, vascular occlusion, uveitic retinal disease; uveal effusion; retinal infective and infiltrative disease; optic nerve diseases such as acquired optic atrophy. Examples of traumatic injuries include, but are not limited to, histoplasmosis; optic nerve trauma; ocular trauma whichaffects a posterior ocular site or location; retinal trauma; viral infection of the eye; viral infection of the optic nerve; a posterior ocular condition caused by or influenced by an ocular laser treatment; posterior ocular conditions caused by or influenced by a photodynamic therapy; photocoagulation, radiation retinopathy; and sympathetic ophthalmia.

[0141] In preferred embodiments, the ocular disease is a VEGF-associated (e.g., VEGF-A- associated) ocular disease. In some embodiments, the VEGF-associated 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 diabetic retinopathy 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; myopia 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, genetic retinal and / or choroidal disease, tumors of the eye, corneal and iris neovascularization.

[0142] In related aspects, a pharmaceutical composition comprising a nucleic acid or vector as herein described for use in the treatment of a VEGF-associated ocular disease (e.g., a VEGF-A-associated ocular disease) or for the manufacture of a medicament for the treatment of a VEGF-associated ocular disease is provided. In other related aspects, an rAAV comprising a nucleic acid as herein described for use in the treatment of a VEGF-associated ocular disease or for the manufacture of a medicament for the treatment of a VEGF-associated ocular disease is provided. In preferred embodiments, the rAAV comprises a capsid sequence of SEQ ID NO:361 or sequence comprising at least 90% identity thereto, and a nucleic acid encoding one or more gene products and comprising one or more miRNA binding sequences selected from those listed at Table 1 and / or Table 2. In some preferred embodiments, the rAAV or pharmaceutical composition comprising same is for intravitreal administration to a subject to treat a VEGF- associated ocular disease.

[0143] In certain preferred embodiments, a method is provided for the treatment and / or prevention of wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema following retinal vein occlusion; diabetic retinopathy; or myopic choroidal neovascularization and all other forms of abnormal ocular and retinal angiogenesis, including but not limited to idiopathic retinal neovascularization, neovascular glaucoma, retinopathy of prematurity, radiation retinopathy, central serous retinopathy, diabetic vitreous hemorrhage, pseudoxanthoma elasticum, Coat's and other forms of peripheral retinal neovascularization in a subject (e.g., human subject) by administering to the subject an effective amount of a pharmaceutical composition comprising an rAAV, said rAAV comprising a nucleic acid as herein described. Preferably the rAAV comprises a capsid protein of SEQ ID NO:361 or sequence comprising at least 90% identity thereto and the nucleic acid comprises one or more miRNA binding sites selected from those listed at Table 1 and Table 2. In particularly preferred embodiments, a method is provided for the treatment of wet age-related macular degeneration.

[0144] In some preferred embodiments, the method of treatment comprises administering to a subject a pharmaceutical composition comprising an rAAV, wherein said rAAV comprises (i) capsid protein of SEQ ID NO:361 or a sequence at least 90% identical thereto and (ii) a nucleic acid comprising a nucleotide sequence encoding aflibercept and comprising one or more miRNA binding sequences selected from those set forth at Table 1 and Table 2, preferably wherein the one or more miRNA binding sequences are located in a 5’ UTR and / or 3’ UTR of the aflibercept coding sequence.

[0145] In some preferred embodiments, the method of treatment comprises administering to a subject a pharmaceutical composition comprising an rAAV, wherein said rAAV comprises (i) capsid protein of SEQ ID NO:361 or a sequence at least 90% identical thereto and (ii) a nucleic acid comprising a nucleotide sequence encoding aflibercept and a nucleotide sequence encoding an interfering RNA that reduces expression of VEGF-C, VEGFR-3, wherein the nucleic acid comprises one or more miRNA binding sequences selected from those set forth at Table 1 and Table 2, preferably wherein one or more of the miRNA binding sequences are located in a 5’ UTR and / or 3’ UTR of the aflibercept coding sequence.EXAMPLES

[0146] The following examples illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention in any way. While this invention has been described in relation to its preferred embodiments, various modifications thereof will be apparent to one skilled in the art from reading this application.Example 1

[0147] Retinal pigment epithelium (RPE) cells are the clinical target for many therapeutic ocular genes. Undesirable off-target expression in ciliary body epithelium (CE) cells is problematic as the two cells types quite similar and expression of therapeutic genes in CE cells can contribute to adverse effects (e.g., inflammation) in human patients receiving gene therapy for ocular disease. To alleviate this off-target expression, selective microRNA (miRNA) binding sequences were selected and added to the untranslated region (UTR) of a reporter gene. Expression of the reporter gene in ocular target cells (here, retinal pigment epithelium (RPE) cells) and off-target cells (here, ciliary body epithelium (CE) cells) was assessed following transfection of RPE and CE cells with nucleic acid comprising the reporter gene with and without the miRNA binding sequences.

[0148] Briefly, two copies of the potential miRNA targeting sequences were cloned in the 3 ’UTR region between the transgene coding sequence and polyA signal sequences (see FIG. 1A). The resulting constructs were sequence verified and normalized to the same concentration. These constructs were then transfected to on- and off-target cell types including ARPE19 (human RPE cell line) hTERT-RPE (human RPE cell line), and primary Ciliary Body Epithelial cells (CE) (see FIG. IB). Transfected cells were then imaged and detached for flow cytometry analysis 48 hours post-transfection. The mean fluorescence intensities of the samples were normalized to a construct containing random sequences with no match to the human genome. The results are shown in FIGS. 2A and 2B.

[0149] Table 3 below lists the miRNA binding sequences tested and their selectivity according to the assay described above.Table 3miRNA ARPE19 hTERT CE vs ARPE19 CE vs hTERT Binding strength strengthSequenceSEQ ID NO: 1 1.02 0.93 0.20 0.20SEQ ID NO:2 1.24 1.21 0.24 0.25SEQ ID NO:3 1.10 0.99 0.23 0.26 SEQIDNO:4 1.03 1.07 0.29 0.27 SEQIDNO:5 1.17 1.13 0.34 0.35SEQ ID NO:6 1.05 0.95 0.32 0.36SEQ ID NO:7 1.06 1.28 0.29 0.24SEQ ID NO:8 1.13 1.10 0.31 0.33SEQ ID NO:9 0.96 1.08 0.31 0.27SEQ ID NO: 10 0.99 0.94 0.33 0.34SEQ ID NO: 11 1.61 1.41 0.37 0.42SEQ ID NO: 12 1.02 0.92 0.35 0.38SEQ ID NO: 13 1.27 1.13 0.40 0.46SEQ ID NO: 14 0.85 0.89 0.35 0.35SEQ ID NO: 15 1.22 1.14 0.37 0.37SEQ ID NO: 16 1.24 1.16 0.38 0.42SEQ ID NO: 17 1.17 1.01 0.35 0.40SEQ ID NO: 18 1.09 1.33 0.44 0.36SEQ ID NO: 19 1.30 1.21 0.40 0.41SEQ ID NO:20 1.01 1.30 0.50 0.39SEQ ID NO:21 0.91 0.87 0.52 0.55SEQ ID NO:22 0.84 0.86 0.46 0.45SEQ ID NO:23 1.07 0.99 0.48 0.51

[0150] Out of the miRNA binding sequences tested, the majority display desired selectivity properties. Specifically, 23 candidates are highlighted in Table 3 above. In on-target (RPE) tissues, these candidates are able to maintain transgene expression over 80% compared to a control with a random sequence insert. In the off-target tissues (CE), these candidates reduce transgene expression by over 40% compared to a control with a random sequence insert.

[0151] Recombinant adeno-associated virus (rAAV) comprising a capsid with the amino acid sequence of SEQ ID NO:361 and a heterologous nucleic acid comprising a reporter gene with two copies of each of the miRNA binding sequences in bold in Table 3 above (i.e., those with the best results in the assay described above) in the UTR region were generated and used to transduce RPE and CE cells. On (RPE)- and off-target (CE) cells were transduced at differentmultiplicities of infection (MOI). Cells were cultured in serum-free media for 24 hours before the media was replaced with serum-containing media. Seven days post-transduction, cells were detached and analyzed with flow cytometry. The mean fluorescence intensities of the samples were normalized to a construct with a C AG promoter driving transgene expression without the miRNA binding sequences. The results are shown in FIGS. 3A-3C. Higher than the control normalized GFP mean was observed for some of the candidates in RPE cells. Multiple candidates showed lower normalized GFP mean in CE with lower MOI.

