Promoters for specific expression of genes in cone photoreceptors

Abstract

Disclosed herein are synthetic cone-specific promoters that can be used in nucleic acids, vectors, or host cells to drive expression of heterologous polypeptides or RNA in retinal cells, particularly cones.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 397,183, filed August 11, 2022, and U.S. Provisional Patent Application No. 63 / 399,824, filed August 22, 2022, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] 1. Background technology Blindness cripples millions of people worldwide and is a major health problem. One common cause of blindness is dysfunction of the retina, particularly photoreceptor cells. The most common forms of retinal blindness are retinitis pigmentosa (RP) and macular degeneration (AMD), which cause degeneration of photoreceptor cells and the resulting loss of light sensitivity. There is a need to be able to prevent problems associated with this degeneration of photoreceptors or the loss of light sensitivity. This can be preferably achieved by specifically expressing therapeutic molecules, particularly polypeptides, kinins, trophic factors, channels, opsins or receptors, or nucleic acid molecules, in cone photoreceptor cells.

[0003] For expression, recombinant or exogenous or heterologous genes are usually transfected into target cells, cell populations, or tissues as cDNA constructs, typically in the context of an active expression cassette that enables transcription of the heterologous gene. The DNA construct is recognized by the cellular transcription machinery in a process that involves the activity of numerous trans-acting transcription factors (TFs) at cis-regulatory elements (including enhancers, silencers, insulators, and promoters, all collectively referred to herein as "regulatory elements"). Gene promoters are involved in all of these levels of regulation and function as determinants in gene transcription by integrating the effects of DNA sequence, transcription factor binding, and epigenetic features. These determine, for example, the strength of transgene expression and the cell type(s) in which the transgene is expressed.

[0004] Common promoters used to drive heterologous gene expression in mammalian cells are the human and mouse cytomegalovirus (CMV) major immediate-early promoters. These confer strong expression and have been shown to be robust in several cell types. Other viral promoters, such as the SV40 immediate-early promoter and the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, are also frequently used in expression cassettes. Cellular promoters can also be used instead of viral promoters. Among the well-known promoters are those derived from housekeeping genes encoding abundantly transcribed cellular transcripts, such as beta-actin, elongation factor 1 alpha (EF-1 alpha), or ubiquitin. Compared to viral promoters, eukaryotic gene expression is more complex and requires the precise coordination of many different factors.

[0005] Concerns regarding the use of endogenous regulatory elements for transgene expression include the generation of stable mRNA and the possibility of expression occurring in the natural environment of the host cell, where trans-acting transcription factors are accordingly provided. Because eukaryotic gene expression is controlled by a complex system of cis- and trans-acting regulatory elements, the lack of detailed functional characterization of most cellular promoters presents a challenge. A portion of a eukaryotic promoter is typically located immediately upstream of the transcribed sequence and serves as a transcription initiation site. A core promoter is located immediately adjacent to a transcription start site (TSS) that is sufficient for recognition by the transcription machinery. A proximal promoter includes the region upstream of the core promoter and contains the TSS and other sequence features required for transcriptional regulation. Transcription factors act in a sequence-specific manner by binding to regulatory motifs in promoter and enhancer sequences.

[0006] Some promoters can act in a cell-specific manner and can be used to express transgenes in specific cell types or subsets of cells. However, the development of promoters for retinal cells such as cone cells has been hampered by their relatively low specificity (i.e., the promoter can also drive relatively high expression in other cell types, such as rods).

[0007] Therefore, there is a need for promoters with improved specificity for expression in cone cells. Summary of the Invention

[0008] 2. Overview The present disclosure relates to an isolated nucleic acid comprising a cone-specific promoter. The promoter comprises a first nucleotide sequence of at least about 150 nucleotides that may have at least 70% identity to a sequence of equal length from the sequence of SEQ ID NO: 1, and a second nucleotide sequence of at least about 260 nucleotides that has at least 70% identity to a sequence of equal length from the sequence of SEQ ID NO: 2. In various embodiments, the promoter comprises a first nucleotide sequence of at least about 150 nucleotides that may have at least 70% identity to a sequence of equal length from the sequence of SEQ ID NO: 1, and a second nucleotide sequence of at least about 370 nucleotides that has at least 70% identity to a sequence of equal length from the sequence of SEQ ID NO: 2.

[0009] The first nucleotide sequence may have at least 70% identity to a sequence of an equal length from the 3' end of SEQ ID NO: 1. The first nucleotide sequence may have at least 90% identity to a sequence of an equal length from the 3' end of SEQ ID NO: 1, and the second nucleotide sequence may have at least 90% identity to a sequence of an equal length from the 3' end or the 5' end, or both, of SEQ ID NO: 2. The first nucleotide sequence may comprise from about 150 nucleotides to about 395 nucleotides from the 3' end of SEQ ID NO: 1. The first nucleotide sequence may comprise SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0010] The second nucleotide sequence can comprise about 260 nucleotides to about 895 nucleotides from the 3' end of SEQ ID NO: 2, or about 895 nucleotides from the 5' end of SEQ ID NO: 2. The second nucleotide sequence can comprise SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 30. The second nucleotide sequence can comprise SEQ ID NO: 39.

[0011] An isolated nucleic acid can comprise, from 5' to 3', a first nucleotide sequence and a second nucleotide sequence, with no sequence between them. An isolated nucleic acid can comprise, from 5' to 3', a second nucleotide sequence and a first nucleotide sequence, with no sequence between them. An isolated nucleic acid can comprise two or more first nucleotide sequences, two or more second nucleotide sequences, or both.

[0012] The isolated nucleic acid can further comprise a nucleotide sequence encoding a heterologous polypeptide or heterologous RNA, wherein the promoter and the nucleotide sequence encoding the polypeptide are operably linked.

[0013] The isolated nucleic acid can further comprise a regulatory element, wherein the promoter, the regulatory element, and, if present, the nucleotide sequence encoding the heterologous polypeptide or RNA are operably linked.

[0014] The regulatory element can be a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0015] The isolated nucleic acid can further comprise a nucleotide sequence encoding a polyadenylation signal (PolyA) 3' to the nucleotide sequence encoding the regulatory element, wherein the nucleotide sequence encoding PolyA and the nucleotide sequence encoding the regulatory element are operably linked.

[0016] The isolated nucleic acid can include a first AAV inverted terminal repeat (ITR) 5' to the promoter and a second AAV ITR 3' to the regulatory elements and preferably 3' to the PolyA signal.

[0017] The isolated nucleic acid can further comprise a nucleotide sequence encoding an AAV inverted terminal repeat (ITR).

[0018] The present disclosure also relates to an isolated nucleic acid comprising a cone-specific promoter, wherein the promoter comprises a first nucleotide sequence of at least about 150 nucleotides and no more than 499 nucleotides, which may have at least 70% identity to a sequence of equal length from the sequence of SEQ ID NO: 1. For example, the cone-specific promoter may be SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or a sequence having at least 70% identity to any of the foregoing. For example, the cone-specific promoter may be SEQ ID NO: 40, or a sequence having at least 70% identity to any of the foregoing.

[0019] The present disclosure further relates to vectors comprising any of the isolated nucleic acids described herein.

[0020] The vector may further comprise a viral capsid.

[0021] The viral capsid can be an adeno-associated viral vector (AAV) capsid selected from the group consisting of an AAV8-BP2 capsid, an AAV-PHP.B capsid, an AAV-PHP.eB capsid, an AAV5 capsid, or an AAV-NHP26 capsid.

[0022] Disclosed herein are host cells containing the vectors described herein.

[0023] The present disclosure also relates to kits comprising the isolated nucleic acids, vectors, host cells, or two or more thereof.

[0024] The present disclosure also relates to the method for expressing heterologous polypeptide or heterologous RNA in retinal organoid, retinal explant or mammalian subject.This method can include administering to retinal organoid, retinal explant or mammalian subject isolated nucleic acid, vector, host cell, or two or more thereof. [Brief explanation of the drawings]

[0025] 3. Brief description of the drawings [Figure 1] 1 shows a schematic representation of the nucleic acid constructs in which ProA7 and truncated ProA7 promoters were tested in Example 1. [Figure 2] Shown are maximum intensity Z projections of live whole organoid images of human retinal organoids transduced with AAV delivering constructs containing ProA7 or truncated ProA7 promoters, as indicated. [Figure 3] A-C show quantitative plots of live imaging of whole organoids for constructs containing ProA7 or truncated ProA7 promoters, as indicated. A shows the number of GFP-expressing cells detected per well. B shows the calculated density of GFP-expressing cells per mm². C shows the average GFP intensity in detected cells in relative fluorescence units (RFU). Experiments were performed in triplicate, except for ProA7 (six replicates). [Figure 4] Spinning disk confocal microscopy images of cross sections of human retinal organoids infected with AAV-ProA7-GFP (top row), AAV-ProA7 5'3-GFP (middle row), or AAV-ProA7 5'6-GFP (bottom row). Left row: GFP (green). Middle row, immunostaining with the cone marker CAR (magenta). Right row: GFP and cone marker (signal overlap visible in white). [Figure 5] Schematic representation of the nucleic acid for AAV production: pAAV-{ProA7 / Pro572 / Pro573 / Pro572.2 / Pro573.2}-GFP-WPRE-hGHpA. [Figure 6] A-C show quantitative plots of live imaging of whole organoids for constructs containing ProA7 or truncated ProA7 / rod-specific promoters, as indicated. A shows the number of GFP-expressing cells detected per well. B shows the calculated density of GFP-expressing cells per mm². C shows the average GFP intensity in detected cells in relative fluorescence units (RFU). Experiments were performed in triplicate. [Figure 7]Spinning disk confocal microscopy images of cross sections of human retinal organoids infected with AAV-Pro572-GFP and AAV-Pro573-GFP, respectively. Left: GFP (green). Center left, immunostaining with the cone marker CAR (magenta). Center right: GFP and cone marker (signal overlap visible in white). Right: GFP and cone marker plus nuclear staining (Hoechst, white). [Figure 8] (A) and (B) are graphs showing that transgene expression of hybrid promoter variants maintains specificity for cone photoreceptors. (A) Quantitative plots of expression in cross-sections of human retinal organoids infected with AAV-{Pro572 / Pro572.2 / Pro573 / Pro573.2}-GFP and AAV-ProA7-GFP. Quantitation of GFP+ cell density as a percentage of cone photoreceptor density; values ​​are means ± sem from n = 10 confocal images. (B) Quantitation of AAV targeting specificity, shown as the percentage of cone (black, cone (CAR+)) and minor cell types (gray, other) cell types or classes of cells expressing GFP in organoids infected with AAV-Pro572.2-GFP and AAV-Pro573.2-GFP, respectively. [Figure 9](A-C) Graphs showing the results of capsid screening using AAV5 and AAVPHP.eB capsids and a promoter driving expression of an EGFP transgene after transduction of human retinal explants. The vectors tested were: AAV5-Pro572.2-EGFP-WPRE, AAVPHP.eB-Pro572.2-EGFP-WPRE, AAV5-Pro573.2-EGFP-WPRE, AAVPHP.eB-Pro573.3-EGFP-WPRE, AAV5-ProSC-EGFP-WPRE, and AAV5-ProA7-EGFP-WPRE. (A and B) Quantification of the percentage of rod and cone photoreceptors expressing GFP under the control of the hybrid promoters Pro572.2 or Pro573.2 or the cone-specific promoters ProSC or ProA7 in human retinal explants. The promoters Pro572.2, Pro573.2, and ProSC are highly effective (>95%) in targeting cone photoreceptors. (C) The mean intensity of the GFP signal in human retinal explants treated with AAV5-Pro573.2-EGFP-WPRE is threefold higher than that in those treated with AAV5-ProSC-EGFP-WPRE. [Figure 10]Quantitative plots of live imaging of GFP-expressing cells in whole human retinal organoids after transduction with AAV5 and AAVPHP.eB vectors containing various numbers of promoter copies at doses of 1E10 and 1E11 viral genomes (vg) per well are shown. The vectors tested were as follows: AAVPHP.eB-ProA7-EGFP-WPRE, AAVPHP.eB-ProSC-EGFP-WPRE, AAVPHP.eB-2xProSC-EGFP-WPRE, AAVPHP.eB-3xProSC-EGFP-WPRE, AAVPHP.eB-4xProSC-EGFP-WPRE, AAV5-ProA330-EGFP-WPRE, AAVPHP.eB-2xmin330-EGFP-WPRE, AAVPhP.eB-3xmin330-EGFP-WPRE, AAVPHP.eB-4xmin330-EGFP-WPRE, AAVPHP.eB-330-3del1del5-EGFP-WPRE, and AAVPHP.eB-3del1del6-EGFP-WPRE. Negative controls included no AAV (Ctrl-noAAV) and AAV5-noPro-EGFP-WPRE (Ctrl-noP). [Figure 11] (A) and (B) are graphs showing that AAVPHP.eB-4xProSC-EGFP-WPRE, which contains a 4x multimerized ProSC promoter, maintains specificity for cone photoreceptors. (A) Quantitative plots of expression in whole mount cultured human retinal explants infected with AAVPHP.eB-ProA7-EGFP-WPRE and AAVPHP.eB-4xProSC-EGFP-WPRE. Quantitation of GFP+ cell density as a percentage of cone photoreceptor density. (B) Quantitative plots of AAV targeting specificity, shown as the percentage of major (circle, cone) and minor (square, other) cell types or classes of cells expressing GFP in human retinas infected with AAVPHP.eB-ProA7-EGFP-WPRE and AAVPHP.eB-4xProSC-EGFP-WPRE, respectively. [Figure 12]Spinning disk confocal microscopy images of cross sections of human retinal organoids transduced with AAVPHP.eB-ProSC-EGFP-WPRE and AAVPHP.eB-4xProSC-EGFP-WPRE are shown. Left: GFP. Center left, immunostaining with the cone marker CAR. Center right, immunostaining with the rod marker NRL, GFP, and a cone marker. Right: GFP and cone markers, as well as nuclear staining. DETAILED DESCRIPTION OF THE INVENTION

[0026] 4. Detailed Description All publications and patents cited in this disclosure are incorporated herein by reference in their entirety. In the event that any material incorporated by reference conflicts or contradicts this specification, the present specification supersedes all such material. The citation of any reference herein is not an admission that such reference is prior art to the present disclosure. When ranges of values ​​are expressed, embodiments using any specific value within that range are included. Furthermore, reference to values ​​stated in ranges includes every value within that range. All ranges are inclusive and combinable. When values ​​are expressed as approximations, by use of the antecedent "about," it is intended that the particular value form another embodiment.

[0027] Various terms relating to aspects of the present specification are used throughout the specification and claims. Unless otherwise specified, such terms shall be given their ordinary meaning in the art. Other terms that are specifically defined shall be interpreted in a manner consistent with the definitions provided herein. The techniques and acts described or referenced herein are generally well understood and commonly employed using conventional methodologies by those of skill in the art, such as, for example, widely used molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Unless otherwise specified, acts involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and conditions, where appropriate.

[0028] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Terms such as "including," "e.g.," are intended to indicate an open-ended inclusion unless expressly stated otherwise.

[0029] Unless otherwise stated, the terms "at least," "less than," and "about," or similar terms, preceding a series or range of elements, are to be understood to refer to every element in that series or range. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0030] As used herein, the term "subject" refers to any animal, including, but not limited to, humans, non-human primates, rodents, mammals commonly kept as pets (e.g., dogs and cats, among others), livestock (e.g., cows, sheep, goats, pigs, horses, and camels, among others), etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0031] The term "cone-specific" used herein refers to a promoter that has a promoter activity that is at least about 90% selective for human cone cells compared to human rod cells in human cone photoreceptors.For example, the promoter can have at least about 91% selectivity, at least about 92% selectivity, at least about 93% selectivity, at least about 94% selectivity, at least about 95% selectivity, at least about 96% selectivity, at least about 97% selectivity, at least about 98% selectivity, at least about 99% selectivity, or 100% selectivity for cone cells compared to rod cells.The selectivity of a promoter in human cone photoreceptors compared to other cells, such as human rod photoreceptors, can be determined using a suitable method well known to those skilled in the art. In one preferred method, retinal organoids or tissues containing both cone and rod cells are transfected with a nucleic acid construct comprising a promoter operably linked to a nucleic acid sequence encoding a reporter molecule such as tdTomato, enhanced yellow fluorescent protein (EYFP), citrine, green fluorescent protein (GFP), cyan fluorescent protein, red fluorescent protein, or functional variants thereof. The organoids are sectioned and immunostained to identify cone and / or rod cells using cell-type-specific antibodies (e.g., anti-human cone arrestin for cone cells and anti-human rhodopsin for rod cells). Promoter activity is assessed by counting cells expressing the reporter molecule and determining the proportion of cells expressing the reporter co-labeled with each cell-type-specific antibody. Cone specificity is calculated as the proportion or percentage of cone cells among all cells expressing the reporter.

[0032] The inventors have observed that the cone-specific synthetic promoter disclosed herein is cone-specific in humans and at least one species of non-human primate (macaque), and that the promoter may have low selectivity for cone cells in non-primate mammals.

[0033] The term "rod-specific" used herein refers to a promoter that has a promoter activity that is at least about 90% selective for human rod cells compared to human cone cells in human rod photoreceptors.For example, a promoter can have at least about 91% selectivity, at least about 92% selectivity, at least about 93% selectivity, at least about 94% selectivity, at least about 95% selectivity, at least about 96% selectivity, at least about 97% selectivity, at least about 98% selectivity, at least about 99% selectivity, or 100% selectivity for rod cells compared to cone cells.The selectivity of a promoter in human rod photoreceptors compared to other cells, such as human cone photoreceptors, can be determined using a suitable method well known to those skilled in the art. In one preferred method, retinal organoids or tissues containing both rod and cone cells are transfected with a nucleic acid construct containing a promoter operably linked to a nucleic acid sequence encoding a reporter molecule such as tdTomato, enhanced yellow fluorescent protein (EYFP), citrine, green fluorescent protein (GFP), cyan fluorescent protein, red fluorescent protein, or functional variants thereof. The organoids are sectioned and immunostained to identify rod and / or cone cells using cell-type-specific antibodies (e.g., anti-human cone arrestin for cone cells and anti-human rhodopsin for rod cells). Promoter activity is assessed by counting cells expressing the reporter molecule and determining the proportion of cells expressing the reporter co-labeled with each cell-type-specific antibody. Rod specificity is calculated as the proportion or percentage of rod cells among all cells expressing the reporter.

[0034] The level of promoter activity in target cell type can be determined by transducing a suitable cell population (such as human retinal organoid) with a nucleic acid construct comprising a promoter operably linked to the nucleic acid sequence encoding a reporter molecule such as tdTomato, enhanced yellow fluorescent protein (EYFP), Citrine, green fluorescent protein (GFP), cyan fluorescent protein, red fluorescent protein or their functional variants, sectioning the organoid, and using cell type-specific antibody (for example, anti-human cone arrestin for cone cells, anti-human rhodopsin for rod cells) to identify the cell of target cell type, counting the number of cells of target cell type, and counting the number of cells of target cell type that express reporter molecule.The promoter activity in target cell type is calculated as the proportion or percentage of cells of target cell type that express reporter molecule.

[0035] Further description of the method and guidance for its implementation is provided herein.

[0036] A. Promoter The present disclosure relates to synthetic promoters that are cone-specific and capable of driving expression of a desired nucleic acid (e.g., a transgene) in cone photoreceptor cells. The synthetic promoter can include a first component (first nucleotide sequence) derived from a cone-specific promoter, such as (SEQ ID NO: 1), and a second component (second nucleotide sequence) derived from a rod-specific promoter, such as SEQ ID NO: 2, wherein the first and second components are operably linked. The inventors have surprisingly discovered that such synthetic promoters are unexpectedly cone-specific and have enhanced promoter activity in human cone cells compared to the cone-specific promoter (e.g., SEQ ID NO: 1) from which the first component (first nucleotide sequence) is derived.

[0037] The cone-specific synthetic promoters disclosed herein may comprise an active fragment of the cone-specific promoter of SEQ ID NO: 1, provided that the synthetic promoter of the present disclosure is not SEQ ID NO: 1. Such synthetic promoters do not comprise nucleotide sequences derived from rod-specific promoters, such as SEQ ID NO: 2. The inventors have unexpectedly determined that such cone-specific synthetic promoters may have enhanced promoter activity in human cone cells compared to SEQ ID NO: 1.

[0038] The synthetic promoter of the present disclosure comprises a first nucleotide sequence having cone-specific promoter activity. Specific examples of the first nucleotide sequence include SEQ ID NO: 1 and a sequence of at least about 150 nucleotides having at least 70% identity to SEQ ID NO: 1 over the entire length of the first nucleotide sequence. The first nucleotide sequence is described in more detail herein. The synthetic promoter can consist essentially of the first nucleotide sequence, i.e., it can include the first nucleotide sequence and omit the second nucleotide sequence as described herein.

[0039] A synthetic promoter can include a first nucleotide sequence as described herein and can further include a second nucleotide sequence as described herein operably linked to the first nucleotide sequence. The operably linked first and second nucleotide sequences function as a single promoter for expression in cone photoreceptors. A promoter that includes both the first and second nucleotide sequences is sometimes referred to herein as a hybrid promoter.

[0040] The first and second nucleotide sequences are typically obtained or derived from distinct cone-specific and rod-specific promoters, respectively. As described and exemplified herein, the inventors have surprisingly discovered that such hybrid promoters have cone-specific activity and can confer higher levels of expression in cone photoreceptor cells compared to the cone-specific promoter from which the first nucleotide sequence is derived. As shown herein, when the first polynucleotide is the cone-specific promoter of SEQ ID NO: 1 or a functional sequence fragment from the 3' region of SEQ ID NO: 1, and the second nucleotide sequence is a rod-specific promoter (SEQ ID NO: 2) or a functional sequence fragment thereof, the hybrid promoter is cone-specific and drove higher reporter (GFP) expression in cone cells than SEQ ID NO: 1 alone. See, e.g., Figure 6. Accordingly, the present disclosure relates to synthetic promoters, including but not limited to hybrid promoters, nucleic acids comprising synthetic promoters, and methods of using such nucleic acids.

[0041] a. a first nucleotide sequence The nucleic acids disclosed herein can include a synthetic promoter comprising a first nucleotide sequence of at least about 150 nucleotides that has at least 70% identity to SEQ ID NO:1 over the entire length of the first nucleotide sequence.

[0042] The ProA7 promoter (SEQ ID NO: 1) is known to be useful for driving the expression of desired genes in the retina of various species. ProA7 is cone-specific and does not drive substantial expression in other retinal cells. See, for example, Juettner et al., Nature Neuroscience 22, 1345-1356 (2019).

[0043] Therefore, the first nucleotide sequence has promoter activity specific to cone cell, and can drive expression in human cone cell, for example, the cone cell that is in resting state and no longer responds to light.The first nucleotide sequence can have promoter activity in human cone cell, for example, the cone cell of in vivo retina, retinal explant or retinal organoid.In some cases, the first nucleotide sequence does not have substantial promoter activity in other human retinal cells, for example, rod cell (for example, in non-cone cell, promoter activity is found to be less than about 10%).

[0044] The ProA7 promoter sequence of SEQ ID NO: 1 is described in International Publication No. WO2017046084. The first nucleotide sequence may comprise a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 1. The first sequence may have a nucleic acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to SEQ ID NO:1 over the entire length of the first nucleotide sequence.

[0045] If desired, the first nucleotide sequence, alone or in combination with an optional second nucleotide sequence, can be a variant of SEQ ID NO: 1 that retains cone-specific promoter activity. The first nucleotide sequence can include about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or more nucleic acid changes, e.g., substitutions or deletions, compared to SEQ ID NO: 1, or, in the case of a functional fragment of ProA7, compared to the corresponding portion of SEQ ID NO: 1.

[0046] The promoter may comprise a first nucleotide sequence comprising a sequence of at least 150 nucleotides having at least 70% identity to SEQ ID NO: 1, preferably the 3' end of SEQ ID NO: 1. As shown herein, such fragments of SEQ ID NO: 1 retain cone-specific promoter activity.

[0047] The first nucleotide sequence may comprise a sequence having at least 70% identity to a sequence of equal length from the 3' end of SEQ ID NO: 1. The first sequence may be considered a 5' truncated version of SEQ ID NO: 1, since nucleotides have been removed from the 5' end of SEQ ID NO: 1 to form the first sequence.

[0048] The first nucleotide sequence may be a 5' truncated version of SEQ ID NO: 1 and may contain less than 500 nt of ProA7 due to the deletion of nucleotides at the 5' end. For example, the first nucleotide sequence may be identical to nucleotides 386-500 of SEQ ID NO: 1, or may be identical to nucleotides 2-500 of SEQ ID NO: 1. Such a first nucleotide sequence may contain any number of nucleotides from 150-499 (i.e., one less than the length of SEQ ID NO: 1).

[0049] For example, the first nucleotide sequence is about 150 nucleotides (nt), about 151 nt, about 152 nt, about 153 nt, about 154 nt, about 155 nt, about 156 nt, about 157 nt, about 158 nt, about 159 nt, about 160 nt, about 161 nt, about 162 nt, about 163 nt, about 164 nt, about 165 nt, about 166 nt, about 167 nt, about 168 nt, about 169 nt, about 170 nt, about 171 nt, about 172 nt, about 173 nt, about 174 nt, about 175 nt, about 176 nt, about 177 nt, about 178 nt, about 179 nt, about 180 nt, about 181 nt, about 182 nt, about 183 nt, about 184 nt, about 185 nt, about 186 nt, about 187 nt, about 188 nt, about 189 nt, about 190 nt, about 191 nt, about 192 nt, about 193 nt, about 194 nt, about 195 nt, about 196 nt, about 197 nt, about 198 nt, about 199 nt, about 200 nt, about 201 nt, about 202 nt, about 203 nt, about 204 nt, about 205 nt, about 206 nt, about 207 nt, about 208 nt, about 209 nt, about 210 nt, about 211 nt, about 212 nt, about 213 nt, about 214 nt, about 215 nt, about 216 nt, about 217 nt, about 218 nt, about 219 nt, about 220 nt, about 221 nt, about 222 nt, about 223 nt, about 224 nt, about 225 nt, about 226 nt, about 227 nt, about 228 nt, about 229 nt, about 230 nt, about 231 nt, about 232 nt, about 233 nt, about 234 nt, about 235 nt, about 236 nt, about 237 nt, about 238 nt, about 239 nt, about 240 nt, about 241 nt, about 242 nt, about 243 nt, about 244 nt, about 245 nt, about 246 nt, about 247 nt, about 248 nt, about 249 nt, about 250 nt, about 251 nt, about 252 nt, about 253 nt, about 254 nt, about 255 nt, about 256 nt, about 257 nt, about 258 nt, about 259 nt, about 260 nt, about 261 nt, about 262 nt, about 263 nt, about 264 nt, about 265 nt, about 266 nt, about 267 nt, about 268 nt, about 269 nt, about 270 nt, about 271 nt, about 272 nt, about 273 nt, about 274 nt, about 275 nt, about 276 nt, about 277 nt, about 278 nt, about 279 nt, about 280 nt, about 281 nt, about 282 nt, about 283 nt, about 284 nt, about 285 nt, about 286 nt, about 287 nt, about 288 ntabout 289nt, about 290nt, about 291nt, about 292nt, about 293nt, about 294nt, about 295nt, about 296nt, about 297nt, about 298nt, about 299nt, about 300nt, about 301nt, about 302nt, about 303nt, about 304nt, about 305nt, about 306nt, about 307nt, about 308nt, about 309nt, about 310nt, about 311nt, about 312nt, about 313nt, about 314nt, about 315nt, about 316nt, about 317nt, about 318nt, about 319nt, about 320nt, about 321nt, about 322nt, about 323nt, about 324nt t, about 325nt, about 326nt, about 327nt, about 328nt, about 329nt, about 330nt, about 331nt, about 332nt, about 333nt, about 334nt, about 335nt, about 336nt, about 337nt, about 338nt, about 339nt, about 340nt, about 341nt, about 342nt, about 343nt, about 344nt, about 345nt, about 346nt, about 347nt, about 348nt, about 349nt, about 350nt, about 351nt, about 352nt, about 353nt, about 354nt, about 355nt, about 356nt, about 357nt, about 358nt, about 359nt, about 360nt 0nt, about 361nt, about 362nt, about 363nt, about 364nt, about 365nt, about 366nt, about 367nt, about 368nt, about 369nt, about 370nt, about 371nt, about 372nt, about 373nt, about 374nt, about 375nt, about 376nt, about 377nt, about 378nt, about 379nt, about 380nt, about 381nt, about 382nt, about 383nt, about 384nt, about 385nt, about 386nt, about 387nt, about 388nt, about 389nt, about 390nt, about 391nt, about 392nt, about 393nt, about 394nt, about 395nt, about 404nt, about 405nt, about 406nt, about 407nt, about 408nt, about 409nt, about 410nt, about 411nt, about 412nt, about 413nt, about 414nt, about 415nt, about 416nt, about 417nt, about 418nt, about 419nt, about 420nt, about 421nt, about 422nt, about 423nt, about 424nt, about 425nt, about 426nt, about 427nt, about 428nt, about 429nt, about 430nt, about 431nt,Approximately 432nt, approximately 433nt, approximately 434nt, approximately 435nt, approximately 436nt, approximately 437nt, approximately 438nt, approximately 439nt, approximately 440nt, approximately 441nt, approximately 442nt, approximately 443nt, approximately 444nt, approximately 445nt, approximately 446nt, approximately 447nt, approximately 448nt, approximately 44 9nt, about 450nt, about 451nt, about 452nt, about 453nt, about 454nt, about 455nt, about 456nt, about 457nt, about 458nt, about 459nt, about 460nt, about 461nt, about 462nt, about 463nt, about 464nt, about 465nt, about 466nt, It may comprise about 467 nt, about 468 nt, about 469 nt, about 470 nt, about 471 nt, about 472 nt, about 473 nt, about 474 nt, about 475 nt, about 476 nt, about 477 nt, about 478 nt, about 479 nt, about 480 nt, about 481 nt, about 482 nt, about 483 nt, about 484 nt, about 485 nt, about 486 nt, about 487 nt, about 488 nt, about 489 nt, about 490 nt, about 491 nt, about 492 nt, about 493 nt, about 494 nt, about 495 nt, about 496 nt, about 497 nt, about 498 nt, about 499 nt, or 500 nt.

[0050] Some preferred first nucleotide sequences used in promoters have a nucleotide sequence comprising about 150 to about 395 nucleotides from the 3' end of SEQ ID NO:1.

[0051] The first nucleotide sequence can comprise a nucleic acid sequence having at least 70% sequence identity over the entire length of the first nucleotide sequence to SEQ ID NO: 1. The first nucleotide sequence can have a sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to SEQ ID NO: 1 over the entire length of the first nucleotide sequence. Preferably, the first nucleotide sequence may have at least 90% identity to SEQ ID NO: 1 over the entire length of the first nucleotide sequence.

[0052] The first nucleotide sequence can contain about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or more nucleic acid changes, e.g., substitutions or deletions, compared to an aligned sequence of the same length from SEQ ID NO:1.

[0053] An exemplary first nucleotide sequence may comprise an approximately 395 nt sequence having at least 70% identity to the 3' end of SEQ ID NO:1. For example, the first nucleotide sequence may comprise SEQ ID NO:12. The first nucleotide sequence may comprise a nucleotide sequence having at least 70% identity to SEQ ID NO:12. An exemplary first nucleotide sequence may comprise an approximately 290 nt sequence having at least 70% identity to the 3' end of SEQ ID NO:1. For example, the first nucleotide sequence may comprise SEQ ID NO:13. The first nucleotide sequence may comprise a nucleotide sequence having at least 70% identity to SEQ ID NO:13. An exemplary first nucleotide sequence may comprise an approximately 185 nt sequence having at least 70% identity to the 3' end of SEQ ID NO:1. For example, the first nucleotide sequence may comprise SEQ ID NO:14. The first nucleotide sequence may comprise a nucleotide sequence having at least 70% identity to SEQ ID NO:14. An exemplary first nucleotide sequence may comprise an approximately 150 nt sequence having at least 70% identity to the 3' end of SEQ ID NO: 1. For example, the first nucleotide sequence may comprise SEQ ID NO: 15. The first nucleotide sequence may comprise a nucleotide sequence having at least 70% identity to SEQ ID NO: 15. In some embodiments, the promoter comprises a first nucleotide sequence comprising SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and an optional second nucleotide sequence, as described herein. In some embodiments, the promoter comprises a first nucleotide sequence comprising SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or a nucleotide sequence having at least 70% identity to any of the foregoing, and an optional second nucleotide sequence, as described herein.