[0152] Conclusion

[0153] Existing miRNA databases were explored for CE cells / tissue to identify differentially-expressed miRNA. Applying this strategy, miRNA target site candidates were identified that reduce expression in the ciliary body and maintain expression in RPE cells. In CE cells, most of the identified miRNA binding sequences resulted in decreased transgene expression compared to the CAG-only control. The effect was more significant at lower MOI. Since the miRNA concentration is limited in the cells, the regulation effect diminishes when a higher amount of miRNA target sites are introduced to the cells. In Iris cells, the miRNA binding sequences didn’t show a significant effect. Most transgene expressions in Iris cells were similar between the miRNA target sites and control across different MOL Overall, a reduction in transgene expression off-target tissue (CE), with expression maintained in on-target tissue (RPE) was observed. Particularly, candidate 5 (comprising a microRNA binding site of SEQ ID NO: 18) showed over 50% transgene expression reduction in CE and maintained over 100% expression in RPE across different MOL Surprisingly, in RPE cells, the presence of the miRNA binding sequences increased transgene expression when compared to the CAG-only control. The effect was more significant at higher MOL Without wishing to be bound by theory, this may be caused by an increase in mRNA stability.Example 2

[0154] The experiments in Example 1 utilized public miRNA databases to determine candidate microRNA (miRNA) binding sequences that could improve selectivity of gene expression in RPE cells relative to ciliary body epithelial cells. However, it is also desirable tosuppress gene expression in other off-target ocular cells such as iris pigment epithelium cells. As such, the aim of the present experiments was to identify candidate sequences that could reduce expression in both the ciliary body and the iris while maintaining expression in both RPE and neural retina.

[0155] However, there is no existing database available containing human iris miRNA expression profiles. Additionally, not a single database contains comprehensive miRNA expression profiles across all ocular tissue types of interest. Moreover, due to data acquisition method differences, comparisons between different tissue types' expression profiles from different databases are typically less accurate. Ideally, expression profiles of different tissues generated with the same method will result in a more accurate comparison. Therefore, a comprehensive miRNA expression profile database across all the tissue types of interest was developed. This database not only enabled identification of potential candidates that fit the current selection criteria, but it can also be used to search for candidates with different criteria in the future. Expression profiles of other tissue types of interest can also be added to the database in the future expansion.

[0156] Human ocular tissue miRNA library construction

[0157] Postmortem human donor eyes were dissected and different tissue types including neural retina, retinal pigment epithelium (RPE), iris, and ciliary body were isolated. miRNAs were extracted from these tissues with commercial miRNA extraction kits. Sequencing libraries were prepared for each sample by incorporating next-generation sequencing adapters and amplifying potential miRNA sequences. The sequencing libraries were then cleaned up, and the quality of the libraries was evaluated with a Bioanalyzer. Sequencing of these libraries was performed on an Illumina MiSeq instrument. A total of 6 pairs of eyes from donors without ocular diseases reported were sequenced. In addition, 4 pairs of eyes from diabetic donors and 4 pairs of eyes from non-human primates (NHP) were also processed in the same manner.

[0158] Differentially expressed miRNA identification

[0159] Over IM reads were obtained for each miRNA library. The adapter sequences of the sequencing output were trimmed off and the pair-end reads were merged into a single read file. A human miRNA reference was constructed based on the miRBase miRNA database. The single merged files of each library were then aligned to the human miRNA reference with the Bowtie aligner. The unaligned reads were realigned to the human reference genome to ensure miRNA sequences were all counted. These two alignment results were then summarized and fed into differentiated expression analysis. Differentiated expression analyses were performed with DeSeq2. In summary, miRNA counts in each library were normalized based on the total number of reads in the library. The normalized read counts were then compared between different tissue types. Candidate miRNAs that have lower expression in RPE and neural retina compared to the ciliary body or iris in 5 out of 6 donors were identified. The corresponding miRNA target sites for these candidates were used in the subsequent screening. A total of 155 miRNA target sites (miRT) were identified and used for downstream lentiviral-based miRNA target site screening (see Table 2).

[0160] A lentiviral screening construct was constructed by incorporating a reporter gene of interest between the CAG promoter and SV40 polyA signal. The screening construct also contains a copGFP reference gene driven by a CMV promoter. The miRTs were incorporated between the reporter gene and the SV40 polyA signal. A repeat of 4 miRTs was included for each construct. Lentiviruses were produced by transfecting these screening constructs and other packaging plasmids in HEK293 cells. The crude viruses were then purified and titered based on their transduction level in HEK293 cells. Three cell types were tested in this screening, including ARPE19 (on-target cell type), primary iris epithelial cell (off-target cell type), and primary ciliary body iris cell (off-target cell type). The screening lentiviruses were transduced into these cells at low MOI to ensure no more than 1 copy of the lentivirus construct was introduced into each cell. 4 days post-transduction, the supernatant of the cell culture was collected for reporter gene ELISA, and the cells were collected for DNA / RNA extraction and flow cytometry.Reporter gene ELISA data were normalized to GFP medium fluorescence intensity (MFI) in flow cytometry to control transduction differences between the wells. DNA and RNA levels of the reporter gene, the GFP, and the housekeeping gene were measured on ddPCR. DNA / RNA signals were first normalized to housekeeping genes to control sampling differences and thennormalized to GFP to control transduction differences. Data of each miRT candidate was then compared to a control without miRT sites to generate the relative expression level. The ideal candidate should be high in on-target cell type (>80%) and low in off-target cell types (<50%). Top candidates were selected based on their expression profile across these cell models. The selectivity of each candidate was calculated by averaging the ELISA relative expression level in off-target tissue and dividing by the level in on-target tissue. Top candidates were then ranked based on their selectivity.

[0161] Candidate miRNA target site validation in AAV

[0162] Eleven top candidates were selected based on their performance in the lentivirus screening and their bio-informatic profile for validation in AAV format. To validate these candidates in AAV format, a 4X repeat of each candidate was incorporated in an AAV construct between an aflibercept gene and an SV40 polyA signal sequence. Briefly, the AAV construct comprises a CAG promoter, with an artificial microRNA sequence (ACCAATTACATGTGGAATAATC; SEQ ID NO:364) targeting VEGF-C located within the intron region of the CAG promoter, driving expression of aflibercept protein. AAV were produced by transfecting these constructs with helper plasmids. Crude AAV were purified and their titers were measured with ddPCR. AAV validation was performed in three cell types, iPSC-derived RPE, primary iris epithelial cell, and primary ciliary body iris cell. 7 days posttransduction, tissue culture supernatant was collected for ELISA and the cells were harvested for DNA / RNA quantification. Data was obtained similarly to the previously described lentivirus screening. However, data were not normalized to GFP since it is not presented in the AAV construct, but were normalized with DNA ddPCR data.

[0163] Materials

[0164] miRNA library construction

[0165] Human post-mortem donor eyes were sourced from VisionGift and Eversight, with tissues received within 48 hours of collection. Desired tissue types were carefully dissected, and RNA was isolated using the miRNeasy Tissue / Cells Advanced Mini Kit. Small RNAs, includingmiRNAs, were then purified using the Zymo Research RNA Clean and Concentrator Kit. RNA quality was assessed using the Agilent 2100 Bioanalyzer. miRNA libraries were prepared with the NEBNext Multiplex Small RNA Prep Kit for Illumina, and sequencing was carried out on the Illumina MiSeq system.

[0166] Lentivirus screening

[0167] The target sites of the desired miRNAs were cloned into a lentiviral backbone, and the lentivirus was produced and titrated. Lentiviral particles were transduced into representative on-target (ARPE19 cell line, ATCC) and off-target (primary ciliary body epithelial cells and primary pigmented iris epithelial cells from ScienCell) cell types. On the day of harvesting, cell supernatants were collected for transgene expression analysis using the Eagle Biosciences ELISA Assay Kit. Cells were then harvested for flow cytometry analysis (BD Biosciences Flow Cytometer) and DNA / RNA isolation using the Zymo Research Quick-DNA / RNA MagBead Kit. Droplet Digital PCR was performed on isolated DNA and RNA with the QX200 Droplet Digital PCR System.

[0168] AAV validation

[0169] The miRNA target sites with the desired profile from lentiviral screening were cloned into an AAV backbone, followed by AAV production and titration for further validation. The AAVs were transduced into representative on-target cell types (ARPE19 cell line and iPSC-derived retinal pigment epithelial cells) and off-target cell types (primary ciliary body epithelial cells and primary pigmented iris epithelial cells from ScienCell). On the day of harvest, cell supernatants were collected for transgene expression analysis using the Eagle Biosciences ELISA Assay Kit. Cells were harvested for flow cytometry analysis (BD Biosciences Flow Cytometer) and DNA / RNA isolation using the Zymo Research Quick-DNA / RNA MagBead Kit. Droplet Digital PCR was performed on isolated DNA and RNA using the QX200 Droplet Digital PCR System.

[0170] Conclusions

[0171] Bioinformatic analysis of ocular tissue miRNA database

[0172] Significant different miRNA expression levels were observed between the miRNA database of different ocular tissues. Ideal miRNA candidates should achieve a high expression level in off-target tissue types while the expression level of on-target tissue types should remain low. Interestingly, some miRNA achieved 100-700 fold expression differences between the on- target tissue compared to an off-target tissue. Based on the bioinformatic profile of each candidate, 155 candidates were selected to further investigate in lentiviral format. These candidates exhibit a high expression level in at least one of the off-target tissue and maintain low expression in both on-target tissues. This pattern is observed in at least 5 out 6 pair of eyes tested.

[0173] Lentiviral-based miRNA target site screening

[0174] The ideal candidate should exhibit a high relative expression level in on-target tissue and a low relative expression level in off-target tissue. Interestingly, this expression pattern is observed in most of the candidates tested (fig.4). Based on a combination of their performance in lentiviral screening and bioinformatic data, top candidates were selected. The top candidates were as follows: hsa-mir-184 (SEQ ID NO:47), hsa-mir-145-5p (SEQ ID NO:48), hsa-mir-133a- 3p (SEQ ID NO:49), hsa-mir-143-3p (SEQ ID NO:50), hsa-mir-892a (SEQ ID NO:71), hsa-let- 7e-3p (SEQ ID NO: 165), hsa-mir-338-5p (SEQ ID NO:168), hsa-mir-3184-5p (SEQ ID NO:149), hsa-mir-744-3p (SEQ ID NO:177), hsa-mir- 14a-5p (SEQ ID NO:160) and hsa-mir-296-5p (SEQ ID NO: 172). Some candidates, such as hsa-miR-3184-5p, were able to achieve over 100% expression in on-target tissue and reduce off-target tissue expression to below 30%. The 11 top candidates were further validated in AAV format.

[0175] AAV validation

[0176] Similar to lentiviral-based screening, top candidates were identified as maintaining expression in the on-target cell type (RPE) and reduce expression in the off-target cell type (IPE and CE) in the context of AAV transduction. Thus far, one of die potential candidates - hsa-mir- 143-3p (SEQ ID NO:50) - showed the desired profile (see Fig. 5). Fig. 5 compares theexpression level of a construct containing the 4X miRNA binding site repeat (test construct) to a control construct without the repeat in the same cells. "1" indicate the expression level is the same between the test construct and the control. Lower than "1" indicates reduced expression and higher than ” 1 " indicates enhanced expression in the specific cell type.Example 3

[0177] Most of the AAV-based ocular gene therapies target tissues in the posterior compartment of the eye. Limiting AAV transduction and therapeutic payload expression in the anterior eye tissues could further increase specificity and enhance safety of a gene therapy product. To identify tissue-specific miRTs achieving this goal, a comprehensive miRNA profile database across ocular tissues is needed. Though several human ocular miRNA profile databases exist today, there is not a single comprehensive one available encompassing both the posterior and anterior tissues of the eye. To address this knowledge gap, a human ocular miRNA profile database including key posterior and anterior ocular tissues was constructed (Figure 7A). This database was then used to discover differentially expressed miRNAs that met the pre-defined expression criteria (Figure 7B). Next, the corresponding miRTs were incorporated into lentiviral constructs to evaluate the potential of these miRTs to regulate transgene expression in multiple cell models representing ocular tissue types (Figure 7C). Selected candidates that demonstrated the desired expression patterns across different cell types were further evaluated in AAV format (Figure 7D). Several miRTs were identified that were able to reduce transgene expression in anterior ocular cell models while maintaining the expression in posterior ocular cell models. The lead miRT, miRT-143-3p reduces expression by over 70% in anterior ocular cell models. Further investigation showed that the presence of miRT-143-3p has a limited impact on the cellular miRNA and mRNA profile of transduced cells. These findings provided a platform for further ocular tissue-selective miRT discovery and highlighted the potential of utilizing miRTs to finetune therapeutic transgene expression across ocular and other tissue types in AAV therapeutic cassettes.

[0178] Results

[0179] Development of a human ocular tissue miRNA profile database and identification of differentially expressed miRNAs across ocular tissues

[0180] A comprehensive human ocular tissue miRNA profile database is critical to identify potential miRNAs that are differentially expressed in various ocular tissues. Unfortunately, the currently available public human ocular tissue miRNA profile datasets do not include both posterior and anterior ocular tissue types. Therefore, human eyes from six post-mortem donors with no reported ocular diseases were sourced and dissected (Table 4) and Illumina short-read next-generation sequencing was performed on small RNAs isolated from four different tissue types, ciliary body, iris, neural retina, and RPE, key tissues in ocular gene therapies. The sequencing data were used to construct an ocular tissue miRNA profile database that can be analyzed further to identify miRNAs with different expression patterns across these tissue types with gene-differential expression programs.Table 4, Human donor descriptions _Donor ID Gender Age Cause of death Death to Preservation timeDonor 1 Male 39 Leukemia 8h51minDonor 2 Male 70 Respiratory Failure 13h32minDonor 3 Male 70 Respiratory Failure 18h43minDonor 4 Male 70 Kidney Cancer 7h30minDonor 5 Male 54 Colon Cancer 3hl5minDonor 6 Female 68 Cardiac Arrest lh50min

[0181] When the resulting sequences were aligned with a well-annotated human microRNA database (Figure 8A), of the 2656 previously annotated miRNAs, 2517 were identified.Approximately 200 to 600 differentially expressed miRNAs were identified between two distinct tissue types (Figure 8B). Among them, the biggest miRNA profile difference was observed between the iris tissue and the neural retina tissue. 633 miRNAs were differentially expressed with an adjusted p-value lower than 0.05 between these two tissue types (Figure 9C).

[0182] As proof-of-concept, anterior ocular tissues, such as the ciliary body and iris, were defined as off-target tissues and posterior tissues, such as the neural retina and RPE, weredefined as on-target tissues. When using an intravitreal route of administration, one of the most successful routes of administration to introduce drug products to the posterior segment of the eye, there is a potential for all four of these tissue types come in contact with a gene therapy product, especially when capsids with board tropism is used, such as AAV8 and AAV7m8. Therefore, incorporating a payload regulation element into a gene therapy product can further refine its expression across these tissues. To achieve a transgene expression reduction in the anterior ocular tissues with corresponding miRT, bioinformatic analysis of the database was employed to find miRNAs that were expressed at a higher level in ciliary body and iris compared to neural retina and RPE. In the ciliary body, 75 miRNAs were identified that expressed at a higher level than in the neural retina and RPE tissues. When accounting for donor-to-donor differences, 68 of these miRNAs were more enriched in the ciliary body in 5 out of 6 donors (Figure 8C). Similarly, in the iris tissue, 132 miRNAs were expressed at a higher level than in the neural retina and RPE tissues. Among them, this expression patterns were found in 113 miRNAs in 5 out of 6 donors. Given that the goal was to downregulate transgene expression in both the ciliary body and iris tissue, an ideal miRNA candidate would display a higher expression level in both of these tissue types compared to the retina and RPE. Between the higher expressed miRNAs in the ciliary body and the iris, 25 miRNAs are common between both groups (Figure 8C), suggesting that these candidates may achieve the desired transgene regulation profile across all tissue types of interest. Alternatively, miRTs that will only be functional in either ciliary body or iris could be combined in a single construct to achieve the same regulation of expression. From the above candidates, 43 candidates were identified that are unique in the ciliary body tissue, and 87 candidates are unique in the iris tissues (Figure 8C).

[0183] In brief, by analyzing the newly developed ocular miRNA profile database, 25 miRNA candidates that met all the desired expression profile and 130 miRNA candidates met the profile in either ciliary body alone or iris alone were identified (Table 5). The miRTs corresponding to these top 155 miRNA candidates were then incorporated into a transgene¬ containing screening construct to evaluate their function in relevant cell types.Table 5. miRNA Candidate summaryCiliary body Ciliary body vs Iris Iris vs RPE vs vs Neural retina Neural retina RPE Total differentially expressed 559 196 633 347 miRNACandidates meeting all on-targetrequirements in only one off- 43 87target tissueCandidates that meet all on / off- target requirements 25

[0184] Top miRTs candidates exhibit the desired expression profile in ciliary body epithelial, iris epithelial, and RPE cells during the lentiviral screening

[0185] Lentivirus was selected as the platform to screen the miRT candidates in the transgene-expression cassettes, due to its large packaging capacity, straightforward production process, and high transduction efficiency across diverse cell types (Figure 10A). As an anti- VEGF therapeutic transgene, AFLB can be used to treat multiple ocular diseases, such as wet age-related macular degeneration (wet AMD), diabetic retinopathy (DR), and diabetic macular edema (DME). Currently, multiple AFLB-based gene therapy clinical trials are ongoing, with some already in the pivotal phase of the trial. Therefore, Aflibercept (AFLB) was selected as the reporter for this screen to mimic a therapeutic relevant construct. The screening construct has 2 expression cassettes, the first is comprised of the AFLB gene driven by a CAG ubiquitous promoter (CAG) and followed by an SV40 poly adenylation (poly A) termination signal. The second expression cassette comprises a copGFP gene driven by an EFla promoter, which served as a control for normalization of the transduction efficiency. The two expression cassettes were designed to be transcribed in opposite directions to minimize any transcriptional interference. Typically, miRT sites are incorporated between the stop codon and the polyA termination signal in a transgene construct mimicking the endogenous target sites for miRNA, which are frequently located in the 3’UTR. A similar design was adopted in these lentiviral-based screening constructs by including 4 copies of the miRT candidate between the stop codon of AFLB and theSV40 polyA termination signal, following one of the common designs for miRT containing constructs.

[0186] All lentivirus constructs were cloned and manufactured in parallel. Lentivirus titer was determined by p24 ELISA assays and by qPCR following lentiviral transduction in HEK cells. In this screen, the impact of 155 miRTs on the transgene expression level was evaluated in ARPE-19 cell line, primary ciliary body epithelial (CE) cells, and iris pigmented epithelial (IPE) cells considering both the availability and the relevance of the cell types. The multiplicity of infection (MOI) was controlled at approximately 0.3. When excess miRTs are present in a cell, they might exhaust the miRNA pool and exceed the regulation capacity. This current design ensured that no more than one copy of the viral genome was received in a transduced cell, therefore minimized transduction variation and limited regulation saturation. Reporter and control gene expressions at the mRNA and protein level were evaluated 5-days post transduction (Figure 10B). AFLB protein expression was measured by cell culture media supernatant ELISA and copGFP protein expression was measured by flow cytometry. AFLB and copGFP expression at the mRNA level were quantified by ddPCR. To understand the effect of the miRT on AFLB expression at the protein level, AFLB ELISA results were first normalized to copGFP fluorescence intensity of the corresponding sample and then compared to the control construct lacking miRTs to determine the relative expression. A relative expression lower than 1 indicated the miRT reduced protein expression, while a number equal to or higher than 1 indicated the miRT maintained or increased protein expression. Similarly, AFLB mRNA ddPCR data was normalized to the copGFP mRNA ddPCR data of the corresponding sample and compared to the control lacking miRTs to provide insights on the miRTs’ impact at the mRNA transcript level.