[0054] b. A second nucleotide sequence The promoter may include a second nucleotide sequence derived from a rod-specific promoter and having rod-specific promoter activity as a separate molecule. When such a second nucleotide sequence is included in the hybrid promoter of the present disclosure as described herein, the resulting hybrid promoter has cone-specific promoter activity. Preferably, the second nucleotide sequence comprises at least about 370 nucleotides and has at least 70% identity to a sequence of equal length from SEQ ID NO:2. The second nucleotide sequence may comprise at least about 260 nucleotides and has at least 70% identity to a sequence of equal length from SEQ ID NO:2. Herein, a promoter consisting of SEQ ID NO:2 may be referred to as ProA330.

[0055] The rod-specific promoter component may be a 5'-truncated version, a 3'-truncated version, or both, of SEQ ID NO:2 and may comprise less than 1000 nt of SEQ ID NO:2 due to deletion of nucleotides at the 5' and / or 3' ends. For example, the rod-specific promoter component may be identical to nucleotides 106 to 1000 of SEQ ID NO:2, or may be identical to nucleotides 1 to 895 of SEQ ID NO:2. Preferably, the rod-specific promoter component may comprise or consist of nucleotides 631 to 895 of SEQ ID NO:2 (SEQ ID NO:39, sometimes referred to as min330), which is believed to be the minimum portion of SEQ ID NO:2 required for rod-specific promoter activity.

[0056] The rod-specific promoter component can contain any number of nucleotides from about 260 to about 369 nucleotides of SEQ ID NO: 2. For example, the rod-specific promoter component can contain about 261nt, 262nt, 263nt, 264nt, 265nt, 266nt, 267nt, 268nt, 269nt, 270nt, 271nt, 272nt, 272nt, 274nt, 275nt, 276nt, 277nt, 278nt, 279nt, 280nt, 281nt, 282nt, 283nt, 284nt, 285nt, 286nt, 287nt, 288nt, 289nt, 290nt, 291nt, 292nt, 293nt, 294nt, 295nt, 296nt, 297nt, 298nt, 299nt, 300nt, 301nt, 302nt, 303nt, 304nt, 305nt, 306nt, 307nt, 308nt, 309nt, 310nt, 311nt, 312nt, 313nt, 314nt, 315nt, 316nt, 317nt, 318nt, 319nt, 320nt, 321nt, 322nt, 323nt, 324nt, 325nt, 326nt, 327nt, 328nt, 329nt, 330nt, 331nt, 332nt, 333nt, 334nt, 335nt, 336nt, 337nt 285nt, 286nt, 287nt, 288nt, 289nt, 290nt, 291nt, 292nt, 293nt, 294nt, 295nt, 296nt, 297nt, 298nt, 29 9nt, 300nt, 301nt, 302nt, 303nt, 304nt, 305nt, 306nt, 307nt, 308nt, 309nt, 310nt, 311nt, 312nt, 313nt , 314nt, 315nt, 316nt, 317nt, 318nt, 319nt, 320nt, 321nt, 322nt, 323nt, 324nt, 325nt, 326nt, 327nt, 3 28nt, 329nt, 330nt, 331nt, 332nt, 333nt, 334nt, 335nt, 336nt, 337nt, 338nt, 339nt, 340nt, 341nt, 342n nt, 343nt, 344nt, 345nt, 346nt, 347nt, 348nt, 349nt, 350nt, 351nt, 352nt, 353nt, 354nt, 355nt, 356nt, 357nt, 358nt, 359nt, 360nt, 361nt, 362nt, 363nt, 364nt, 365nt, 366nt, 367nt, 368nt, or about 369nt. In some preferred embodiments, the rod-specific promoter comprises or consists of SEQ ID NO:39, or a sequence having at least about 70% identity to SEQ ID NO:39.

[0057] For example, the second nucleotide sequence can have a nucleic acid sequence that has at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to SEQ ID NO: 2 over the entire length of the second nucleotide sequence. Preferably, the second nucleotide sequence can have at least 90% identity to SEQ ID NO: 2 over the entire length of the second nucleotide sequence. These embodiments include, but are not limited to, those in which the first nucleotide sequence has at least 90% identity to a sequence of equal length from the 3' end of SEQ ID NO:1.

[0058] Exemplary second nucleotide sequences that can be used in the promoters of the present disclosure include SEQ ID NO:2 and nucleotide sequences having at least about 90% identity to SEQ ID NO:2 over the entire length of the second nucleotide sequence.

[0059] The second nucleotide sequence can contain about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or more nucleic acid changes, e.g., substitutions or deletions, compared to an aligned sequence of the same length from SEQ ID NO:2.

[0060] The second nucleotide sequence may comprise a sequence having at least 70% identity to a sequence of an equal length from the 3' end of SEQ ID NO: 2. The second nucleotide sequence according to this paragraph may be considered a 5' truncated version of SEQ ID NO: 2, since nucleotides have been removed from the 5' end of SEQ ID NO: 2 to form the second nucleotide sequence.

[0061] The second nucleotide sequence may comprise a sequence having at least 70% identity to a sequence of an equal length from the 5' end of SEQ ID NO: 2. The second nucleotide sequence according to this paragraph may be considered a 3' truncated version of SEQ ID NO: 2, since nucleotides have been removed from the 3' end of SEQ ID NO: 2 to form the second nucleotide sequence.

[0062] The second nucleotide sequence can be a 5'-truncated version, a 3'-truncated version, or both, of SEQ ID NO:2 and can contain less than 1000 nt of SEQ ID NO:2 due to deletion of nucleotides at the 5' and / or 3' ends. For example, the second nucleotide sequence can be identical to nucleotides 106 to 1000 of SEQ ID NO:2, or can be identical to nucleotides 1 to 895 of SEQ ID NO:2.

[0063] The second nucleotide sequence can contain any number of nucleotides from 370 to 1000. For example, the second nucleotide sequence can contain about 370 nt, about 371 nt, about 372 nt, about 373 nt, about 374 nt, about 375 nt, about 376 nt, about 377 nt, about 378 nt, about 379 nt, about 380 nt, about 381 nt, about 382 nt, about 383 nt, about 384 nt, about 385 nt, about 386 nt, about 387 nt, about 388 nt, about 389 nt, about 390 nt, about 391 nt, about 392 nt, about 393 nt, about 394 nt, about 395 nt, about 396 nt, about 397 nt, about 398 nt, about 399 nt, about 400 nt, or about 401 nt. , about 402nt, about 403nt, about 404nt, about 405nt, about 406nt, about 407nt, about 408nt, about 409nt, about 410nt, about 411nt, about 412nt, about 413nt, about 414nt, about 415nt, about 416nt, about 417nt, about 418nt, 419nt, 420nt, 421nt, 422nt, 423nt, 424nt, 425nt, 426nt, 427nt, 428nt, 429nt, 430nt, 431nt, 432nt, 433nt, 434nt, 435nt, 4 36nt, about 437nt, about 438nt, about 439nt, about 440nt, about 441nt, about 442nt, about 443nt, about 444nt, about 445nt, about 446nt, about 447nt, about 448nt, about 449nt, about 450nt, about 451nt, about 452nt, about 45 3nt, about 454nt, about 455nt, about 456nt, about 457nt, about 458nt, about 459nt, about 460nt, about 461nt, about 462nt, about 463nt, about 464nt, about 465nt, about 466nt, about 467nt, about 468nt, about 469nt, about 470 nt, about 471nt, about 472nt, about 473nt, about 474nt, about 475nt, about 476nt, about 477nt, about 478nt, about 479nt, about 480nt, about 481nt, about 482nt, about 483nt, about 484nt, about 485nt, about 486nt, about 487n t, about 488nt, about 489nt, about 490nt, about 491nt, about 492nt, about 493nt, about 494nt, about 495nt, about 496nt, about 497nt, about 498nt, about 499nt, about 500nt, about 501nt, about 502nt, about 503nt, about 504nt,about 505nt, about 506nt, about 507nt, about 508nt, about 509nt, about 510nt, about 511nt, about 512nt, about 513nt, about 514nt, about 515nt, about 516nt, about 517nt, about 518nt, about 519nt, about 520nt, about 521nt, about 522nt, about 523nt, about 524nt, about 525nt, about 526nt, about 527nt, about 528nt, about 529nt, about 530nt, about 531nt, about 532nt, about 533nt, about 534nt, about 535nt, about 536nt, about 537nt, about 538nt, about 539nt, about 540nt t, about 541nt, about 542nt, about 543nt, about 544nt, about 545nt, about 546nt, about 547nt, about 548nt, about 549nt, about 550nt, about 551nt, about 552nt, about 553nt, about 554nt, about 555nt, about 556nt, about 557nt, about 558nt, about 559nt, about 560nt, about 561nt, about 562nt, about 563nt, about 564nt, about 565nt, about 566nt, about 567nt, about 568nt, about 569nt, about 570nt, about 571nt, about 572nt, about 573nt, about 574nt, about 575nt, about 576nt 6nt, about 577nt, about 578nt, about 579nt, about 580nt, about 581nt, about 582nt, about 583nt, about 584nt, about 585nt, about 586nt, about 587nt, about 588nt, about 589nt, about 590nt, about 591nt, about 592nt, about 593nt, about 594nt, about 595nt, about 596nt, about 597nt, about 598nt, about 599nt, about 600nt, about 601nt, about 602nt, about 603nt, about 604nt, about 605nt, about 606nt, about 607nt, about 608nt, about 609nt, about 610nt, about 611nt, about about 612nt, about 613nt, about 614nt, about 615nt, about 616nt, about 617nt, about 618nt, about 619nt, about 620nt, about 621nt, about 622nt, about 623nt, about 624nt, about 625nt, about 626nt, about 627nt, about 628nt, about 629nt, about 630nt, about 631nt, about 632nt, about 633nt, about 634nt, about 635nt, about 636nt, about 637nt, about 638nt, about 639nt, about 640nt, about 641nt, about 642nt, about 643nt, about 644nt, about 645nt, about 646nt, about 647nt,about 648nt, about 649nt, about 650nt, about 651nt, about 652nt, about 653nt, about 654nt, about 655nt, about 656nt, about 657nt, about 658nt, about 659nt, about 660nt, about 661nt, about 662nt, about 663nt, about 664nt, about 665nt, about 666nt, about 667nt, about 668nt, about 669nt, about 670nt, about 671nt, about 672nt, about 673nt, about 674nt, about 675nt, about 676nt, about 677nt, about 678nt, about 679nt, about 680nt, about 681nt, about 682nt, about 683nt t, about 684nt, about 685nt, about 686nt, about 687nt, about 688nt, about 689nt, about 690nt, about 691nt, about 692nt, about 693nt, about 694nt, about 695nt, about 696nt, about 697nt, about 698nt, about 699nt, about 700nt, about 701nt, about 702nt, about 703nt, about 704nt, about 705nt, about 706nt, about 707nt, about 708nt, about 709nt, about 710nt, about 711nt, about 712nt, about 713nt, about 714nt, about 715nt, about 716nt, about 717nt, about 718nt, about 719nt 9nt, about 720nt, about 721nt, about 722nt, about 723nt, about 724nt, about 725nt, about 726nt, about 727nt, about 728nt, about 729nt, about 730nt, about 731nt, about 732nt, about 733nt, about 734nt, about 735nt, about 736nt, about 737nt, about 738nt, about 739nt, about 740nt, about 741nt, about 742nt, about 743nt, about 744nt, about 745nt, about 746nt, about 747nt, about 748nt, about 749nt, about 750nt, about 751nt, about 752nt, about 753nt, about 754nt, about about 755nt, about 756nt, about 757nt, about 758nt, about 759nt, about 760nt, about 761nt, about 762nt, about 763nt, about 764nt, about 765nt, about 766nt, about 767nt, about 768nt, about 769nt, about 770nt, about 771nt, about 772nt, about 773nt, about 774nt, about 775nt, about 776nt, about 777nt, about 778nt, about 779nt, about 780nt, about 781nt, about 782nt, about 783nt, about 784nt, about 785nt, about 786nt, about 787nt, about 788nt, about 789nt, about 790nt,about 791nt, about 792nt, about 793nt, about 794nt, about 795nt, about 796nt, about 797nt, about 798nt, about 799nt, about 800nt, about 801nt, about 802nt, about 803nt, about 804nt, about 805nt, about 806nt, about 807nt, about 808nt, about 809nt, about 810nt, about 811nt, about 812nt, about 813nt, about 814nt, about 815nt, about 816nt, about 817nt, about 818nt, about 819nt, about 820nt, about 821nt, about 822nt, about 823nt, about 824nt, about 825nt, about 826nt t, about 827nt, about 828nt, about 829nt, about 830nt, about 831nt, about 832nt, about 833nt, about 834nt, about 835nt, about 836nt, about 837nt, about 838nt, about 839nt, about 840nt, about 841nt, about 842nt, about 843nt, about 844nt, about 845nt, about 846nt, about 847nt, about 848nt, about 849nt, about 850nt, about 851nt, about 852nt, about 853nt, about 854nt, about 855nt, about 856nt, about 857nt, about 858nt, about 859nt, about 860nt, about 861nt, about 862nt 2nt, about 863nt, about 864nt, about 865nt, about 866nt, about 867nt, about 868nt, about 869nt, about 870nt, about 871nt, about 872nt, about 873nt, about 874nt, about 875nt, about 876nt, about 877nt, about 878nt, about 879nt, about 880nt, about 881nt, about 882nt, about 883nt, about 884nt, about 885nt, about 886nt, about 887nt, about 888nt, about 889nt, about 890nt, about 891nt, about 892nt, about 893nt, about 894nt, about 895nt, about 896nt, about 897nt, about 898nt, about 899nt, about 900nt, about 901nt, about 902nt, about 903nt, about 904nt, about 905nt, about 906nt, about 907nt, about 908nt, about 909nt, about 910nt, about 911nt, about 912nt, about 913nt, about 914nt, about 915nt, about 916nt, about 917nt, about 918nt, about 919nt, about 920nt, about 921nt, about 922nt, about 923nt, about 924nt, about 925nt, about 926nt, about 927nt, about 928nt, about 929nt, about 930nt, about 931nt, about 932nt, about 933nt,Approximately 934nt, approximately 935nt, approximately 936nt, approximately 937nt, approximately 938nt, approximately 939nt, approximately 940nt, approximately 941nt, approximately 942nt, approximately 943nt, approximately 944nt, approximately 945nt, approximately 946nt, approximately 947nt, approximately 948nt, approximately 949nt, approximately 950nt, 951nt, 952nt, 953nt, 954nt, 955nt, 956nt, 957nt, 958nt, 959nt, 960nt, 961nt, 962nt, 963nt, 964nt, 965nt, 966nt, 967nt, approx. 968 nt, about 969 nt, about 970 nt, about 971 nt, about 972 nt, about 973 nt, about 974 nt, about 975 nt, about 976 nt, about 977 nt, about 978 nt, about 979 nt, about 980 nt, about 981 nt, about 982 nt, about 983 nt, about 984 nt, about 985 nt, about 986 nt, about 987 nt, about 988 nt, about 989 nt, about 990 nt, about 991 nt, about 992 nt, about 993 nt, about 994 nt, about 995 nt, about 996 nt, about 997 nt, about 998 nt, about 999 nt, or about 1000 nt.

[0064] Some preferred second nucleotide sequences for use in promoters of the present disclosure have a nucleotide sequence comprising about 370 to about 1000 nucleotides from the 3' end of SEQ ID NO: 2. Some preferred second nucleotide sequences for use in promoters of the present disclosure have a nucleotide sequence comprising about 895 to about 1000 nucleotides from the 5' end of SEQ ID NO: 2. A particularly preferred second nucleotide sequence comprises SEQ ID NO: 39, or a sequence having at least about 70% identity to SEQ ID NO: 39.