[0187] When exploring the relative protein expression level in ARPE19 and CE, over 70% of the candidates (112 / 155) showed lower relative expression in CE than in ARPE-19 (Figure 10C). Interestingly, for 17 candidates, AFLB expression level was maintained or increased in ARPE19, while it decreased to 0.5 or lower in CE compared to the control construct, indicating a strong selection preference. On the other hand, greater than 90 % (145 / 155) of the candidates preferentially reduced expression in IPE over ARPE-19 (Figure 10D). Similar to CE, 30 candidates have AFLB expression levels at or higher than 1 in ARPE-19 and at or lower than 0.5in IPE. In total, 108 candidates expressed at higher levels in ARPE-19 compared to both CE and IPE (Figure 11). In order to get an intuitive illustration of the expression levels across the three cell types, the average relative expression of CE and IPE over the relative expression in ARPE- 19 was designated as a candidate’s expression preference parameter. A candidate with expression preference lower than 1 exhibits a lower relative expression level in the CE and IPE compared to ARPE-1 (Figure 10E). When selecting top candidates for further evaluation, the relative expression in each cell type, the overall expression preference, and their representation in the human ocular miRNA profile database were evaluated. Among the 25 candidates that met all the desired expression profile requirements in the ocular miRNA profile database, five were selected for further evaluation. And additional six were chosen from the candidates who met the desired profile in either ciliary body alone or iris alone in the ocular miRNA profile database. In total, eleven miRT candidates underwent further characterization. The relative expression level of these candidates in ARPE-19 cells ranges from 0.8 to 2, while their relative expression level was between 0.1 and 0.5 in CE and IPE cells (Figure 10F), highlighting the potential of these candidates to achieve substantial differential expression across different ocular tissues. Given that the most important criteria were to regulate transgene protein expression, top candidate selection was primarily based on protein expression data. However, a similar expression pattern across these cell types was also found in the RNA transcript data. (Figure 12)

[0188] The miRT-143-3p within an AAV payload construct decreased transgene expression in ciliary body and iris cell types while maintaining expression in RPE cell types

[0189] One of the applications of these miRT candidates is to modulate transgene expression for AAV-based ocular gene therapy constructs. Therefore, this group of eleven miRT candidates was incorporated into an AAV vector construct for further evaluation (Figure 13 A). Similar to the lentiviral screening constructs, the AAV constructs encoded the AFLB transgene driven by a CAG promoter and regulated by an SV40 polyA termination signal. To mimic a multi-functional therapeutic payload, a miRNA pri-miRNA transcript was embedded in the intron region of the CAG promoter. Four copies of the miRT candidate were placed between the stop codon of the AFLB transcript and the SV40 polyA termination signal. In this characterization study, induced pluripotent stem cells (iPSC) derived RPE were used as opposed to ARPE-19 cell line given thatfewer number of candidates to characterize in addition to CE and IPE. Cells were transduced at a MOI of 5,000 and harvested 7-days post-transduction (Figure 13B). AFLB protein expression was measured by cell culture media supernatant ELISA. AFLB expression at the mRNA level and the transduced viral genome level were quantified by ddPCR. Both the protein and the mRNA expression data were first normalized to transduced viral genome level of each sample to account for transduction differences between samples, followed by comparison to the control lacking miRTs to evaluate relative expression. A relative expression lower than 1 indicated the miRT reduced protein expression, while a number equal to or higher than 1 indicated the miRT maintained or increased protein expression. At the protein level, a reduction in protein expression in one or both off-target cell types (CE and IPE) was observed for 8 out of 11 of the miRT candidates, including miR-143-3p, miR-145-5p, let-7e-3p, miR-338-5p, miR-3184-5p, miR-744-3p, miR-514a-5p, and miR-296-5p (Figure 13D). Three of the eight, miR-143-3p, miR- 145-5p, and miR-338-5p, showed reduction in both off-target cell types (Figure 13D). In addition, for both miR-143-3p and miR-338-5p the protein expression level in the on-target cell type, iPSC-derived RPE, was maintained. When exploring the expression preference data, the expression preference for both miR-143-3p and miR-338-5p is below 0.5 suggesting a strong expression preference in on-target tissue over off-target tissues (Figure 13C). Particularly, miR-143-3p had an expression preference of 0.22, indicating over 4-fold expression differences between the on-target tissue and the off-target tissues. Similar to the relative protein expression data, an expression preference of 0.3 was observed for miR-143-3p based on the relative RNA expression data (Figure 14).

[0190] Minimal impact on the cellular miRNA and mRNA profiles when incorporating the miRT-143-3p in an AAV payload construct

[0191] MicroRNAs (miRNA) play an important role in maintaining cellular homeostasis by regulating miRNA and mRNA levels. Given the interesting expression profile that miRT-143-3p achieved in the on and off-target cell types following AAV transduction, additional studies were performed to better understand its impact on cellular miRNA and mRNA profiles. In this study, the miRT-143-3p containing AAV and the previous AAV control lacking miRT site were transduced into iPSC-derived RPE, CE and IPE. Following transduction, cellular small RNA andmRNA were isolated for next-generation sequencing. The sequencing results were aligned to the miRNA reference or mRNA reference, respectively, and differentially expressed genes were identified by comparing samples transduced with the miRT-containing construct and the control construct.

[0192] Principal component analysis (PCA) for both the miRNA and mRNA profiles demonstrated that samples from the same cell type, with or without the miRT, cluster closely together (Figure 15A and 15B). This suggested that the incorporation of the miRT did not cause substantial deviation in the miRNA and mRNA profiles. In iPSC-derived RPE, CE and IPE no miRNA was found to have log2-fold-change > 2 and padj < 0.0001, when comparing between the miRT-containing sample and the control (Figure 16A, 16B, 16C). This result illustrated that the miRT did not introduce notable miRNA changes. Similarly, no mRNA was found to have log2-fold-change > 2 and padj < 0.0001 in iPSC-RPE (Figure 16F). However, one mRNA in CE and eleven mRNA in IPE were discovered to have log2-fold-change > 2 and padj < 0.0001 in cells transduced with the miRT-containing construct compared to control (Figure 16D, 16E).

[0193] In CE, the one differentially regulated gene was SELP. However, based on the public miRNA target database, SELP is not one of the targets for miR-143-3p. Therefore, the differentiated expression should not be a direct outcome of changing miR-143-3p level in the cell. To understand the functional implications of the eleven genes that were differentially expressed in IPE, gene ontology analysis was performed based on the biological processes related to these genes. Most of the differentially expressed genes are not involved in biological processes associated with IPE functions. However, among these genes, CDKN1 A, and TYMS, do play a role in epithelial cell maturation (Figure 15C and Table 6). In summary, the number of differentially expressed genes is relatively small, but further investigation is needed to better understand the potential impact of these gene expression changes on CE and IPE cellular functions.

[0194] To understand the specificity of the miRTs, an analysis was carried out to identify other miRNAs that may target miRT-143-3p. Since sequences containing a perfect match of the seed region of a miRNA have the potential to be targeted by this miRNA, a comprehensive search was performed for the miRT site to identify seed regions of any annotated humanmiRNAs. The seed regions for four additional miRNAs were found in miRT-143-3p (Figure 17A). Investigation of the expression levels for these potential unintended targeting miRNAs in the human ocular miRNA profile databases generated herein illustrated that their expression levels were at least two orders of magnitude lower than hsa-miR-143-3p in any of the tissues of interest(Figure 17B). These results suggest that any potential unintended targeting miRNAs would have minimal contribution to the transgene expression regulation in the ocular tissues.[00195 J Discussion

[0196] In AAV gene therapy, a diverse group of tissues comes into contact with the therapeutic vector regardless of the route of administration. Refining virus transduction and therapeutic transgene expression to specific tissues improves therapeutic precision and minimizes undesired effects stemmed from off-target transduction or expressions. Engineered AAV capsids can increase the specificity of gene therapy products that utilize them by achieving selective tissue transduction. Complementarity, integrating rniRT sequences is an attractive payload cassette design strategy for targeted gene expression, owing to its small size and no requirement for exogenous effectors. When tissue-specific engineered AAV capsids are combined with targeted payload cassette design, both the viral transduction and therapeutic transgene expression can be substantially limited in off-target tissues. This dual approach reduces the risk of undesired effects arising from viral exposure as well as therapeutic transgene production outside of the targeted tissues. In addition, platform strategy in AAV therapeutics enables rapid clinical development, by leveraging common components (such as capsid with robust clinical data) while customizing other elements (such as transgenes and regulatory elements). In this case, integrating appropriate regulatory elements can leverage clinically validated AAV vectors while providing specificity required for an effective therapeutic product.

[0197] In recent years, ocular tissues have been attractive targets for AAV based gene therapies. In addition to Luxtuma, the approved AAV gene therapy treating inherited retinal dystrophy, multiple AAV based therapeutics for ocular diseases are currently being evaluated in the clinic. Given that a diverse group of tissue and cell types are present in the ocular system, refining therapeutic transgene expression to specific tissues and cells enhances therapeutic precision and minimizes any potential undesired effects stemmed from undesired expressions.Incorporating miRT sequences in the AAV transgene cassette is an attractive technique to achieve differential expression across tissues, owing to its small size and no requirement for exogenous effectors. However, existing miRNA profile databases for ocular tissues are limited and miRNA expression profiles in some key ocular tissue types are not included. To overcome this barrier, this study describes the generation of the first human ocular tissue miRNA expression profile database, including ocular tissues of the anterior and posterior segments of the eye, using human donor ocular tissues. From this database, miRT candidates that met the predefined expression profiles were identified by bioinformatic analysis and screened in lentivirus format. Lead miRT candidates’s expression profile were further characterized in an AAV format. The top miRT candidate, miRT-143-3p, achieved over 70% reduction of transgene protein expression was observed in the off-target cell types (CE and IPE), while the expression in the on-target cell (iPSC-RPE) remained constant

[0198] The ocular miRNA profile database generated herein matches closely with previously published databases. In the ciliary body, miRNAs highly expressed in the previous database, such as hsa-miR-143-3p, hsa-miR-148a-3p and hsa-miR-26a-5p, are identified as some of the most abundant miRNAs in the database generated herein. In fact, out of the top eleven most abundant miRNAs of the previous database, seven of them were found in the top eleven most abundant list and all of them were found in the top thirty-five most abundant list in the database generated herein. Similarly, out of the top twenty most abundant miRNA of the previous database, all of them were found in the top fifty-five most abundant list of the neural retina database generated herein.

[0199] Lentivirus constructs enable high-throughput screening of a large number of candidates before advancing to AAV characterization. However, differences in expression profiles between two systems are to be expected. Some of the sources of these differences include transduction MOIs, vector expression mechanisms, transgene construct variations, experimental cell models and experimental settings. Taken together, this workflow identified candidates that fulfilled the pre-defined expression requirements despite the minor differences between these experimental approaches. For future studies, performing the initial screening in AAV format could minimize some of the variability between the screening and characterizationstages. However, performing high-throughput array screening in an AAV format presents its own challenges, such as vector production.

[0200] During AAV characterization, the miR-143-3p expression preference at the mRNA level was found to be slightly higher than the protein level. It raises the possibility that the miRNA is not only regulating the transgene mRNA amount within the cell through mRNA degradation, but it is also modulating the translation process of these mRNA, which has been previously reported as part of the mRNA's natural functions. In AAV characterization of other candidates, such as miRT-338-5p, the transgene expression in the on-target cells is slightly higher when a miRT site is incorporated into the construct compared to the control without miRTs. This observation has also been previously reported in other miRT-containing constructs. While the exact mechanism has not been elucidated, it could potentially be caused by mRNA transcript stabilization by miRT repeats. However, further studies are needed to verify this hypothesis,|00201 ] Introduction of the lead miRT- 143-3p did not cause any substantial changes to the miRNA profile in iPSC-RPE, CE or IPE. Particularly, it is encouraging to see the introduction of miRT-143-3p does not cause substantial changes of miR-143-3p level among the cell models in this study, given that miR-143-3p is crucial for many cellular pathways in different ocular tissues, such as regulating proliferation of the lens epithelial cells and sternness of the corneal epithelial cells. In IPE cells, GREM1, OGN, TIMP1, TYMS, and CDKN1A were the most differentially expressed mRNA transcripts. Overall, the expression levels of this group of genes were relatively low in the ocular systems compared to other tissue types in the body, suggesting that their contributions to the ocular tissue function can be limited. Nevertheless, additional studies are needed to further illustrate the impact of miRNA and gene expression changes. It is worth noting that other than the introduction of miRT site, the reduction of AFLB expression may also be a contributor to these changes, since it inhibits VEGF signaling pathway by acting as a decoy receptor.65Evaluation of these miRT within another transgene construct would not only provide clarification on this question but also illustrate the potential universality of these constructs.

[0202] When investigating the miRNA profile comparison across the cell models used in this study, some miRNA profile differences were observed between different RPE representing cells (Figure 18A). However, the expression levels of hsa-miR-143-3p remain at a much lower level in all RPE representative cells compared to off-target cell types (Figure 18B), suggesting that miRT-143-3p is likely to function effectively across RPE cell models and in RPE tissue under physiological conditions. Other than RPE, ciliary body and iris cell models are also included in this study. Another important model to be considered for these studies was neural retina cell models. However, a very limited number of human neural retina cell models are commercially available and most of them are poor representations of human neural retina cells in the physiological condition.66Although it is unlikely that these miRT candidates would down-regulate transgene expression in neural retina, given that the corresponding miRNA for all the miRT candidates screened were expressed at low levels in neural retina tissues. Ultimately, in vivo studies are needed to elucidate the performance of top candidates across different ocular cell types.

[0203] Overall, these data demonstrate that miRT can refine transgene expression profiles across ocular cell types and it supports the further investigation of these lead constructs in a more physiologically relevant model, such as in vivo animal studies. Furthermore, continued optimization of the current lead candidates could result in constructs with superior properties. Some further optimization strategies include combination of different miRTs in a single construct, alteration of the miRT copy number or adjustment of the miRT location within the construct. Also, the general miRT development roadmap described here could easily be adapted and applied to other on and off-target tissue of interest. By building the miRNA database for target tissues, screening and characterizing miRT of differentially expressed miRNA, therapeutic constructs that meet the desired expression profiles can be quickly developed for downstream preclinical evaluation.

[0204] Materials and Methods:

[0205] Human ocular tissue miRNA library construction

[0206] Human post-mortem donor eyes were sourced from VisionGift (Portland, Oregon) and Eversight (Ann Arbor, Michigan), with tissues received within 48 hours of collection. Desired tissue types, including neural retina, retinal pigment epithelium (RPE), iris, and ciliary body, were carefully dissected. Total RNA from these tissues was isolated using the miRNeasy Tissue / Cells Advanced Mini Kit (Qiagen, Cat. No. 217604, Venlo, Netherlands). Small RNAs, including miRNAs, were then purified using the RNA Clean and Concentrator Kit (Zymo Research, Cat. No. RIO 14, Irvine, California). RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, California). Sequencing libraries for miRNA were prepared with the NEBNext® Multiplex Small RNA Prep Kit for Illumina® (New England Biolabs, Cat. No. E7330L, Ipswich, Massachusetts) to incorporate the next-generation sequencing adapters and amplify potential miRNA sequences. The sequencing libraries were then cleaned up, and the quality of these libraries were evaluated on an Agilent 2100 Bioanalyzer. The final pooled library was quantified on Qubit (Thermo Fisher Scientific, Waltham, Massachusetts) before it was sequenced on the MiSeq system (Illumina, San Diego, California). A total of 6 pairs of eyes from human donors were sequenced.

[0207] Differentially expressed miRNA identification

[0208] The miRNA library sequencing data was processed using an established pipeline.67,68First, the sequence data qualities of the miRNA libraries were evaluated with FastQC (v0.12.1).69The adapter sequences of the sequencing output were then trimmed off with cutadapt (v5.0),7° and the pair-end reads were merged into a single read file with PEAR (Paired- End reAd merger, v0.9.11).71A human miRNA reference was constructed based on the miRBase miRNA database.38,39The single merged files of each library were then aligned to the human miRNA reference with the Bowtie aligner (v1.3.1).72The unaligned reads were realigned to the human reference genome to ensure miRNA sequences were all counted. These two alignment results were then counted with Samtools (v1.21) and fed into differentiated expression analysis. Differentiated expression analyses were performed with DESeq2 (vl.48.1).37In brief, miRNA counts in each library were normalized using the median of ratios method. The normalized read counts were then compared between different tissue types. The expression pattern of each miRNA was evaluated based on four pre-defined criteria (CB > NR, CB > RPE,Iris > NR, Iris > RPE). Candidates that met both criteria for the CB, or both criteria for the Iris, or all four criteria, in 5 out of 6 donors were selected. The corresponding miRTs for these candidates were used in the subsequent screening. A total of 155 miRTs were identified and used for downstream screening.