[0065] The second nucleotide sequence can comprise a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 2 over the entire length of the second nucleotide sequence. The second nucleotide sequence can have a sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to SEQ ID NO: 2 over the entire length of the second nucleotide sequence. Preferably, the second nucleotide sequence may have at least 90% identity to SEQ ID NO:2 over the entire length of the second nucleotide sequence.

[0066] The second nucleotide sequence can contain about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or more nucleic acid changes, e.g., substitutions or deletions, compared to an aligned sequence of the same length from SEQ ID NO:2.

[0067] An exemplary second nucleotide sequence may comprise an approximately 895 nt sequence having at least 70% identity to the 3' end of SEQ ID NO:2. For example, the second nucleotide sequence may comprise SEQ ID NO:21. An exemplary second nucleotide sequence may comprise an approximately 790 nt sequence having at least 70% identity to the 3' end of SEQ ID NO:2. For example, the second nucleotide sequence may comprise SEQ ID NO:22. An exemplary second nucleotide sequence may comprise an approximately 685 nt sequence having at least 70% identity to the 3' end of SEQ ID NO:2. For example, the second nucleotide sequence may comprise SEQ ID NO:23. An exemplary second nucleotide sequence may comprise an approximately 580 nt sequence having at least 70% identity to the 3' end of SEQ ID NO:2. For example, the second nucleotide sequence may comprise SEQ ID NO:24. An exemplary second nucleotide sequence may comprise an approximately 475 nt sequence having at least 70% identity to the 3' end of SEQ ID NO:2. For example, the second nucleotide sequence may comprise SEQ ID NO:25. An exemplary second nucleotide sequence may comprise an approximately 390 nt sequence having at least 70% identity to the 3' end of SEQ ID NO: 2. For example, the second nucleotide sequence may comprise SEQ ID NO: 26. An exemplary second nucleotide sequence may comprise an approximately 895 nt sequence having at least 70% identity to the 5' end of SEQ ID NO: 2. For example, the second nucleotide sequence may comprise SEQ ID NO: 30.

[0068] In some embodiments, the second nucleotide sequence comprises SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:30. In some embodiments, the second nucleotide sequence comprises a sequence having at least 70% identity to SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:30. In preferred embodiments, the second nucleotide sequence comprises SEQ ID NO:39, or a sequence having at least about 70% identity to SEQ ID NO:39.

[0069] c. Hybrid promoter structure and location As described and exemplified herein, a hybrid promoter comprising a first nucleotide sequence derived from a cone-specific promoter (SEQ ID NO: 1) and a second nucleotide sequence derived from a rod-specific promoter (SEQ ID NO: 2) has two unexpected properties: it is cone-specific and has increased promoter activity compared to full-length SEQ ID NO: 1 alone.

[0070] A hybrid promoter comprises a first nucleotide sequence and a second nucleotide sequence (both as described herein). If desired, the promoter can further comprise one or more other rod- or cone-specific promoter sequences. In some embodiments, the promoter can further comprise nucleotide sequences derived from one or more other cone-specific promoters, such as hG1.7 (SEQ ID NO: 20) or PR1.7 (SEQ ID NO: 19).

[0071] The first nucleotide sequence, the second nucleotide sequence, and any other rod-specific or cone-specific promoter sequences (if included) can each be included in the promoter as a single copy or multiple copies. For example, each first nucleotide sequence and each second nucleotide sequence can be present in 1 to about 10 copies (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies). In some embodiments, a promoter can include two or more first nucleotide sequences, two or more second nucleotide sequences, or both. When two or more first nucleotide sequences are included in a promoter, the two first nucleotide sequences do not need to be identical, as long as each individually satisfies the criteria described herein for the first nucleotide sequence. Similarly, when two or more second nucleotide sequences are included in a promoter, the two second nucleotide sequences do not need to be identical, as long as each individually satisfies the criteria described herein for the second nucleotide sequence.

[0072] In the hybrid promoters of the present disclosure, the first nucleotide sequence(s) and the second nucleotide sequence(s) can be positioned in any desired order, with or without other sequences between them. As exemplified herein, hybrid promoters containing the same first nucleotide sequence and the same second nucleotide sequence in different orders are effective for driving expression of a reporter gene in human cone photoreceptors.

[0073] A promoter can include a first nucleotide sequence linked to a second nucleotide sequence from 5' to 3', with no sequence between them. A promoter can include a second nucleotide sequence linked to a first nucleotide sequence from 5' to 3', with no sequence between them. In another example, a promoter can include two first nucleotide sequences (first sequence A and first sequence B) and one second nucleotide sequence in the following order from 5' to 3': first sequence A-second nucleotide sequence-first sequence B.

[0074] In some embodiments, a promoter may comprise one or more promoter units, each comprising a first nucleotide sequence and a second nucleotide sequence, in any order, and each promoter unit as a separate molecule having cone-specific promoter activity. For example, a promoter may comprise from 1 to about 4 promoter units. In another example, a promoter may comprise 4 to 10 promoter units. A promoter may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 promoter units, which may be the same or different.

[0075] Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:1 and a second nucleotide sequence comprising SEQ ID NO:2. A particular such promoter, Pro572, has SEQ ID NO:3. In some embodiments, the first nucleotide sequence comprises a sequence having at least 70% identity to SEQ ID NO:3. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:2 and a first nucleotide sequence comprising SEQ ID NO:1. A particular such promoter, Pro573, has SEQ ID NO:5. In some embodiments, the first nucleotide sequence comprises a sequence having at least 70% identity to SEQ ID NO:5. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:12 and a second nucleotide sequence comprising SEQ ID NO:2. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:2 and a first nucleotide sequence comprising SEQ ID NO:12. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 13 and a second nucleotide sequence comprising SEQ ID NO: 2. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 2 and a first nucleotide sequence comprising SEQ ID NO: 13. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 14 and a second nucleotide sequence comprising SEQ ID NO: 2. A particular such promoter, Pro572.2, has SEQ ID NO: 4. In some embodiments, the nucleotide sequence of the promoter comprises a sequence having at least 70% identity to SEQ ID NO: 4.

[0076] In some embodiments, a promoter may comprise, 5' to 3', a second nucleotide sequence comprising SEQ ID NO:2 and a first nucleotide sequence comprising SEQ ID NO:14. A particular such promoter, Pro573.2, has SEQ ID NO:6. In some embodiments, the nucleotide sequence of the promoter comprises a sequence having at least 70% identity to SEQ ID NO:6. Some particular promoters may comprise, 5' to 3', a first nucleotide sequence comprising SEQ ID NO:15 and a second nucleotide sequence comprising SEQ ID NO:2. Some particular promoters may comprise, 5' to 3', a second nucleotide sequence comprising SEQ ID NO:2 and a first nucleotide sequence comprising SEQ ID NO:15. Some particular promoters may comprise, 5' to 3', a first nucleotide sequence comprising SEQ ID NO:1 and a second nucleotide sequence comprising SEQ ID NO:21. Some particular promoters may comprise, 5' to 3', a second nucleotide sequence comprising SEQ ID NO:21 and a first nucleotide sequence comprising SEQ ID NO:1. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:1 and a second nucleotide sequence comprising SEQ ID NO:22. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:22 and a first nucleotide sequence comprising SEQ ID NO:1. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:1 and a second nucleotide sequence comprising SEQ ID NO:23. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:23 and a first nucleotide sequence comprising SEQ ID NO:1. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:1 and a second nucleotide sequence comprising SEQ ID NO:24. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:24 and a first nucleotide sequence comprising SEQ ID NO:1. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:1 and a second nucleotide sequence comprising SEQ ID NO:25.Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:25 and a first nucleotide sequence comprising SEQ ID NO:1. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:1 and a second nucleotide sequence comprising SEQ ID NO:26. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:26 and a first nucleotide sequence comprising SEQ ID NO:1. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:1 and a second nucleotide sequence comprising SEQ ID NO:30. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:30 and a first nucleotide sequence comprising SEQ ID NO:1. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:12 and a second nucleotide sequence comprising SEQ ID NO:21. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:21 and a first nucleotide sequence comprising SEQ ID NO:12. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:12 and a second nucleotide sequence comprising SEQ ID NO:22. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:22 and a first nucleotide sequence comprising SEQ ID NO:12. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:12 and a second nucleotide sequence comprising SEQ ID NO:23. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:23 and a first nucleotide sequence comprising SEQ ID NO:12. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:12 and a second nucleotide sequence comprising SEQ ID NO:24. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:24 and a first nucleotide sequence comprising SEQ ID NO:12.Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 12 and a second nucleotide sequence comprising SEQ ID NO: 25. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 25 and a first nucleotide sequence comprising SEQ ID NO: 12. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 12 and a second nucleotide sequence comprising SEQ ID NO: 26. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 26 and a first nucleotide sequence comprising SEQ ID NO: 12. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 12 and a second nucleotide sequence comprising SEQ ID NO: 30. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 30 and a first nucleotide sequence comprising SEQ ID NO: 12. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 13 and a second nucleotide sequence comprising SEQ ID NO: 21. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 21 and a first nucleotide sequence comprising SEQ ID NO: 13. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 13 and a second nucleotide sequence comprising SEQ ID NO: 22. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 22 and a first nucleotide sequence comprising SEQ ID NO: 13. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 13 and a second nucleotide sequence comprising SEQ ID NO: 23. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 23 and a first nucleotide sequence comprising SEQ ID NO: 13. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:13 and a second nucleotide sequence comprising SEQ ID NO:24.Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:24 and a first nucleotide sequence comprising SEQ ID NO:13. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:13 and a second nucleotide sequence comprising SEQ ID NO:25. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:25 and a first nucleotide sequence comprising SEQ ID NO:13. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:13 and a second nucleotide sequence comprising SEQ ID NO:26. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:26 and a first nucleotide sequence comprising SEQ ID NO:13. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:13 and a second nucleotide sequence comprising SEQ ID NO:30. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:30 and a first nucleotide sequence comprising SEQ ID NO:13. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:14 and a second nucleotide sequence comprising SEQ ID NO:21. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:21 and a first nucleotide sequence comprising SEQ ID NO:14. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:14 and a second nucleotide sequence comprising SEQ ID NO:22. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:22 and a first nucleotide sequence comprising SEQ ID NO:14. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO:14 and a second nucleotide sequence comprising SEQ ID NO:23. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO:23 and a first nucleotide sequence comprising SEQ ID NO:14.Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 14 and a second nucleotide sequence comprising SEQ ID NO: 24. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 24 and a first nucleotide sequence comprising SEQ ID NO: 14. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 14 and a second nucleotide sequence comprising SEQ ID NO: 25. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 25 and a first nucleotide sequence comprising SEQ ID NO: 14. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 14 and a second nucleotide sequence comprising SEQ ID NO: 26. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 26 and a first nucleotide sequence comprising SEQ ID NO: 14. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 14 and a second nucleotide sequence comprising SEQ ID NO: 30. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 30 and a first nucleotide sequence comprising SEQ ID NO: 14. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 15 and a second nucleotide sequence comprising SEQ ID NO: 21. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 21 and a first nucleotide sequence comprising SEQ ID NO: 15. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 15 and a second nucleotide sequence comprising SEQ ID NO: 22. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 22 and a first nucleotide sequence comprising SEQ ID NO: 15. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 15 and a second nucleotide sequence comprising SEQ ID NO: 23. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 23 and a first nucleotide sequence comprising SEQ ID NO: 15. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 15 and a second nucleotide sequence comprising SEQ ID NO: 24. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 24 and a first nucleotide sequence comprising SEQ ID NO: 15. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 15 and a second nucleotide sequence comprising SEQ ID NO: 25. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 15 and a second nucleotide sequence comprising SEQ ID NO: 25. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 15 and a second nucleotide sequence comprising SEQ ID NO: 26. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 26 and a first nucleotide sequence comprising SEQ ID NO: 15. Some particular promoters may comprise, from 5' to 3', a first nucleotide sequence comprising SEQ ID NO: 15 and a second nucleotide sequence comprising SEQ ID NO: 30. Some particular promoters may comprise, from 5' to 3', a second nucleotide sequence comprising SEQ ID NO: 30 and a first nucleotide sequence comprising SEQ ID NO: 15. In certain preferred embodiments, the hybrid promoter comprises no more than about 700 nucleotides, no more than about 600 nucleotides, or more preferably no more than about 500 nucleotides.

[0077] d. A cone-specific promoter that is a truncated version of SEQ ID NO: 1 In other embodiments, the cone-specific synthetic promoter disclosed herein comprises an active fragment of the cone-specific promoter of SEQ ID NO: 1, with the proviso that the synthetic promoter of this embodiment is not SEQ ID NO: 1. Nucleic acids disclosed herein can include promoters comprising a cone-specific promoter of at least about 150 nucleotides and no more than 499 nucleotides having at least 70% identity to SEQ ID NO: 1 over the entire length of the cone-specific promoter. Such promoters may lack a second nucleotide sequence, as described herein.

[0078] As disclosed and exemplified herein, the truncated version of SEQ ID NO: 1 has cone-specific promoter activity and can drive expression in human cone cells, for example, cone cells that are resting and no longer respond to light.Furthermore, such promoter can drive higher expression in cone cells than full-length SEQ ID NO: 1 alone.Such promoter can have promoter activity in human cone cells, for example, in vivo retinal cone cells, retinal explants or retinal organoids.In some cases, cone-specific promoter does not have promoter activity in other human retinal cells, such as rod cells.

[0079] A cone-specific promoter may comprise a nucleic acid sequence having at least 70% sequence identity to an equivalent length sequence from SEQ ID NO:1 (ie, at least about 150 nt and no more than 499 nt). The first sequence may have a nucleic acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to SEQ ID NO:1 over the entire length of the first nucleotide sequence.

[0080] A cone-specific promoter can contain about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or more nucleic acid changes, e.g., substitutions or deletions, compared to the corresponding portion of SEQ ID NO:1.

[0081] A cone-specific promoter may comprise at least 150 nucleotides having at least 70% identity to SEQ ID NO: 1, preferably the 3' end of SEQ ID NO: 1. As shown herein, such fragments of SEQ ID NO: 1 retain cone-specific promoter activity.

[0082] The cone-specific promoter may comprise at least 150 nucleotides having at least 70% identity to a sequence of equal length from the 3' end of SEQ ID NO: 1. The cone-specific promoter according to this paragraph may be considered a 5' truncated version of SEQ ID NO: 1, since nucleotides have been removed from the 5' end of SEQ ID NO: 1 to form the first sequence.

[0083] The cone-specific promoter can be a 5' truncated version of SEQ ID NO: 1 and can include at least 150 nt but less than 500 nt of ProA7 due to a deletion of nucleotides at the 5' end. For example, the cone-specific promoter can be nucleotides 386-500 of SEQ ID NO: 1 or nucleotides 2-500 of SEQ ID NO: 1.