[0209] Lentiviral-based miRNA target site screening

[0210] A lentiviral screening construct was built by incorporating a reporter gene of interest (AFLB) between the CAG promoter and SV40 polyA signal. The screening construct also contains a copGFP reference gene driven by an EFla promoter. The miRT candidates, which are the DNA version of the reverse-complement of the miRNA sequences, were incorporated between the reporter gene and the SV40 polyA signal. A repeat of 4 miRTs was included for each construct. Lentiviruses were produced by Genewiz (Azenta Life Sciences, Burlington, Massachusetts). In brief, screening constructs and other packaging plasmids were transfected in HEK293 cells for virus production. The crude viruses were purified and titrated with p24 ELISA and qPCR. Three cell types were tested in this screening, including an on-target cell type ARPE19 (ATCC, Cat. No. CRL-2302, Manassas, Virginia), an off-target cell type primary iris pigmented epithelial cell (IPE, ScienCell, Cat. No. 6560, Carlsbad, California), and an off-target cell type primary ciliary body epithelial cell (CE, ScienCell, Cat. No. 6580, Carlsbad, California). These cells were transduced with the lentiviruses at an MOI of approximately 0.3 to ensure only 1 vector copy was introduced into each cell. Four days post-transduction, the supernatant of the cell culture was collected for reporter gene ELISA, and the cells were collected for DNA and RNA extraction and flow cytometry. The AFLB ELISA was performed with AFLB ELISA Assay Kit (Eagle Biosciences, Cat. No. IG-AA115, Amherst, New Hampshire) according to manufacturer’s instructions, and the flow cytometry was performed on FACSCelesta (BD Biosciences, Franklin Lakes, New Jersey). Detached cells were suspended in PBS with 5% FBS prior to the flow cytometry. Data was analyzed using FlowJo (v10.10.0, BD Biosciences, Franklin Lakes, New Jersey). Reporter AFLB ELISA data were normalized to GFP medium fluorescence intensity (MFI) in flow cytometry to control transduction differences between the wells. DNA and RNA were isolated with Quick-DNA / RNA MagBead Kit (Zymo Research, Cat No. R2131, Irvine, California). RNA levels of the reporter gene, the copGFP, andthe housekeeping gene were measured on Droplet Digital PCR (Bio-rad, QX200 Droplet Digital PCR System, Cat. No. 1864003, Hercules, California). RNA signals were first normalized to the housekeeping gene to control sampling differences and then normalized to GFP RNA levels to control for transduction differences. Data of each miRT candidate was then compared to a control without miRT sites to generate the relative expression level. The relative expression of ideal candidates should be high in on-target cell type and low in off-target cell types. To further compare miRT candidates, the expression preference of each candidate was calculated by averaging the expression levels in off-target cell types and dividing by the level in on-target cell type. The formula of expression preference is below:r> ■ r CE releative expresslon+IPE relative expression

[0211] Expression preference — - 2 xRPE relative expression

[0212] Characterization of the miRT sites in AA V format

[0213] The miRNA target sites with the desired profile from lentiviral screening were cloned into an AAV backbone, followed by AAV production and titration for further characterization. During the cloning of the AAV cassette plasmid, 4X repeats of these miRT candidates were incorporated in an AAV construct between the reporter AFLB gene and the SV40 polyA signal sequence. The recombinant AAV were then produced via PEI-mediated triple transfection into HEK293 cells. Briefly, HEK 293 cells were cultured in DMEM supplemented with FBS and maintained at 37 °C in a 5% CO2 environment. For virus production, these cells were seeded into CellSTACK vessels and transfected with the pHelper, the Rep-Cap, and the various payload cassette plasmids. Three to four days following transfection, cells and supernatant were collected, lysed, endonuclease treated, then clarified via sterile filtration prior to storage at -80 C. For further purification, harvest material was thawed, clarified and loaded onto affinity resins. Elution was performed at low pH and immediately neutralized. Purified lots were formulated in a Tris-based buffer system containing NaCl and 0.005% Pluronic F-68. Virus titers were measured with ddPCR. Purified lots were stored at -80 °C until use.

[0214] AAV characterization was performed in three human cell types: iPSC-derived RPE cell, primary iris pigmented epithelial cell, and primary ciliary body epithelial cell. RPE cellswere maintained in X-VIVO- 10 medium (Lonza, Cat. No. BEBP02-055Q, Walkersvill, Maryland) supplemented with 10. M RHO / ROCK pathway inhibitor Y-27632 (Stemcell Technologies, Inc., Cat. No. 72304, Vancouver, Canada) at 37 °C and 5% CO2 in normoxic conditions until maturation (30-33 days post-seeding). When the cultures were mature (RPE cell) or confluent (primary cells), cells were transduced at an MOI of 5000. Media was changed 3 days later, and 7 days post-transduction culture supernatant was collected for ELISA, and cells were harvested for DNA / RNA quantification similar to the previously described lentivirus screening, except for no cells were collected for flow cytometry. Both protein and RNA expression data were normalized to the viral genome number to control for the transduction level differences. Data for each miRT candidate was then compared to the control without miRT sites to generate the relative protein or RNA expression level. The relative expressions of miRT- 143-3p construct between on-target tissue and each off-target tissues were compared using two way- ANOVA test.

[0215] Further evaluation of the impact of miRT-143-3p

[0216] The evaluations of miRNA and mRNA profile alterations were performed in the same cell types used for the AAV characterization study, iPSC-derived RPE cell, primary iris epithelial cell, and primary ciliary body iris cell. 7 days post-transduction with the AAV construct containing miRT-143-3p and AAV control construct without miRTs, cells were harvested for total RNA purification with miRNeasy Tissue / Cells Advanced Mini Kit (Qiagen, Cat. No. 17604, Venlo, Netherlands). Small and large RNAs were then purified with the RNA Clean and Concentrator Kit (Zymo Research, Cat. No. R1014, Irvine, California). RNA quality was assessed using the Agilent 2100 Bioanalyzer. Sequencing libraries for miRNA were prepared with the NEBNext® Multiplex Small RNA Prep Kit for Illumina® (New England Biolabs, Cat. No. E7490L, Ipswich, Massachusetts) to incorporate the next-generation sequencing adapters and amplify potential miRNA sequences. NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Cat. No. E7770L, Ipswich, Massachusetts) was used to enrich the mRNA sequences and NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (New England Biolabs, Cat. No. E7770L, Ipswich, Massachusetts) was used to generate the mRNA sequencing libraries. They were then cleaned up, and the quality of thelibraries was evaluated on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, California). The final pooled library was quantified on Qubit before it was sequenced on the Illumina system. A process similar the one described in the Differentially expressed miRNA identification section was used to analyze miRNA and mRNA profile data to determine differential expressed miRNAs and mRNAs, except for mRNA reference was used to align the mRNA reads. Differentially expressed mRNAs were compared to a list of miR-143-3p targets53to determine the potential relationship between the mRNAs and miR-143-3p. Gene ontology study of the differentially expressed mRNA in IPE were performed with topGO (V2.60.1).73

[0217] For unintended targeting miRNA analysis, seed regions of all annotated miRNA were aligned with miRT-143-3p. The miRNA was considered a potential unintended targeting miRNA if the reverse complement of their seed region could be found in the miRT-143-3p. Four potential unintended targeting miRNA were identified. The sequence alignment was generated with ggmsa (v1.15.1).74The normalized expression level of miRNA-143-3p and the potential unintended targeting miRNAs were identified across different tissues in the ocular miRNA database and plotted.

[0218] While the materials and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the method 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.

Claims

CLAIMS1. A vector comprising a heterologous nucleic acid, said nucleic acid comprising a polynucleotide sequence comprising one or more miRNA target sequences as set forth in SEQ ID NOs:50, 49, 47, 48, 51-201 and 1-23 or a reverse complement thereof, operably linked to a transgene, and wherein the polynucleotide sequence further comprises a promoter operably linked to the transgene.

2. The vector according to claim 1, wherein the polynucleotide sequence comprises at least one miRNA target site selected from the group consisting of an hsa-mir-143-3p (GAGCTACAGTGCTTCATCTCA; SEQ ID NO:50), hsa-mir-133a-3p (CAGCTGGTTGAAGGGGACCAAA; SEQ ID NO:49), hsa-mir-184 (ACCCTTATCAGTTCTCCGTCCA; SEQ ID NO:47), hsa-mir-145-5p (AGGGATTCCTGGGAAAACTGGAC; SEQ ID NO:48), hsa-mir-892a (CTACGCAGAAAGGACACAGTG; SEQ ID NO:71), hsa-let-7e-3p (GGAAAGCTAGGAGGCCGTATAG; SEQ ID NO: 165), hsa-mir-338-5p (CACTCAGCACCAGGATATTGTT: SEQ ID NO: 198), hsa-mir-3184-5p (AAAAGCTCGGTCTGAGGCCCCTCA; SEQ ID NO: 149), hsa-mir-744-3p (AGGTTGAGGTTAGTGGCAACAG; SEQ ID NO: 177), hsa-mir-514a-5p (CATGATTGTCACTCTCCAGAGTA; SEQ ID NO: 160) and hsa-mir-296-5p (ACAGGATTGAGGGGGGGCCCT; SEQ ID NO: 172).

3. The vector according to claim 1 or 2, wherein the polynucleotide sequence comprises two, three, four or five miRNA target sites.

4. The vector according to claim 3, wherein the miRNA target sites are separated by spacer sequences.

5. The vector according to any one of claims 1 -4, wherein the polynucleotide sequence comprises at least one hsa-mir-143-3p target site (GAGCTACAGTGCTTCATCTCA; SEQ ID NO:50), preferably wherein the polynucleotide comprises two, three, four or more hsa-mir-143-3p target sites.

6. The vector according to any one of claims 1 -5, wherein expression of the transgene in ciliary body epithelium and / or iris pigment epithelium cells is at least 25% lower, at least 50% lower, or at least 75% lower than the expression of the transgene in retinal pigment epithelial cells under the same conditions and / or wherein expression of the transgene in ciliary body epithelium and / or iris pigment epithelium cells is at least 25% lower, at least 50% lower, or at least 75% lower than the expression of the transgene in ciliary body epithelium and / or iris pigment epithelium cells from an otherwise identical polynucleotide that does not comprise the one or more miRNA target sequences under the same conditions.

7. The vector according to any one of claims 1 -6, wherein the one or more miRNA target sites are located after the transgene in the 5’ to 3’ direction, preferably between a stop codon and a polyadenylation signal.

8. The vector according to any of claims 1 to 6, wherein the one or more miRNA target sites are located between the promoter and the transgene on the nucleic acid.

9. The vector according to any of claims 1 to 6, wherein the one or more miRNA target sequences are located within a 5’ untranslated region (UTR) or within a 3’ UTR on the nucleic acid.

10. The vector according to any of claims 1 to 9, wherein the promoter is a ubiquitous promoter or a tissue-specific promoter.

11. The vector according to any of claims 1 to 10, wherein the promoter is a ubiquitous promoter, preferably a CAG promoter.

12. The vector according to any of claims 1 to 13, wherein the transgene is selected from ADP-ribosylation factor-like 6 (ARL6); BBSome interacting protein 1(BBIP1); BBSome protein 1 (BBS1); BBSome protein 2 (BBS2); BBSome protein 4 (BBS4); BBSome protein 5 (BBS5); BBSome protein 7 (BBS7); BBSome protein 9(BBS9); BBSome protein 10 (BBS 10); BBSome protein 12 (BBS 12); centrosomal protein 290 kDa (CEP290); intraflagellar transport protein 172 (IFT172); intraflagellar transport protein 27 (IFT27); inositol polyphosphate-5-phosphatase E(INPP5E); inwardly-rectifying potassium channel subfamily J member 13 (KCNJ13); leucine zipper transcription factor like-1 (LZTFL1); McKusick-Kaufman syndrome protein (MKKS); Meckel syndrome type 1 protein(MK. S1); nephronophthisis 3 protein (NPHP1); serologically-defined colon cancer antigen 8 (SDCCAG8); tripartite motif-containing protein 32 (TRJM32); tetratricopeptide repeat domain 8 (TTC8); Batten disease protein (CLN3); Rab escort protein 1 (CHM); (PRDM13); (RGR; (TEAD1); aiylhydrocarbon-interacting receptor protein-like 1 (AIPL1); cone-rod otx-like photoreceptor homeobox transcription factor (CRX); guanylate cyclase activating protein 1 A (GUCA1 A); retinal-specific guanylate cyclase (GUCY2D); phosphatidylinositol transfer membrane-associated family member 3 (PITPNM3); prominin 1 (PROMI); peripherin (PRPH); peripherin 2 (PRPH2); regulating synaptic membrane exocytosis protein 1 (RIMS1); semaphorin 4A (SEMA4A); human homolog of C. elegans uncl 19 protein (UNCI 19); ATP-binding cassette transporter - retinal (ABCA4); ADAM metallopeptidase domain 9 (ADAM9); activating transcription factor 6 (ATF6); chromosome 21 open reading frame 2 (C21orf2); chromosome 8 open reading frame 37 (C8orf37); calcium channel; voltagedependent; alpha 2 / delta subunit 4 (CACNA2D4); cadherin-related family member 1 (protocadherin 21) (CDHR1); ceramide kinase-like protein (CERKL); cone photoreceptor cGMP-gated cation channel alpha subunit (CNGA3); cone cyclic nucleotide-gated cation channel beta 3 subunit (CNGB3); cyclin M4 (CNNM4); guanine nucleotide binding protein (G protein); alpha transducing activity polypeptide 2 (GNAT2); potassium channel subfamily V member 2 (KCNV2); Phosphodiesterase 6C (PDE6C); Phosphodiesterase 6H (PDE6H); proteome of centriole 1 centriolar protein B (POC1B); RAB28 member of RAS oncogene family (RAB28); retina and anterior neural fold homeobox 2 transcription factor (RAX2); 11-cis retinol dehydrogenase 5 (RDH5); RP GTPase regulator-interacting protein 1 (RPGRIP1); tubulin tyrosine ligase-like family member 5 (TTLL5); L-type voltage-gated calcium channel alpha-1 subunit (CACNA1F); retinitis pigmentosa GTPase regulator (RPGR);(GNAT1); (PDE6B); (RHO); CABP4); GPR179); (GRK1); GRM6); LRIT3);SLC24A1); TRPM1); NYX); OPN1LW); OPN1MW); blue cone opsin (OPN1SW); frataxin (FXN); (IMPDH1); (OTX2); CRB1); DTHD1GDF6); IFT140); IQCB1); LCA5); LRAT); NMNAT1); RD3); RDH12); RPE65); SPATA7); TULPI); mitochondiral genes (KSS, LHON, MT-ATP6, MT-TH, MT-TL1, MT-TP, MT-TS2, mitochondrially encoded NADH dehydrogenases [MT-ND]);(BEST1); C1QTNF5EFEMP1); ELOVL4); FSCN2); GUCA1B); HMCN1); IMPG1); RP1L1); TIMP3); DRAM2); MFN2); NR2F1); optic atrophy 1 (OPA1); TMEM126A); TIMM8A); CA4); HK1); KLHL7); NR2E3); NRL); OR2W3); PRPF3); PRPF4);PRPF6); PRPF8); PRPF31); R0M1); retinitis pigmentosa protein 1 (RP1); RP9);SNRNP200); SPP2); TOPORS); ARL2BP); C2orf71); CLRN1); CNGA1); CNGB1); CYP4V2); DHDDS); DHX38); EMC1); EYS); FAM161A); GPR125); HGSNAT); IDH3B); IMPG2); KIAA1549); KIZ); MAK); MERTK); MVK); NEK2); NEURODI); PDE6A); PDE6G); PRCD); RBP3); RLBP1); SLC7A14); USH2A); ZNF408);ZNF513); OFD1); RP2); retmoschisin (RSI); ABHD12); CDH23); CEP250); CIB2); DFNB31); GPR98); BARS); MYO7A); PCDH15); USH1C); USH1G); NDP); PGK1); CAPN5); FZD4); ITM2B); LRP5); MIR204); RBI); TSPAN12); C12orf65); CDH3); MFRP); OAT); PLA2G5); RBP4); RGS9); RGS9BP); ARMS2; ERCC6); FBLN5); HTRA1); TLR3); TLR4); opsin; rhodopsin; channel rhodopsin; halo rhodopsin; aflibercept; ranibizumab; brolucizumab; bevacizumab; soluble fins-like tyrosine kinase 1 (sFLTl); endostatin; tumstatin; angiostatin; pigment epithelium-derived factor (PEDF); a VEGF-specific antibody or antagonist; an ANG2-specific antibody or antagonist, a wild type or genetically engineered complement factor (e.g., complement factor H or a biologically active fragment thereof), and an interfering RNA.

13. The vector according to any of claims 1 to 12, wherein the transgene is for the treatment of an ophthalmic disease selected from Bardet-Biedl syndrome; Batten’s Disease; Bietti’s Crystalline Dystrophy; choroideremia; chorioretinal atrophy; chorioretinal degeneration; cone or cone-rod dystrophies (autosomal dominant, autosomal recessive, and X-linked); congenital stationary night blindness (autosomal dominant, autosomal recessive, and X-linked); disorders of color vision, includingachromatopsia (including ACHM2, ACHM3, ACHM4, and ACHM5), protanopia, deuteranopia, and tritanopia; Friedreich’s ataxia; Leber’s congenital amaurosis (autosomal dominant and autosomal recessive), including, but not limited to, LCA1, LCA2, LCA3, LCA4, LCA6, LCA7, LCA8, LCA12, and LCA15; Leber’s Hereditary Optic Neuropathy; macular dystrophy (autosomal dominant and autosomal recessive), including, but not limited to, acute macular degeneration, Best vitelliform macular dystrophy, pattern dystrophy, North Carolina Macular Dystrophy, inherited drusen, Sorsby’s fundus dystrophy, malattia levantanese, and genetically-determined retinopathy of prematurity; ocular-retinal developmental disease; ocular albinism; optic atrophies (autosomal dominant, autosomal recessive, and X-linked); retinitis pigmentosa (autosomal dominant, autosomal recessive, X-linked, and mitochondrially-inherited traits), examples of which include RP1, RP2, RP3, RP10, RP20, RP38, RP40, and RP43; X-linked retinoschisis; Stargardt disease; and Usher syndrome, including, but not limited to, USH1B, USH1C, USH1D, USH1F, USH1G, USH2A, USH2C, USH2D, AND USH3. Examples of complex genetic diseases include, but are not limited to, glaucoma (open angle, angle-closure, low-tension, normal-tension, congenital, neovascular, pigmentary, pseudoexfoliation); age-related and other forms of macular degeneration, both exudative and non-exudative forms (autosomal dominant and autosomal recessive), such as acute macular degeneration, vitelliform macular degeneration; retinopathy of prematurity; and Vogt Koyanagi-Harada (VKH) syndrome. Examples of acquired diseases include, but are not limited to, acute macular neuroretinopathy; anterior ischemic optic neuropathy and posterior ischemic optic neuropathy; Behcet's disease; branch retinal vein occlusion; choroidal neovascularization; diabetic retinopathy, including proliferative diabetic retinopathy and associated complications; diabetic uveitis; edema, such as macular edema, cystoid macular edema and diabetic macular edema; epiretinal membrane disorders; macular telangiectasia; multifocal choroiditis; non-retinopathy diabetic retinal dysfunction; ocular tumors; optic atrophies; retinal detachment; retinal disorders, such as central retinal vein occlusion, proliferative vitreoretinopathy (PVR), retinal arterial and venous occlusive disease, vascular occlusion, uveitic retinal disease; uveal effusion; retinal infective and infiltrative disease; optic nerve diseases such as acquired optic atrophy.Examples of traumatic injuries include, but are not limited to, histoplasmosis; optic nerve trauma; ocular trauma which affects a posterior ocular site or location; retinal trauma; viral infection of the eye; viral infection of the optic nerve; a posterior ocular condition caused by or influenced by an ocular laser treatment; posterior ocular conditions caused by or influenced by a photodynamic therapy; photocoagulation, radiation retinopathy; and sympathetic ophthalmia, preferably wherein the ophthalmic 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 diabetic retinopathy 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; myopia 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, genetic retinal and / or choroidal disease, tumors of the eye, corneal and iris neovascularization,14. The vector according to any of claims 1 to 14, wherein the vector is a viral vector.