[0084] For example, the cone-specific promoter is, among SEQ ID NO: 1, about 150 nucleotides (nt), about 151 nt, about 152 nt, about 153 nt, about 154 nt, about 155 nt, about 156 nt, about 157 nt, about 158 nt, about 159 nt, about 160 nt, about 161 nt, about 162 nt, about 163 nt, about 164 nt, about 165 nt, about 166 nt, about 167 nt, about 168 nt, about 169 nt, about 170 nt, about 171 nt, about 172 nt, about 173 nt, about 174 nt, about 175 nt, about 176 nt, about 177 nt, about 178 nt, about 179 nt, about 180 nt, about 181 nt, about 182 nt, about 183 nt, about 184 nt, about 185 nt, about 186 nt, about 187 nt, about 188 nt, about 189 nt, about 190 nt, about 191 nt, about 192 nt, about 193 nt, about 194 nt, about 195 nt, about 196 nt, about 197 nt, about 198 nt, about 199 nt, about 200 nt, about 201 nt, about 202 nt, about 203 nt, about 204 nt, about 205 nt, about 206 nt, about 207 nt, about 208 nt, about 209 nt, about 210 nt, about 211 nt, about 212 nt, about 213 nt, about 214 nt, about 215 nt, about 216 nt, about 217 nt, about 218 nt, about 219 nt, about 220 nt, about 221 nt, about 222 nt, about 223 nt, about 224 nt, about 225 nt, about 226 nt, about 227 nt, about 228 nt, about 229 nt, about 230 nt, about 231 nt, about 232 nt, about 233 nt, about 234 nt, about 235 nt, about 236 nt, about 237 nt, about 238 nt, about 239 nt, about 240 nt, about 241 nt, about 242 nt, about 243 nt, about 244 nt, about 245 nt, about 246 nt, about 247 nt, about 248 nt, about 249 nt, about 250 nt, about 251 nt, about 252 nt, about 253 nt, about 254 nt, about 255 nt, about 256 nt, about 257 nt, about 258 nt, about 259 nt, about 260 nt, about 261 nt, about 262 nt, about 263 nt, about 264 nt, about 265 nt, about 266 nt, about 267 nt, about 268 nt, about 269 nt, about 270 nt, about 271 nt, about 272 nt, about 273 nt, about 274 nt, about 275 nt, about 276 nt, about 277 nt, about 278 nt, about 279 nt, about 280 nt, about 281 nt, about 282 nt, about 283 nt, about 284 nt, about 285 nt, about 286 nt, about 287 nt,about 288nt, about 289nt, about 290nt, about 291nt, about 292nt, about 293nt, about 294nt, about 295nt, about 296nt, about 297nt, about 298nt, about 299nt, about 300nt, about 301nt, about 302nt, about 303nt, about 304nt, about 305nt, about 306nt, about 307nt, about 308nt, about 309nt, about 310nt, about 311nt, about 312nt, about 313nt, about 314nt, about 315nt, about 316nt, about 317nt, about 318nt, about 319nt, about 320nt, about 321nt, about 322nt, about 323nt t, about 324nt, about 325nt, about 326nt, about 327nt, about 328nt, about 329nt, about 330nt, about 331nt, about 332nt, about 333nt, about 334nt, about 335nt, about 336nt, about 337nt, about 338nt, about 339nt, about 340nt, about 341nt, about 342nt, about 343nt, about 344nt, about 345nt, about 346nt, about 347nt, about 348nt, about 349nt, about 350nt, about 351nt, about 352nt, about 353nt, about 354nt, about 355nt, about 356nt, about 357nt, about 358nt, about 359nt 9nt, about 360nt, about 361nt, about 362nt, about 363nt, about 364nt, about 365nt, about 366nt, about 367nt, about 368nt, about 369nt, about 370nt, about 371nt, about 372nt, about 373nt, about 374nt, about 375nt, about 376nt, about 377nt, about 378nt, about 379nt, about 380nt, about 381nt, about 382nt, about 383nt, about 384nt, about 385nt, about 386nt, about 387nt, about 388nt, about 389nt, about 390nt, about 391nt, about 392nt, about 393nt, about 394nt, about about 409nt, about 410nt, about 411nt, about 412nt, about 413nt, about 414nt, about 415nt, about 416nt, about 417nt, about 418nt, about 419nt, about 420nt, about 421nt, about 422nt, about 423nt, about 424nt, about 425nt, about 426nt, about 427nt, about 428nt, about 429nt, about 430nt, about 431nt, about 432nt, about 433nt, about 434nt, about 435nt, about 436nt, about 437nt, about 438nt, about 439nt, about 440nt, about 441nt, about 442nt, about 443approx. 431nt, approx. 432nt, approx. 433nt, approx. 434nt, approx. 435nt, approx. 436nt, approx. 437nt, approx. 438nt, approx. 439nt, approx. 8nt, approx. 449nt, approx. 450nt, approx. 451nt, approx. 452nt, approx. 453nt, approx. 454nt, approx. 455nt, approx. 456nt, approx. 457nt, approx. It may contain about 466 nt, about 467 nt, about 468 nt, about 469 nt, about 470 nt, about 471 nt, about 472 nt, about 473 nt, about 474 nt, about 475 nt, about 476 nt, about 477 nt, about 478 nt, about 479 nt, about 480 nt, about 481 nt, about 482 nt, about 483 nt, about 484 nt, about 485 nt, about 486 nt, about 487 nt, about 488 nt, about 489 nt, about 490 nt, about 491 nt, about 492 nt, about 493 nt, about 494 nt, about 495 nt, about 496 nt, about 497 nt, about 498 nt, or about 499 nt.

[0085] Some preferred cone-specific promoters of this aspect of the disclosure have a nucleotide sequence comprising about 150 nucleotides to about 395 nucleotides from the 3' end of SEQ ID NO:1.

[0086] As discussed above, a cone-specific promoter may comprise a nucleic acid sequence having at least 70% sequence identity over the entire length of the cone-specific promoter to SEQ ID NO: 1. A cone-specific promoter may have a sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to SEQ ID NO: 1 over the entire length of the cone-specific promoter. Preferably, the cone-specific promoter may have at least 90% identity with SEQ ID NO: 1 over the entire length of the cone-specific promoter.

[0087] A cone-specific promoter can contain about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or more nucleic acid changes, e.g., substitutions or deletions, compared to an aligned sequence of the same length from SEQ ID NO:1.

[0088] An exemplary cone-specific promoter may comprise an approximately 395-nt sequence having at least 70% identity to the 3' end of SEQ ID NO: 1. For example, a cone-specific promoter may comprise SEQ ID NO: 12. A cone-specific promoter may comprise a nucleotide sequence having at least 70% identity to SEQ ID NO: 12. An exemplary cone-specific promoter may comprise an approximately 290-nt sequence having at least 70% identity to the 3' end of SEQ ID NO: 1. For example, a cone-specific promoter may comprise SEQ ID NO: 13. A cone-specific promoter may comprise a nucleotide sequence having at least 70% identity to SEQ ID NO: 13. An exemplary cone-specific promoter may comprise an approximately 185-nt sequence having at least 70% identity to the 3' end of SEQ ID NO: 1. For example, a cone-specific promoter may comprise SEQ ID NO: 14 (sometimes referred to as ProSC). A cone-specific promoter may comprise a nucleotide sequence having at least 70% identity to SEQ ID NO: 14. An exemplary cone-specific promoter may comprise a sequence of about 150 nt having at least 70% identity to the 3' end of SEQ ID NO: 1. For example, a cone-specific promoter may comprise SEQ ID NO: 15. A cone-specific promoter may comprise a nucleotide sequence having at least 70% identity to SEQ ID NO: 15.

[0089] In some embodiments, the promoter comprises a cone-specific promoter comprising SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0090] The cone-specific promoter may be present in one or more copies. Alternatively, or in addition, sequences from one or more other cone-specific promoters may be included. Examples of such other promoters include hG1.7 (SEQ ID NO: 20) and PR1.7 (SEQ ID NO: 19).

[0091] The cone-specific promoter and any other cone-specific promoter sequences (if included) may each be included in the promoter as a single copy or multiple copies. In some embodiments, a promoter may include two or more cone-specific promoters. When two or more cone-specific promoters are included in a promoter, the two cone-specific promoters do not need to be identical, as long as each individually meets the criteria described herein for cone-specific promoters. For example, each cone-specific promoter may be present in 1 to about 10 copies (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies).

[0092] In promoters lacking a second nucleotide sequence and comprising two or more cone-specific promoters, the cone-specific promoter(s) may be positioned in any desired order, and with or without other sequences between them.

[0093] In some embodiments, a promoter can include one or more units, each unit including one or more cone-specific promoters in any desired order. For example, a promoter can include from 1 to about 4 promoter units of cone-specific promoter(s).

[0094] In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:12 and SEQ ID NO:12 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:12 and SEQ ID NO:13 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:13 and SEQ ID NO:12 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:12 and SEQ ID NO:14 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:14 and SEQ ID NO:12 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:12 and SEQ ID NO:15 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:15 and SEQ ID NO:12 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:13 and SEQ ID NO:12 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:12 and SEQ ID NO:13 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:13 and SEQ ID NO:13 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising, from 5' to 3', SEQ ID NO: 13 and SEQ ID NO: 14. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising, from 5' to 3', SEQ ID NO: 14 and SEQ ID NO: 13.In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:13 and SEQ ID NO:15 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:15 and SEQ ID NO:13 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:14 and SEQ ID NO:12 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:12 and SEQ ID NO:14 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:14 and SEQ ID NO:13 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:13 and SEQ ID NO:14, from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:14 and SEQ ID NO:14, from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:14 and SEQ ID NO:15, from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:15 and SEQ ID NO:14, from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO:15 and SEQ ID NO:12, from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising, from 5' to 3', SEQ ID NO: 12 and SEQ ID NO: 15. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising, from 5' to 3', SEQ ID NO: 15 and SEQ ID NO: 13.In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO: 13 and SEQ ID NO: 15 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO: 15 and SEQ ID NO: 14 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO: 14 and SEQ ID NO: 15 from 5' to 3'. In some embodiments, the promoter may comprise about 1, about 2, about 3, or about 4 units, each comprising SEQ ID NO: 15 and SEQ ID NO: 15 from 5' to 3'.

[0095] B. Promoter-Containing Nucleic Acid The present disclosure also relates to nucleic acids comprising a synthetic promoter comprising a first nucleotide sequence (including a synthetic promoter further comprising a second nucleotide sequence) and / or a synthetic promoter comprising a cone-specific promoter sequence, as described herein. The nucleic acid is typically designed for expression of a desired protein and / or nucleic acid (typically encoded by a transgene in the nucleic acid) in cone photoreceptors. The nucleic acid may be in the form of a vector, such as, for example, an AAV vector, a lentiviral vector, or other suitable vector. Thus, the nucleic acid comprises a hybrid promoter described herein and a transgene encoding the desired protein or nucleic acid. The nucleic acid also typically includes regulatory elements, which are well known in the art and are selected based on the desired expression level, delivery vector, and other considerations. For example, the transgene is typically operably linked to a polyA signal. Illustrative, non-limiting examples of regulatory elements are briefly described herein.

[0096] a. Post-transcriptional regulatory elements An exemplary regulatory element is a post-transcriptional regulatory element, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Regulatory elements are nucleic acid sequences that contribute to regulating the expression of the DNA sequence in which they reside. Regulatory elements can sometimes contain three components (alpha, beta, and gamma). The activity of a regulatory element can depend on the number of components present.

[0097] As disclosed and exemplified herein, a WPRE element in combination with a promoter of the present disclosure can result in high level expression of a heterologous polypeptide or heterologous RNA (e.g., encoded by a transgene) in human cone cells.

[0098] The regulatory element may be operably linked to nucleotides encoding a heterologous polypeptide or RNA, as well as other expression control elements, such as a promoter and polyadenylation (PolyA) signal.

[0099] Any suitable regulatory element can be used, such as a naturally occurring WPRE or a WPRE containing one or more mutations in the X region. For example, a suitable WPRE with a mutation in the X region is disclosed in U.S. Patent No. 7,419,829 and provided herein as SEQ ID NO:8.

[0100] Suitable WPREs are described in U.S. Publication No. US2021 / 0032656. Generally, a WPRE can have a nucleotide sequence comprising SEQ ID NO: 7. A WPRE can comprise a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 7. A WPRE can have a nucleotide sequence having at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to SEQ ID NO: 7.

[0101] A WPRE can contain about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or more nucleic acid changes, e.g., substitutions or deletions. The nucleic acid substitutions can be conservative or non-conservative, but are preferably conservative.

[0102] b. Polyadenylation signal The nucleic acid may also contain a nucleotide sequence encoding a suitable PolyA signal. Desirably, the nucleotide sequence encoding polyA is operably linked to the nucleotide sequence encoding the desired protein or nucleic acid for expression. Any suitable PolyA signal can be used, such as the SV40 PolyA signal, rabbit beta globin PolyA signal, human growth hormone (hGH) PolyA signal, bovine growth hormone PolyA signal, etc. Human growth hormone (hGH) polyA (SEQ ID NO: 9) is a preferred PolyA.

[0103] The PolyA can be in any suitable position, but preferably the PolyA is 3' to any regulatory element.

[0104] PolyA can have a nucleotide sequence that includes SEQ ID NO: 9. PolyA can comprise a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 9. PolyA can have a nucleotide sequence that has at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to SEQ ID NO: 9.

[0105] PolyA can contain about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or more nucleic acid changes, e.g., substitutions or deletions. The nucleic acid substitutions can be conservative or non-conservative, but are preferably conservative.

[0106] c. Packaging sequence The nucleic acid may further comprise a suitable packaging sequence or signal for packaging into a viral vector such as an AAV or lentiviral vector. For example, the nucleic acid may comprise an inverted terminal repeat ITR, such as an AAV ITR. This may be particularly appropriate when the nucleic acid is incorporated into an AAV vector. Typically, two ITRs are included in the nucleic acid, one at the 5' end and one at the 3' end. For example, the nucleic acid may comprise an AAV ITR at the 5' end of the promoter and an AAV ITR at the 3' end. Specific examples of AAV ITRs are SEQ ID NO: 10 and SEQ ID NO: 11.

[0107] The ITRs can be independently selected from wild-type and, optionally, self-complementary (scAAV) ITRs. Other sequences that are functionally equivalent to the AAV 5' and / or 3' ITRs (e.g., parvovirus terminal repeats) can also be used.

[0108] C. Transgene The nucleic acid may comprise a nucleotide sequence encoding a desired polypeptide or nucleic acid. Typically, the polypeptide is heterologous to the synthetic promoter. This means that the first nucleotide sequence, the second nucleotide sequence (in embodiments in which the synthetic promoter comprises the second nucleotide sequence), the cone-specific promoter sequence, and / or any other promoter sequences that may be included in the synthetic promoter do not naturally drive expression of the polypeptide. The synthetic promoter and the nucleotide sequence encoding the polypeptide are operably linked. The nucleotides may encode any heterologous polypeptide whose expression in cone cells is desired.

[0109] The heterologous polypeptide can be a therapeutic polypeptide, a reporter protein, or an optogenetic actuator, such as those described below.

[0110] In some embodiments, the nucleotide sequence encodes a therapeutic polypeptide, meaning that the nucleotide sequence is capable of rescuing a defective or mutated gene known to cause a retinal disease. Examples of retinal diseases caused by defective or mutated genes and therefore that can be rescued by nucleotide sequences encoding therapeutic polypeptides include, but are not limited to, MT-ND4 (Gene ID: 4538), MT-ND1 (Gene ID: 4535), MT-ND6 (Gene ID: 4541), MT-CYB (Gene ID: 4519), MT-C03 (Gene ID: 4514), MT-ND5 (Gene ID: 4540), MT-ND2 (Gene ID: 4536), MT-COI (Gene ID: 4512), MT-ATP6 (Gene ID: 4508), MT-ND4L (Gene ID: 4539), OPA1 (Gene ID: 4976), OPA3 (Gene ID: 80207), OPA7 (Gene ID: 84233), AC02, and (Gene ID: 50). The nucleotide sequence may encode neurotrophic factors such as GDNF (Gene ID: 2668), CNTF (Gene ID: 1270), FGF2 (Gene ID: 2247), BDNF (Gene ID: 627) and EPO (Gene ID: 2056), anti-apoptotic genes such as BCL2 (Gene ID: 596) and BCL2L1 (Gene ID: 598), anti-angiogenic factors such as endostatin, angiostatin and sFlt, anti-inflammatory factors such as IL10 (Gene ID: 3586), IL1R1 (Gene ID: 3554), TGFBI (Gene ID; 7045) and IL4 (Gene ID: 3565), or rod-derived cone survival factor (RdCVF) (Gene ID: 115861).

[0111] Signal peptides may be added to therapeutic polypeptides to enable, inter alia, directing their uptake by specific organelles (e.g., mitochondria), secreting them from the cell, or inserting them into the cell membrane.

[0112] In another embodiment, the heterologous polypeptide can be an optogenetic actuator, which is a photochemically reactive polypeptide that uses vitamin A or its isoforms as its chromophore. The optogenetic actuator is particularly a light-gated ion pump or channel that absorbs and is activated by light. The optogenetic actuator can be derived from prokaryotes or eukaryotes. In particular, it can be microbial opsin or vertebrate opsin. The optogenetic actuator can be an optogenetic activator or an optogenetic inhibitor.

[0113] Optogenetic activators depolarize cells when exposed to light. Examples of optogenetic activators include rhodopsin, photopsin, melanopsin, pinopsin, parapinopsin, VA opsin, peropsin, neuropsin, encephalopsin, retinochrome, RGR opsin, microbial opsins with red-shifted spectral characteristics, such as ReaChR, Chrimson, or ChrimsonR, vertebrate opsins that can recruit Gi / 0 signaling, such as short-wavelength vertebrate opsin or long-wavelength vertebrate opsin, channelrhodopsins from microalgae of the genus Chlamydomonas, such as channelrhodopsin-1 and channelrhodopsin-2 (from Chlamydomonas reinhardtii), and optimized or functionally improved variants thereof (e.g., codon-optimized variants, mutants, chimeras).

[0114] In some embodiments, the optogenetic actuator may be an optogenetic activator, preferably selected from channelrhodopsin, ChrimsonR and variants thereof, such as hChR2(L132C)-hCatCh and ChrimsonR-tdTomato.

[0115] In some embodiments, the optogenetic actuator is an optogenetic activator, preferably selected from channelrhodopsin and variants thereof, such as hChR2(L132C)-hCatCh.

[0116] Optogenetic inhibitors hyperpolarize cells when exposed to light.Examples of optogenetic inhibitors include but are not limited to halorhodopsins, such as halorhodopsin (NpHR), enhanced halorhodopsin (eNpHR2.0 and eNpHR3.0) and red-shifted halorhodopsin Halo57, archerhodopsin-3 (AR-3), archerhodopsin (Arch), bacteriorhodopsins, such as enhanced bacteriorhodopsin (eBR), proteorhodopsin, xanthorhodopsin, Leptosphaeria maculans fungal opsin (Mac), clax halorhodopsin Jaws, and their optimized or functionally improved variants (for example, codon-optimized variants, mutants, chimeras).

[0117] In some embodiments, the nucleic acid operably linked to the promoter of the present invention can encode a reporter protein. Preferably, the reporter protein can be detectable in living cone photoreceptor cells. By expressing the reporter protein under the control of the promoter of the present disclosure, cone photoreceptor cells can be specifically detected or identified. The reporter protein can be, for example, a fluorescent protein (e.g., GFP), a calcium indicator (e.g., GCamP), luciferase, alkaline phosphatase, β-galactosidase, β-lactamase, horseradish peroxidase, and variants thereof.

[0118] In some embodiments, the reporter protein may be selected from the group consisting of a fluorescent protein, a calcium indicator, alkaline phosphatase, β-galactosidase, β-lactamase, horseradish peroxidase, and variants thereof.

[0119] The nucleic acid may comprise a nucleotide sequence encoding an RNA heterologous to the synthetic promoter. This means that the first nucleotide sequence, the second nucleotide sequence (in embodiments where the synthetic promoter comprises the second nucleotide sequence), the cone-specific promoter sequence, and / or any other promoter sequences that may be included in the synthetic promoter do not naturally drive expression of the RNA. The synthetic promoter and the nucleotide sequence encoding the RNA are operably linked. The nucleotides may encode any heterologous RNA whose expression in cone cells is desired.

[0120] In some embodiments, the heterologous RNA can be a therapeutic RNA. The RNA can be, for example, siRNA, shRNA, RNAi, miRNA, antisense RNA, or ribozyme. In some specific embodiments, the nucleotide sequence encodes a heterologous RNA that, when transcribed from the nucleotide sequence operably linked to a promoter, can treat or prevent the disorder by preventing the translation or transcription of abnormal or excessive proteins associated with eye disease.

[0121] The nucleic acids described herein, or any components thereof (e.g., promoters, sequences encoding depolarizing optogenetic proteins), can be optimized by sequence alterations using well-known methods, e.g., to achieve a desired level of expression, reduce immunogenicity, or for other purposes. Suitable methods for optimizing nucleic acid constructs by sequence alterations, including, e.g., to increase expression, packaging, and / or reduce immunogenicity, are well known in the art, and such modifications of the nucleic acids disclosed herein are considered variants of the particular nucleic acid. For example, the nucleic acids described herein, or any component thereof, can be codon-optimized, CpG-depleted (see, e.g., U.S. Patent No. 11,015,210; YAMedvedeva, et al., Bioinformatics-Trends and Methodologies, 449-472 (2011)), modified to remove repeat and hairpin sequences, modified to eliminate alternative reading frames, modified to remove unwanted splice donor and acceptor sites, modified to add stuffer sequences, modified to include dsRNA or gRNA sequences (see, e.g., Domenger and Grimm, Human Molecular Genetics, 2019, 28:R1-R12), modified to include an inducible regulatory system (e.g., a Tet on / off system) (see, e.g., Gossen et al., Science, 268:1766-1769 (1995); Harvey et al., Bioinformatics-Trends and Methodologies, 449-472 (2011)), modified to include a nucleotide sequence that is specifically targeted to the target gene, such as a nucleotide sequence. al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), or modified to include ITRs with deleted terminal release site (trs) sequences to generate scAAV (see, e.g., McCarty et al., Gene Therapy, 2001, 16:1248-54). The methods for optimizing the nucleic acids disclosed herein are conventional and well known to those of skill in the art.

[0122] D. Vector The present disclosure further relates to recombinant vectors containing the nucleic acids disclosed herein or host cells containing such vectors. AAV vectors may be based on viral genomes from which the capsid and other structural proteins have been removed. The vectors provided herein may be suitable for gene therapy, particularly for targeting human cone cells. As disclosed herein, the nucleic acid may include a synthetic promoter and may include a nucleotide sequence encoding a heterologous polypeptide or RNA, regulatory elements, and a suitable polyA signal. Each of the nucleotide sequences is operably linked.

[0123] Furthermore, a vector may contain additional elements for expression of a nucleic acid, for example, a vector may contain one or more ITRs, ribosome binding elements, terminators, enhancers, selectable markers, introns, polyA signals, and / or origins of replication.

[0124] In some embodiments, the AAV vector is a single-stranded AAV (ssAAV). In some embodiments, the AAV vector is a self-complementary AAV (scAAV).

[0125] Many different viral and non-viral vectors and their delivery methods are known to those skilled in the art, such as adenoviral vectors, AAV vectors, retroviral vectors, lentiviral vectors, herpesvirus vectors, liposomes, naked DNA administration, etc. See, e.g., Wright (1997), Br. J. Ophthalmol., 8(1):620-622. Many suitable vectors are commercially available. Such vectors typically contain additional elements such as an origin of replication, a selectable marker gene (e.g., LEU2, URA3, TRP1, HIS3, GFP), a centromere sequence, etc., as well as a polyadenylation signal in conjunction with a multiple cloning site.

[0126] Suitable vectors for the nucleic acids disclosed herein can be viral vectors such as vectors derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV or SNV, lentiviral vectors (e.g., derived from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), or equine infectious anemia virus (EIAV)), adenovirus (Ad) vectors, AAV vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus, herpesvirus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, and Rous sarcoma virus vectors.

[0127] The vector can be a retroviral vector, such as a lentiviral vector, or a non-pathogenic parvovirus.

[0128] In some specific embodiments, the vector can be an AAV viral vector comprising an AAV capsid.Without being bound by theory, the AAV capsid can improve the selective delivery of nucleic acid to cone cells, and may also improve the expression of heterologous polypeptides or RNA.

[0129] Many suitable AAV capsids and viral backbones are well known in the art, and multiple AAV capsid serotypes are known and may be suitable for the optogenetic constructs disclosed herein. At least 16 AAV serotypes have been described in the literature, referred to as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16. Many engineered capsids and variant capsids are also well known in the art.

[0130] Exemplary AAV capsids include, but are not limited to, AAV8-BP2, AAV-PHP.B, AAV-PHP.eB, AAV5, or AAV-NHP26. Preferred AAV capsid proteins are AAV-PHP.eB, AAV8-BP2, or AAV5.

[0131] Instead of using natural AAV serotypes, artificial AAV serotypes, such as AAVs containing non-naturally occurring capsids, may be used. Such artificial capsids can be produced by any suitable technique using a selected AAV sequence (e.g., a fragment of the VP1 capsid protein) in combination with a heterologous sequence that can be obtained from a different selected AAV serotype, a non-contiguous portion of the same AAV serotype, a non-AAV viral source, or a non-viral source. The artificial AAV serotype can be, but is not limited to, a chimeric AAV capsid or a mutant AAV capsid. A chimeric capsid contains VP capsid proteins from at least two different AAV serotypes, or at least one chimeric VP protein that combines VP protein regions or domains from at least two AAV serotypes.

[0132] AAV capsid proteins may also be mutated, particularly to improve transduction efficiency. Mutant AAV capsids can be obtained from capsid modifications introduced by error-prone PCR and / or peptide insertion, or by including one or more amino acid substitutions. In particular, mutations can be made in any one or more tyrosine residues of native or non-native capsid proteins (e.g., VP1, VP2, or VP3). The mutated residue may be a surface-exposed tyrosine residue. Exemplary mutations include, but are not limited to, tyrosine-to-phenylalanine substitutions, such as Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F, and Y720F.

[0133] The vector can be in any form, including, but not limited to, a viral particle such as an rAAV particle comprising a nucleic acid encoding a depolarizing optogenetic protein described herein. If desired, the nucleic acid encoding a depolarizing optogenetic protein described herein can be combined with other suitable nucleic acid delivery agents for delivery, e.g., complexed with lipids or encapsulated in liposomes.

[0134] Nucleic acids encoding the depolarizing optogenetic proteins disclosed herein can be packaged into viral capsids to produce viral particles, preferably AAV particles.

[0135] The viral particles may be capable of transducing up to about 10% of primary human cone cells. For example, the viral particles may be capable of transducing about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of primary human cone cells.

[0136] Also provided herein are production methods and cultures for the production of viral particles. The cultures may include suitable host cells, including, for example, human-derived cell lines such as HeLa, A549, or HEK293 cells, suitable helper virus functions provided by a wild-type or mutant adenovirus, such as a temperature-sensitive adenovirus, herpesvirus, or a plasmid construct that provides helper functions, AAV rep and cap genes and gene products, a nucleic acid disclosed herein or a vector containing the nucleic acid, and suitable media and media components that support viral particle production as are well known in the art.

[0137] Suitable host cells may include, but are not limited to, mammalian cells, insect cells, plant cells, microorganisms, and yeast. The host cell may also be a packaging cell. Exemplary packaging and producer cells are derived from HEK293, A549, or HeLa cells.

[0138] The host cells disclosed herein can be transformed or transfected with vectors containing the nucleic acids or viral particles disclosed herein. The host cells can be any animal cell, plant cell, bacterial cell, or yeast. The vectors disclosed herein can be transferred into the host cell using any known technique, including viral infection, and can be maintained in the host cell in an ectopic form or integrated into the genome.

[0139] E. Pharmaceutical Compositions The present disclosure also relates to pharmaceutical compositions that may contain nucleic acids, vectors, or host cells. The pharmaceutical compositions can be administered to a subject to restore the light sensitivity of human cone cells, particularly human cone cells that are not activated by light stimuli, so that the subject's vision can be restored. The compositions containing nucleic acids can be suitable for administration to a subject. The pharmaceutical compositions can be provided as a solution, suspension, emulsion, or as a solid form suitable for dissolving or suspending in a liquid before use.

[0140] Pharmaceutical compositions can contain pharmaceutically acceptable carriers, i.e., any carrier that does not impair the effectiveness of the biological activity of the components and is not toxic to the subject to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Such carriers can be formulated by conventional methods and administered to subjects in appropriate dosages. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservatives, emulsifiers, and dispersing agents. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents.

[0141] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippincott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic; however, the formulation may be hypertonic or hypotonic, if desired. Examples of pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions such as Ringer's solution, and dextrose solution. The pH of the solution is generally about 5 to about 8 or about 7 to 7.5. Other carriers include sustained-release preparations, such as semipermeable matrices of solid hydrophobic polymers containing an immunogenic polypeptide. The matrices are in the form of shaped articles, such as films, liposomes, or microparticles. Certain carriers may be more suitable depending, for example, on the route of administration and concentration of the administered composition. Carriers suitable for direct delivery to the eye can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of various tween compounds, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts may be included therein, such as mineral acid salts such as hydrochloride, hydrobromide, phosphate, and sulfate, and organic acid salts such as acetate, propionate, malonate, and benzoate. Most preferably, the composition is combined with saline, Ringer's balanced salt solution (pH 7.4), or the like.

[0142] The pharmaceutical composition may optionally include one or more agents that facilitate delivery of the nucleic acid or vector to the target cell, including, but not limited to, a transfection reagent or components thereof, such as lipids or polymers.

[0143] The pharmaceutical compositions disclosed herein may be formulated for administration to the eye, particularly by intraocular injection, for example, by subretinal and / or intravitreal or suprachoroidal administration. For intravitreal delivery, the pharmaceutical compositions disclosed herein may be directly injected into the vitreous. For subretinal delivery, the pharmaceutical compositions disclosed herein may be delivered to a localized subretinal bleb between the retinal pigment epithelium (RPE) and the photoreceptor layer during a surgical procedure. This can be achieved during pars plana vitrectomy (ppV). Subretinal administration may provide direct access to the photoreceptors and RPE. Suprachoroidal injection may provide access to the photoreceptors through the choroidal layer. Alternatively, the pharmaceutical composition may be delivered into the anterior segment of the eye, particularly into the anterior chamber. Subretinal injection is the preferred mode of administration.

[0144] The amount of the pharmaceutical composition to be administered can be determined by standard procedures well known to those skilled in the art. To determine the appropriate dosage, it is necessary to take into account the physiological data of the patient (e.g., age, size, and weight) and the type and severity of the disease to be treated.

[0145] The pharmaceutical composition may be formulated for administration by injection, for example, subretinal injection, intravitreal injection, or suprachoroidal injection.The preparation for injection may be provided in a unit dosage form, for example, in an ampule or in a multi-dose container.The composition may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents.Alternatively, the active ingredient may be in powder form, which is reconstituted with a suitable vehicle such as sterile pyrogen-free water before use.

[0146] The pharmaceutical compositions disclosed herein can also be formulated as depot preparations or for use in implantable delivery systems.Such long-acting formulations can be administered, for example, by intraocular implantation or intraocular injection.The pharmaceutical compositions can also be formulated as depot preparations for use in implantable drug delivery systems or devices, particularly for repeated refilling of the reservoir of implantable drug delivery systems or devices.Therefore, the pharmaceutical compositions can be formulated with suitable polymers or hydrophobic materials (for example, as emulsions in acceptable oils), or with ion exchange resins, or as sparingly soluble derivatives, for example, as sparingly soluble salts.

[0147] In some embodiments, the pharmaceutical composition disclosed herein can comprise a vector or virus particle comprising the nucleic acid disclosed herein.Preferably, the vector or virus particle is an AAV vector or AAV particle.The pharmaceutical composition can comprise a host cell comprising the nucleic acid disclosed herein, or a virus particle comprising the nucleic acid.

[0148] Optionally, the pharmaceutical composition comprising the host cells may be frozen for storage at any temperature suitable for storage of the cells. The pharmaceutical composition may comprise viral particles, wherein each unit dose contains 10E+8 to 10E+13 viral particles as determined by polymerase chain reaction using a probe specific for the viral genome.

[0149] The pharmaceutical composition may further comprise one or more additional active compounds, such as a corticosteroid, an antibiotic, an analgesic, an immunosuppressant, a nutritional factor, or any combination thereof.

[0150] F. Kit Also disclosed herein are kits comprising the nucleic acids, vectors comprising the nucleic acids, viral particles comprising the nucleic acids, host cells, or pharmaceutical compositions thereof disclosed herein.

[0151] The kit may be in the form of a pharmaceutically acceptable solution, for example, in combination with sterile saline, dextrose solution, or buffer solution, or other pharmaceutically acceptable sterile fluid. Alternatively, the complex may be lyophilized or dehydrated. In this case, the kit optionally further comprises a pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.) in a container for reconstituting the complex to form a solution for injection purposes.