15. The vector according to claim 14, wherein the viral vector is a recombinant adeno- associated virus (rAAV).

16. The vector according to claim 15, wherein the rAAV comprises a capsid protein comprising an amino acid sequence at least 95% identical, at least 98% identical or 100% identical to the entire length of the amino acid sequence set forth as SEQ ID NO:361.

17. The vector according to claim 16, wherein the variant rAAV capsid protein comprises an amino acid sequence having 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:361.

18. The vector according to any one of claims 15 to 17, wherein the rAAV comprises inverted terminal repeats (ITRs) and a polyadenylation sequence and / or a WPRE sequence.

19. The vector according to claim 18, wherein the ITRs are AAV2 ITRs.

20. The vector according to claim 18 or 19, wherein the poly adenylation sequence is an SV40 polyadenylation sequence.

21. A host cell comprising the vector according to any one of claims 1 to 20.

22. A pharmaceutical composition for use in the treatment of an ocular disorder comprising the vector according to any one of claims 12 to 20 and a pharmaceutically acceptable carrier, diluent, excipient or buffer.

23. A method of selectively repressing expression of a heterologous nucleic acid from a vector in the ciliary body and / or iris of a subject comprising:administering to the eye of a subject the vector according to any one of claims 1 to 20 or the pharmaceutical composition of claim 22.

24. A method of treating an ophthalmic disease in a subject comprising:administering to the eye of a subject the vector according to any one of claims 1 to 20 or the pharmaceutical composition of claim 22.

25. The method according to claim 23 or 24, wherein the vector or the pharmaceutical composition is administered to the subject by periocular, intravitreal, suprachoroidal or subretinal administration.

26. The method according to claim 25, wherein the vector or the pharmaceutical composition is administered to the subject by intravitreal administration.

7. The method according to any one of claims 25 or 26, wherein the ophthalmic disease is selected from Bardet-Biedl syndrome; Batten’s Disease; Bietti’s Crystalline Dystrophy; choroideremia; chorioretinal atrophy; chorioretinal degeneration; cone or cone-rod dystrophies (autosomal dominant, autosomal recessive, and X-linked); congenital stationary night blindness (autosomal dominant, autosomal recessive, and X-linked); disorders of color vision, including achromatopsia (including ACHM2, ACHM3, ACHM4, and ACHM5), protanopia, deuteranopia, and tritanopia; Friedreich’s ataxia; Leber’s congenital amaurosis (autosomal dominant and autosomal recessive), including, but not limited to, LCA1, LCA2, LCA3, LCA4, LCA6, LCA7, LCA8, LCA12, and LCA15; Leber’s Hereditary Optic Neuropathy; macular dystrophy (autosomal dominant and autosomal recessive), including, but not limited to, acute macular degeneration, Best vitelliform macular dystrophy, pattern dystrophy, North Carolina Macular Dystrophy, inherited drusen, Sorsby’s fundus dystrophy, malattia levantanese, and genetically-determined retinopathy of prematurity; ocular-retinal developmental disease; ocular albinism; optic atrophies (autosomal dominant, autosomal recessive, and X-linked); retinitis pigmentosa (autosomal dominant, autosomal recessive, X-linked, and mitochondrially-inherited traits), examples of which include RP1, RP2, RP3, RP10, RP20, RP38, RP40, and RP43; X-linked retinoschisis; Stargardt disease; and Usher syndrome, including, but not limited to, USH1B, USH1C, USH1D, USH1F, USH1G, USH2A, USH2C, USH2D, AND USH3. Examples of complex genetic diseases include, but are not limited to, glaucoma (open angle, angle¬ closure, low-tension, normal-tension, congenital, neovascular, pigmentary, pseudoexfoliation); age-related and other forms of macular degeneration, both exudative and non-exudative forms (autosomal dominant and autosomal recessive), such as acute macular degeneration, vitelliform macular degeneration; retinopathy of prematurity; and Vogt Koyanagi-Harada (VKH) syndrome. Examples of acquired diseases include, but are not limited to, acute macular neuroretinopathy; anterior ischemic optic neuropathy and posterior ischemic optic neuropathy; Behcet's disease; branch retinal vein occlusion; choroidal neovascularization; diabetic retinopathy, including proliferative diabetic retinopathy and associated complications; diabeticuveitis; edema, such as macular edema, cystoid macular edema and diabetic macular edema; epiretinal membrane disorders; macular telangiectasia; multifocal choroiditis; non-retinopathy diabetic retinal dysfunction; ocular tumors; optic atrophies; retinal detachment; retinal disorders, such as central retinal vein occlusion, proliferative vitreoretinopathy (PVR), retinal arterial and venous occlusive disease, vascular occlusion, uveitic retinal disease; uveal effusion; retinal infective and infiltrative disease; optic nerve diseases such as acquired optic atrophy. Examples of traumatic injuries include, but are not limited to, histoplasmosis; optic nerve trauma; ocular trauma which affects a posterior ocular site or location; retinal trauma; viral infection of the eye; viral infection of the optic nerve; a posterior ocular condition caused by or influenced by an ocular laser treatment; posterior ocular conditions caused by or influenced by a photodynamic therapy; photocoagulation, radiation retinopathy; and sympathetic ophthalmia, preferably wherein the ophthalmic 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 nonproliferative diabetic retinopathy 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; myopia 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, genetic retinal and / or choroidal disease, tumors of the eye, corneal and iris neovascularization.

28. A method for delivering the vector according to any one of claims 1 to 20 or the pharmaceutical composition of claim 22 to an eye of a subject, wherein the rAAV or the pharmaceutical composition is administered to the subject by periocular, intravitreal, suprachoroidal or subretinal administration.2.

9. The method according to claim 28, wherein the vector or the pharmaceutical composition is administered to the subject by intravitreal administration.

30. A method for identifying one or more microRNA target sequence(s) that decreases expression of a gene in one or more off-target human ocular cell(s) relative to one or more target human ocular cell(s), the method comprising:(i) comparing, in an miRNA expression database encompassing at least one target human ocular tissue and at least one off-target human ocular tissue, miRNA expression levels in the at least one target human ocular tissue and in the at least one off-target human ocular tissue to identify one or more miRNAs that are differentially expressed in the at least one target human ocular tissue relative to the at least one off-target human ocular tissue according to a predetermined expression criteria;(ii) introducing a nucleic acid into one or more target human ocular cells and one or more non-target human ocular cells, said nucleic acid comprising a polynucleotide sequence comprising one or more miRNA target sequences of the one or more miRNAs operably linked to a transgene, and a promoter operably linked to the transgene; and(iii) evaluating an expression level of the transgene in the one or more target human ocular cells and in the one or more off-target human ocular cells to determine whether the identified one or more microRNA target sequence(s) decrease expression of the transgene in at least one off-target human ocular cell relative to at least one target human ocular cell.

31. The method of claim 30, further comprising an initial step of generating a human ocular miRNA database encompassing target and off target ocular tissues, preferably wherein the human ocular miRNA database is generated from ocular tissue obtained from at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more humans without a diagnosed ocular disorder.

32. The method of claim 30 or 31, wherein the human ocular miRNA database encompasses anterior and posterior ocular tissue.

33. The method of claim 32, wherein the target human ocular tissue is a posterior ocular tissue and / or wherein the off-target ocular tissue is an anterior ocular tissue34. The method according to claim 33, wherein the off-target ocular tissue comprises iris tissue and / or ciliary body tissue, wherein the target ocular tissue comprises retinal pigmented epithelium cells.

35. The method of any one of claims 30-34, wherein the nucleic acid introduced into a target human ocular cell and a non-target human ocular cell is comprised within a vector, preferably a lentiviral vector36. The method of any one of claims 30-35, wherein the one or more miRNA target sequences does not substantially reduce the level of transgene expression in the target ocular cell compared to a nucleic acid that that does not comprise the one or more miRNA target sequences and is otherwise identical to the nucleic acid of step (ii) under the same conditions.

37. The method of any one of claims 30-36, wherein at least one target cell is a retinal pigmented epithelial cell and / or neural retina cell, preferably an RPE and neural retinal cell, and at least one off-target cell is an iris cell and / or ciliary body cell, preferably an iris and ciliary body cell.

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