[0152] The kit may further include a needle or syringe, preferably packaged in sterile form, for injecting the conjugate, and / or a packaged alcohol pad. Instructions for administration of the composition by a clinician or patient are optionally included.

[0153] G. Method The present disclosure also relates to the method for expressing heterologous polypeptide or heterologous RNA in cone photoreceptor cells, for example, in culture, in retinal organoid, retinal explant or mammalian subject.This method can comprise administering to retinal organoid, retinal explant or mammalian subject the nucleic acid described herein, vector, host cell, or two or more thereof.Heterologous polypeptide or RNA can be expressed in cone photoreceptor cells.

[0154] This method can be used to test the function of heterologous polypeptides or RNA in organoids or retinal explants.This method can be used in mammalian subjects to study, prevent, or treat mammalian visual diseases or disorders, such as blindness.

[0155] Exemplary methods include those comprising administering to a subject in need thereof an effective amount of a nucleic acid comprising a transgene disclosed herein, a vector comprising a nucleic acid disclosed herein, or a pharmaceutical composition thereof, to treat a retinal disease through expression of the transgene in cone cells of the retina.

[0156] The vector can be an AAV vector.The nucleic acid, vector, or pharmaceutical composition thereof can be administered by subretinal injection.The nucleic acid, vector, or pharmaceutical composition can be administered before, after, or at the onset of retinal disease.

[0157] The methods disclosed herein may be useful for treating or ameliorating blindness. The methods disclosed herein may be useful for restoring vision. The methods disclosed herein may be useful for restoring light sensitivity in human cone photoreceptor cells. The methods disclosed herein may be suitable for treating retinal degeneration.

[0158] The methods disclosed herein may be suitable for treating any disease, disorder, or condition associated with vision loss, including retinitis pigmentosa, rod-cone dystrophy, Leber's congenital amaurosis, Usher syndrome, Bardet-Biedl syndrome, Best disease, retinoschisis, Stargardt disease, untreated retinal detachment, pattern dystrophy, cone-rod dystrophy, color vision deficiency, ocular albinism, S-cone enhancement syndrome, diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, sickle cell retinopathy, congenital stationary night blindness, congenital choroideremia, cone dysfunction after retinal detachment, or pigment epithelial retinal degeneration retinal vein occlusion.

[0159] Additionally, the nucleic acid molecules, vectors, or pharmaceutical compositions may be used to manufacture medicaments and / or to treat patients with diseases, disorders, or conditions associated with vision loss.

[0160] The subject may be a human, dog, cat, horse, or any animal in which restoration of vision is desired.

[0161] The nucleic acids disclosed herein can be administered to a subject in an amount sufficient to at least partially restore vision.

[0162] The nucleic acids provided herein can be administered to a subject by any suitable route, including, but not limited to, intraocular (e.g., subretinal, intravitreal, or suprachoroidal injection), oral, intradermal, intrathecal, intratumoral, intramuscular, intraperitoneal, intravenous, topical, subcutaneous, transdermal, intranasal, and inhalation routes, as well as scarification (e.g., using a bifurcated needle to create a scratch in the top layer of the skin). A preferred administration route is intraocular administration, and more preferred is subretinal injection.

[0163] The dosage of the nucleic acid may depend on the type of composition, as well as the subject's age, weight, body surface area, individual condition, individual pharmacokinetic data, and mode of administration.

[0164] The nucleic acid can be administered to a subject who has a pathological condition associated with vision loss or who is at risk of developing the condition.The nucleic acid can be administered before or after the onset of symptoms of the disease.For example, before or after the partial or complete degeneration of cone cells.The nucleic acid can be administered before or after the partial or complete loss of vision.

[0165] 5. Equivalents It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present invention described herein will be apparent and may be made using suitable equivalents without departing from the scope of the disclosure or embodiments. Having described certain compositions and methods in detail, the same will be more clearly understood by reference to the following examples, which are presented for illustrative purposes only and are not intended to be limiting. [Example]

[0166] 6. Working Example The following are examples of methods and compositions of the present invention. Given the summary provided herein, it will be understood that other various embodiments may be practiced.

[0167] Example 1. Short promoter sequences for specific expression of genes in cone photoreceptors 1.1. Method Vector construction and production The existing cone promoter ProA7, known to drive eGFP expression specifically in cone photoreceptors (Juettner et al., 2019), as well as its 3'- and 5'-truncated fragments, were cloned into the pAAV plasmid, respectively, in front of an optimized Kozak sequence (GCCACC) and the translation start codon of the eGFP coding sequence, followed by the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and SV40 polyadenylation (Figure 1).

[0168] Adeno-associated viral vectors (AAV) of the serotype PHP.eB were produced using a high-throughput version of the general triple transfection method described by Grieger et al. (2006). Briefly, HEK293T cells were seeded in 24-well plates. Each promoter construct was transfected in equimolar ratios with two other plasmids required for AAV production (RepCap gene plasmid, adenovirus helper gene plasmid) using jetPRIME® transfection reagent according to the manufacturer's instructions. Seventy-two hours after transfection, the cell medium was removed, and the cells were lysed by freezing and thawing three times in 50 μL of buffer (150 mmol / L NaCl, 20 mmol / L Tris, pH 8.0).

[0169] Generation of retinal organoids from human induced pluripotent stem cells (Cowan et al., 2020)

[0170] Retinal organoids were generated from human induced pluripotent stem cells (iPSCs) as previously described (Zhong et al., 2014). Briefly, on day 0 of differentiation, floating embryoid bodies (EBs) were generated by dissociating iPSC colonies into small colony fragments from a single well of a 6-well plate (Corning, #3516) using a cell passaging tool (Thermo Fisher Scientific, #23181010). EBs were cultured in suspension in mTesR1 medium supplemented with 10 μmol / L blebbistatin (Sigma, #B0560-5MG) in 3.5 cm untreated Petri dishes (Corning, #351008). On days 1 and 2, one-third of the medium was replaced with "neural induction medium" (NIM) containing DMEM / F12 (GIBCO, #31331-028), 1x N2 supplement (GIBCO, #17502-048), 1% NEAA solution (Sigma, #M7145), and 2 μg / mL heparin (Sigma, #H3149-50KU). On day 3, EBs were allowed to settle by gravity in 15 mL tubes to remove dead cells and debris, washed with NIM, and cultured in NIM in 3.5 cm untreated Petri dishes (Corning, #351008). Half of the NIM was replaced daily. On day 7, one 3.5 cm dish of EBs was plated onto a 6 cm dish (Corning, #430166) coated with growth factor-reduced Matrigel (Corning, #356230) and then maintained with daily changes of NIM.

[0171] AAV infection of human retinal organoids At week 26, retinal organoids were infected with 100 μl of filtered cell culture lysate containing AAV carrying a novel hybrid promoter variant (serotype, AAV5) driving enhanced GFP expression.

[0172] Individual organoids were placed in a single well of an ultra-low attachment U-bottom 96-well plate (Corning, #7007) and maintained at 37°C in 5% CO2 in a mixture of 15 μL of culture medium and 100 μL of AAV-containing cell lysate. After 1 day, 100 μL of fresh medium was added to each well. The solution was completely replaced with fresh medium after 24 hours and every 48 hours thereafter.

[0173] Culturing AAV-infected retinal organoids Infected organoids were cultured for 4 weeks in DMEM (GIBCO, #10569-010) supplemented with 20% Ham's F12 nutrient mixture (GIBCO, #31765-027), 10% heat-inactivated fetal bovine serum (Millipore, #es-009-b), 1% N2 supplement (GIBCO, #17502-048), 1% NEAA solution (Sigma, #M7145), 100 μmol / L taurine (Sigma, #T0625), and 1 μmol / L retinoic acid (Sigma, #R2625).

[0174] Live image acquisition and processing of whole organoids An Olympus "IXplore Spin confocal" spinning disk microscope system was used to record GFP expression and brightfield images of retinal organoids in 96-well plates using a 4x objective.

[0175] Maximum intensity projection images were generated using OLYMPUS CellSens software.

[0176] To evaluate the GFP expression profiles of the different promoter variants, the following three parameters were compared for each retinal organoid: the number of GFP-positive cells, the average GFP intensity of all detected cells, and the cell density (calculated by dividing the number of GFP-positive cells by the organoid area).

[0177] The number of GFP-expressing cells was determined using maximum intensity projections (MIPs) of the GFP channel for each image stack. First, Gaussian filtering was applied to reduce the overall noise level. Next, local maxima in pixel intensity were detected and then filtered in three steps. In the first step, low-contrast local maxima were removed by calculating a local ROI window and increasing its size with each iteration. Otsu thresholding was applied to the ROI window, and if there were any active pixels on the edge, the window size was increased. If there were no active pixels on the edge after Otsu thresholding, the iterations were terminated and the local maximum was accepted as an object ("cell"). The maximum size of the ROI window was set to 70 x 70 pixels, and local maxima larger than this size were ignored. This excluded large fluorescent objects.

[0178] The next step was to separate cells that were close to each other. These cases were first identified by screening the diameter and perimeter / area ratio of each object. Then, a binary shrinkage was applied to separate touching objects.

[0179] In the final filtering step, diameter and perimeter / area ratio were analyzed. The perimeter / area ratio was more suitable for circular-like objects. Objects with diameters between 6 and 100 μm were retained.

[0180] Bright-field images were used to calculate organoid area and cell density. A 0.05 gamma transformation was applied to amplify darker pixels. An Otsu threshold was then applied to determine the pixels that belonged to the organoid region.

[0181] Fixation of retinal organoids Organoids were fixed in 4% PFA in PBS for 4 hours at 4° C. After fixation, samples were washed 3 times with PBS for 30 minutes and cryopreserved overnight in 30% sucrose in PBS at 4° C. Samples were stored at −80° C. until use.

[0182] Preparation and staining of cryosections Cryosections (20–40 μmol / L) were generated for organoids and human retinas embedded in OCT compound (VWR, #25608-930) using a cryostat (MICROM International, #HM560). Sections were mounted on Superfrost Plus slides (Thermo Fisher Scientific, #10149870), dried at room temperature for 4–16 hours, and stored at -80°C until use. Photoreceptor outer segments of retinal organoids were not preserved during OCT embedding. Therefore, for cryosectioning of organoids with preserved photoreceptor outer segments, organoids were embedded in PBS containing 7.5% gelatin and 10% sucrose (Lancaster and Knoblich, 2014).

[0183] For immunostaining of cryosections, slides were first dried at room temperature for 30 min and then rehydrated in PBS for 5–10 min. Next, slides were blocked for 1 h at room temperature with blocking buffer: PBS containing 10% normal donkey serum (Sigma, #S30-100ML), 1% (wt / vol) bovine serum albumin (BSA; Sigma, #05482-25G), 0.5% Triton X-100 (Sigma, #T9284-500ML), and 0.02% sodium azide (Sigma, #S2002-25G). Sections were then incubated with primary antibodies, namely rabbit anti-GFP (Invitrogen; 1:200) and mouse monoclonal anti-human cone arrestin 7G6 (CAR; Zhang et al., 2003), in a humidity chamber.

[0184] Primary antibodies were diluted in 100 μL of "blocking buffer B" (PBS containing 3% normal donkey serum, 1% BSA, 0.5% Triton X-100, and 0.02% sodium azide) per slide overnight at room temperature. Slides were washed 3 times for 15 minutes in PBS containing 0.1% TWEEN 20 (Sigma, #P9416-100ML) and then incubated for 2 hours in the dark at room temperature with secondary antibodies (Thermo Fisher Scientific, donkey secondary antibodies conjugated to Alexa Fluor 488, 568, or 647) diluted 1:500 in blocking buffer and Hoechst diluted 1:1000. Sections were washed 2 times for 15 minutes in PBS containing 0.1% Tween, washed once for 15 minutes in PBS, and coverslipped with ProLong Gold (Thermo Fisher Scientific, #P36934). Images were acquired using a spinning disk microscope (Olympus IXplore Spin confocal spinning disk microscope system).

[0185] 1.2. Results Because none of the 3' truncated fragments showed expression, the 3' truncated fragments were not further analyzed. Quantification of live imaging data from whole organoids showed that AAVs driving GFP expression under the control of ProA7 5'1, 5'2, or 5'3 resulted in comparable numbers of GFP-expressing cells per well, at similar densities as GFP driven by full-length ProA7 (Figure 2). The mean GFP intensity of GFP+ counted cells in organoids infected with the truncated fragments was superior to that in organoids infected with AAV-ProA7-GFP (Figures 2-3).

[0186] To determine which cell types express GFP, organoids infected with ProA7 5'3 and 5'4 were further processed and analyzed. Immunohistochemistry of a cone photoreceptor-specific marker (cone arrestin, CAR) and promoter-driven GFP revealed strong cone-specific expression by ProA7 5'3 in organoid cryosections. In confocal images of organoids infected with ProA7 5'3 (by analyzing at least 500 GFP-positive cells each), GFP expression was 98-100% specific to cone photoreceptors, with no other cell types observed (Figure 4).

[0187] Example 2. Promoter sequences for specific expression of genes in cone photoreceptors 2.1. Method The procedure of Example 1 was followed with the following exceptions.

[0188] Vector construction and production By Gibson assembly, the full-length and truncated 185-bp sequences of the cone-specific promoter ProA7 (Juettner et al., 2019) were fused in different orders to the full-length rod-specific promoter ProA330 (PCT / EP2021 / 068653) and inserted into the pAAV plasmid in front of an optimized Kozak sequence (GCCACC) and the translation start codon of the eGFP coding sequence, followed by the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and SV40 polyadenylation site. These vectors are shown schematically in Figure 5.

[0189] Serotype 5 AAV was produced as described by Grieger et al. (2006). Genome copy (GC) number titration was performed using real-time PCR (Applied Biosystems, TaqMan reagents).

[0190] AAV infection of human retinal organoids At week 26, retinal organoids were infected with AAV (1E+11GC / organoid) carrying a novel hybrid promoter variant (serotype, AAV5) that drives enhanced GFP expression.

[0191] Individual organoids were placed in a single well of an ultra-low attachment U-bottom 96-well plate (Corning, #7007) and maintained in 100 μL of culture medium containing 1E+11GC AAV at 37° C. and 5% CO 2 . Fresh medium was added and replaced as in Example 1.

[0192] Histological analysis Expression density is the total number of cone photoreceptors / mm in cryosections stained with the cone marker cone arrestin (CAR). 2 The density of labeled cone photoreceptors was defined as the percentage of the density of labeled cone photoreceptors relative to the total density of CAR+ cells. All hybrid promoter sequences tested drove expression in greater than 80% of CAR+ cells.

[0193] Expression specificity was quantified by determining the percentage of cone photoreceptors in the total GFP+ cell population expressed by AAV. Expression in cone photoreceptors was identified by the location of cell bodies in the outer nuclear layer of the retina and overlap with CAR marker expression.

[0194] 2.2. Results Quantification of live imaging data from whole organoids showed that AAVs driving GFP expression under the control of Pro572, Pro573, Pro572.2, or Pro573.2 resulted in comparable numbers of GFP-expressing cells per well at similar densities as GFP driven by ProA7. The mean GFP intensity of GFP+ counted cells in organoids infected with AAV-{Pro572 / Pro573 / Pro572.2 / Pro573.2}-GFP was superior to that in organoids infected with AAV-ProA7-GFP (Figure 6).

[0195] To determine which cell types express GFP, organoids infected with AAV-{Pro572 / Pro573 / Pro572.2 / Pro573.2}-GFP were further processed and analyzed.

[0196] Immunohistochemistry for a cone photoreceptor marker (cone arrestin, CAR) and promoter-driven GFP revealed strong cone-specific expression by Pro572, Pro573, Pro572.2, or Pro573.2 in organoid cryosections (Figure 7). Quantitative comparison of GFP expression density confirmed the live imaging data, with over 85% of total cone photoreceptors expressing GFP. This is comparable to the expression density achieved by ProA7 (Figure 8A).

[0197] In confocal images (n = 10) of organoids infected with AAV-Pro572.2-GFP and AAV-Pro573.2-GFP (each analyzed from at least 500 GFP-positive cells), GFP expression was 98–100% specific to cone photoreceptors, with no other cell types observed (Figure 8B).

[0198] Example 3. Efficiency of hybrid cone photoreceptor-specific promoters in human retinal explants 3.1 Method The procedure of Example 1 was followed with the following exceptions.

[0199] Vector construction and production By Gibson assembly, the full-length and truncated 185-bp sequences of the cone-specific promoter ProA7 (Juettner et al., 2019) were fused in different orders to the full-length rod-specific promoter ProA330 (WO2023 / 280388) and inserted into the pAAV plasmid in front of an optimized Kozak sequence (GCCACC) and the translation start codon of the eGFP coding sequence, followed by the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and SV40 polyadenylation site. These vectors are shown schematically in Figure 5.

[0200] AAVs of serotype 5 and PHP.eB were produced as described by Grieger et al. (2006). Genome copy (GC) titration was performed using real-time PCR (Applied Biosystems, TaqMan reagents).

[0201] AAV transduction of human retinal cultures 0.1% BSA, 10 μM O-acetyl-L-carnitine hydrochloride, 1 mM fumaric acid, 0.5 mM galactose, 1 mM glucose, 0.5 mM glycine, 10 mM HEPES, 0.05 mM mannose, 13 mM sodium bicarbonate, 3 mM taurine, 0.1 mM putrescine dihydrochloride, 0.35 μM retinol, 0.3 μM retinyl acetate, Human retinal cultures were maintained at 37°C in 5% CO2 in DMEM / F-12 nutrient medium (Thermo Fisher Scientific) supplemented with 0.2 μM (±)-α-tocopherol, 0.5 mM ascorbic acid, 0.05 μM sodium selenite, 0.02 μM hydrocortisone, 0.02 μM progesterone, 1 μM insulin, and 0.003 μM 3,3',5'-triiodo-L-thyronine (Sigma-Aldrich). For AAV transduction experiments, 20 μL of the same titer of AAV (1e11GC) was used per human retinal explant. Cultures were maintained for 5 weeks before fixation and immunohistochemical staining. Experiments were performed in duplicate.

[0202] Result 3.2 To determine whether the results obtained in retinal organoids using the optimized hybrid promoters could be transferred to human retinas, we transduced cultured human retinas with AAV5 and AAVPHP.eB vector constructs expressing enhanced GFP (eGFP) under the control of the Pro527.2, Pro573.3, ProSC, or ProA7 promoters, and quantified GFP expression. All constructs contained the WPRE element. Histochemical analysis was performed after 5 weeks of culture of human retinas. High efficiency (>95%) of GFP expression targeting cone photoreceptors was observed with all hybrid promoter variants (Figure 9A). ProSC showed little expression in rod photoreceptors, but also showed minimal efficacy in cone photoreceptors (Figure 9B). Human retinas treated with AAV5-Pro573.2-EGFP-WPRE exhibited a mean GFP signal intensity threefold higher than those treated with AAV5-ProSC-EGFP-WPRE (Figure 9C). These results suggest that AAV constructs containing the optimized hybrid promoter combined with the WPRE element are expressed with high efficiency and specificity in cone photoreceptors, regardless of AAV capsid serotype, which is comparable to what was observed in transduction experiments performed in human retinal organoids.

[0203] Example 4. Truncated ProA330 promoter 4.1 Method The procedure of Example 1 was followed with the following exceptions.

[0204] Vector construction and production The existing rod-specific promoter ProA330 (WO2023 / 280388), known to drive eGFP expression specifically in rod photoreceptors, and its 3′-truncated and 5′-truncated fragments were cloned into an AAV plasmid in front of an optimized Kozak sequence (GCCACC) and the translation start codon of the eGFP coding sequence, respectively, followed by a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Pieces of 105 bp were excised at both ends from the original rod promoter ProA330 sequence (1000 bp), yielding 3'1 (895 bp), 3'2 (790 bp), 3'3 (685 bp), and 3'4 (580 bp), 3'5 (475 bp), 3'6 (370 bp), 3'7 (265 bp), 3'8 (160 bp), and 3'9 (55 bp) 3' fragments, and 5'1 (895 bp), 5'2 (790 bp), 5'3 (685 bp), and 5'4 (580 bp), 5'5 (475 bp), 5'6 (370 bp), 5'7 (265 bp), 5'8 (160 bp), and 5'9 (55 bp) 5' fragments, respectively.

[0205] Adeno-associated viral vectors (AAVs) of serotypes AAV5 and AAVPHP.eB were produced in a high-throughput version of the general triple transfection method described by Grieger et al. (2006). AAVs were used to transduce retinal organoids and analyzed for GFP expression.

[0206] 4.2 Results We cloned 3'- and 5'-truncated fragments of Pro330 into an AAV plasmid expressing EGFP, subsequently producing AAV vectors and testing them in human retinal organoids. Live imaging of GFP-expressing cells in whole human retinal organoids was performed after transduction with AAV5 and AAVPHP.eB vectors containing various numbers of promoter copies at doses of 1E10 and 1E11 viral genomes (vg) per well. The vectors tested were as follows: AAVPHP.eB-ProA7-EGFP-WPRE, AAVPHP.eB-ProSC-EGFP-WPRE, AAVPHP.eB-2xProSC-EGFP-WPRE, AAVPHP.eB-3xProSC-EGFP-WPRE, AAVPHP.eB-4xProSC-EGFP-WPRE, AAV5-ProA330-EGFP-WPRE, AAVPHP.eB-2xmin330-EGFP-WPRE, AAVPHP.eB-3xmin330-EGFP-WPRE, AAVPHP.eB-4xmin330-EGFP-WPRE, AAVPHPhP.eB-330-3del1del5-EGFP-WPRE, and AAVPHP.eB-3del1del6-EGFP-WPRE. Negative controls included no AAV (Ctrl-noAAV) and AAV5-noPro-EGFP-WPRE (Ctrl-noP).

[0207] Quantification of live imaging data from whole organoids showed that AAVs driving GFP expression under the control of Pro330 5'1, 5'2, 5'3, 5'4, 5'5, and 5'6 resulted in comparable numbers of GFP-expressing cells per well at similar densities as those driven by full-length Pro330 (Table 1). The 3'-truncated ProA330 3'1 also expressed GFP at levels comparable to those of the full-length ProA330 promoter. Because ProA330 5'6 (370 bp) and ProA330 3'1 (895 bp) still drove rod-specific GFP expression, these results suggest that the critical region of ProA330 driving cone-specific expression resides between base pairs 640 and 895.

[0208] [Table 1]

[0209] Example 5. Multiple copies of promoters enhance gene expression in cone and rod photoreceptors 5.1 Method The procedure of Example 1 was followed with the following exceptions.

[0210] Vector construction and production Promoter duplicates were designed and gene synthesis was ordered using Geneious Prime. For the cone-specific promoter, 1x, 2x, 3x, and 4x ProSC promoter duplicates were cloned into the pAAV-SynP330-EGFP-WPRE plasmid, followed by an optimized Kozak sequence (GCCACC) and the translation start codon of the eGFP coding sequence, followed by a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). For the rod-specific promoter, 1x, 2x, 3x, and 4x min330 duplicates were cloned into the pAAV-SynP330-EGFP-WPRE plasmid, followed by an optimized Kozak sequence (GCCACC) and the translation start codon of the eGFP coding sequence, followed by a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Two deletion mutants of min330, 330-3del1del5 and 330-3del1del6, were also cloned into the pAAV-SynP330-EGFP-WPRE plasmid, followed by the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), in front of the optimized Kozak sequence (GCCACC) and the translation start codon of the eGFP coding sequence.

[0211] AAVs of serotype 5 and PHP.eB were produced as described by Grieger et al. (2006). Genome copy (GC) titration was performed using real-time PCR (Applied Biosystems, TaqMan reagents).

[0212] AAV transduction of human retina and human retinal organoids At week 28, human retinal explants and human retinal organoids were transduced in triplicate with a mixture of AAV-containing cell lysates (serotypes AAV5 and AAVPHP.eB) carrying replication promoter variants driving enhanced GFP expression, at a total of 1E10 and 1E11 v.g per well.

[0213] Analysis of live GFP expression in whole organoids was as described above.

[0214] 5.2 Results To determine the functionality of including multiple copies of rod- and cone-specific promoters on gene expression, AAV vectors containing promoter duplications (2x, 3x, and 4x) of the ProSC and min330 promoters driving eGFP expression were tested in organoid cultures and compared with vectors containing a single copy of the ProSC or min330 promoter. Analysis of raw GFP intensity was quantified in retinal organoids, and enhanced GFP expression was observed after transduction of AAV vectors containing multimerized promoter constructs (Figure 10). The 4xProSC variant retained specificity for cone photoreceptors in human retina (Figures 11A and 11B) and human retinal organoids (Figure 12). These results suggest that multiple copies of cone- and rod-specific promoters, either as single elements or in the context of hybrid promoters, can enhance gene expression after AAV transduction of human retina or human retinal organoids.

[0215] [Literature] Juettner,J.,Szabo,A.,Gross-Scherf,B.,Morikawa,R.K.,Rompani,S.B.,Hantz,P.,&Roska,B.Targeting neuronal and glial cell types with synthetic promoter AAVs in mice,non-human primates and humans.Nature Neuroscience,2019;22(8):1345-1356. Grieger JC,Choi VW,Samulski RJ.Production and characterization of adeno-associated viral vectors.Nat Protoc.2006;1(3):1412-28. Cowan,C.S.,Renner,M.,De Gennaro,M.,Gross-Scherf,B.,Goldblum,D.,Hou,Y.,&Roska,B.Cell types of the human retina and its organoids at single-cell resolution.Cell 2020;182(6):1623-1640. Zhong,X.,Gutierrez,C.,Xue,T.,Hampton,C.,Vergara,M.N.,Cao,L.-H.,Peters,A.,Park,T.S.,Zambidis,E.T.,Meyer,J.S.,et al.Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs.Nat.Commun.2014;5:4047. Zhang, H., et al. Identification and Light-Dependent Translocation of a Cone-Specific Antigen, Cone Arrestin, Recognized by Monoclonal Antibody 7G6. Invest. Ophthalmol. Vis. Sci. 44(2003): 2858 - 2867. Lancaster, M. A., and Knoblich, J. A. Generation of cerebral organoids from human pluripotent stem cells. Nat. Protoc. 2014; 9: 2329 - 2340.

[0216] 7. Sequence Listing TIFF2025528187000003.tif 50142 TIFF2025528187000004.tif 222142 TIFF2025528187000005.tif 222142 TIFF2025528187000006.tif 222142 TIFF2025528187000007.tif

Claims

1. 1. An isolated nucleic acid comprising a cone-specific promoter, said promoter comprising: a first nucleotide sequence of at least about 150 nucleotides having at least 70% identity to a sequence of equal length from the sequence of SEQ ID NO: 1; a second nucleotide sequence of at least about 260 nucleotides having at least 70% identity to a sequence of equal length from the sequence of SEQ ID NO:2; The isolated nucleic acid comprising:

2. 2. The isolated nucleic acid of claim 1, wherein the second nucleotide sequence comprises at least about 370 nucleotides having at least 70% identity to a sequence of equal length from the sequence of SEQ ID NO:

2.

3. 2. The isolated nucleic acid of claim 1, wherein the first nucleotide sequence has at least 70% identity to a sequence of equal length from the 3' end of the sequence of SEQ ID NO:

1.

4. 4. The isolated nucleic acid of any one of claims 1 to 3, wherein the first nucleotide sequence has at least 90% identity to a sequence of equal length from the 3' end of the sequence of SEQ ID NO: 1, and the second nucleotide sequence has at least 90% identity to a sequence of equal length from the 3' end or 5' end or both of the sequence of SEQ ID NO:

2.

5. 5. The isolated nucleic acid of claim 1, wherein the first nucleotide sequence comprises from about 150 nucleotides to about 395 nucleotides from the 3' end of the sequence of SEQ ID NO:

1.

6. 6. The isolated nucleic acid of any one of claims 1 to 5, wherein the first nucleotide sequence comprises SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:

15.

7. 7. The isolated nucleic acid of any one of claims 1 to 6, wherein the second nucleotide sequence comprises from about 370 nucleotides to about 895 nucleotides from the 3' end of SEQ ID NO:2, or about 895 nucleotides from the 5' end of SEQ ID NO:

2.

8. 8. The isolated nucleic acid of any one of claims 1 to 7, wherein the second nucleotide sequence comprises SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:

30.

9. 10. The isolated nucleic acid of any one of the preceding claims, wherein the second nucleic acid is SEQ ID NO:

39.

10. 10. The isolated nucleic acid of any one of claims 1 to 9, comprising, from 5' to 3', the first nucleotide sequence and the second nucleotide sequence, with no sequence therebetween.

11. 11. The isolated nucleic acid of any one of claims 1 to 10, comprising, from 5' to 3', the second nucleotide sequence and the first nucleotide sequence, with no sequence therebetween.

12. 12. The isolated nucleic acid of any one of claims 1 to 11, comprising two or more first nucleotide sequences, two or more second nucleotide sequences, or both.

13. 13. The isolated nucleic acid of any one of claims 1 to 12, further comprising a nucleotide sequence encoding a heterologous polypeptide or a heterologous RNA, wherein the promoter and the nucleotide sequence encoding the polypeptide are operably linked.

14. 14. The isolated nucleic acid of any one of claims 1 to 13, further comprising a regulatory element, wherein the promoter, the regulatory element, and, if present, the nucleotide sequence encoding the heterologous polypeptide or the heterologous RNA are operably linked.

15. 15. The isolated nucleic acid of claim 14, wherein the regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

16. 16. The isolated nucleic acid of claim 14 or 15, further comprising a nucleotide sequence encoding a polyadenylation signal (Poly A) 3' to the nucleotide sequence encoding the regulatory element, wherein the nucleotide sequence encoding Poly A and the nucleotide sequence encoding the regulatory element are operably linked.

17. 17. The isolated nucleic acid of any one of claims 14 to 16, comprising a first AAV inverted terminal repeat (ITR) 5' to the promoter and a second AAV ITR 3' to the regulatory element and preferably 3' to the PolyA signal.

18. 18. The isolated nucleic acid of any one of claims 1 to 17, further comprising a nucleotide sequence encoding an AAV inverted terminal repeat (ITR).

19. 10. The isolated nucleic acid of any one of the preceding claims, comprising from 1 to about 10 first nucleotide sequences and / or from 1 to about 10 second nucleotide sequences.

20. 10. The isolated nucleic acid of any one of the preceding claims, wherein the nucleic acid may comprise one or more of codon optimization, CpG reduction, removal of alternative start sites, removal of repeats, removal of hairpins, removal of unnecessary splice donors and acceptors, deletion of ITR end release sites, addition of stuffer sequences, or addition of miRNA.

21. 1. An isolated nucleic acid comprising a cone-specific promoter, said promoter comprising: A cone-specific promoter of at least about 150 nucleotides and no more than 499 nucleotides having at least 70% identity to a sequence of equal length from the sequence of SEQ ID NO:

1. The isolated nucleic acid comprising:

22. 22. The isolated nucleic acid of claim 21, comprising from 1 to about 10 cone-specific promoters.

23. 1. An isolated nucleic acid comprising a cone-specific promoter, said promoter comprising SEQ ID NO:

40.

24. A vector comprising the isolated nucleic acid of any one of claims 1 to 23.

25. 25. The vector of claim 24, further comprising a viral capsid.

26. 26. The vector of claim 25, wherein the viral capsid is an adeno-associated viral vector (AAV) capsid selected from the group consisting of an AAV8-BP2 capsid, an AAV-PHP.B capsid, an AAV-PHP.eB capsid, an AAV5 capsid, or an AAV-NHP26 capsid.

27. A host cell comprising the vector according to any one of claims 24 to 26.

28. A kit comprising the isolated nucleic acid of any one of claims 1 to 23, the vector of any one of claims 24 to 26, the host cell of claim 27, or two or more of them.

29. 28. A method for expressing a heterologous polypeptide or heterologous RNA in a retinal organoid, a retinal explant, or a mammalian subject, comprising administering to said retinal organoid, said retinal explant, or said mammalian subject an isolated nucleic acid of any one of claims 1 to 23, a vector of any one of claims 24 to 26, a host cell of claim 27, or two or more thereof.

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