Human cone photoreceptor optogenetic constructs

Abstract

The present disclosure relates to a nucleic acid comprising a synthetic cone-specific promoter, a depolarizing optogenetic protein and optionally a reporter molecule, and a Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element (WPRE), which can be used in a vector or host cell to drive expression of the depolarizing optogenetic protein in retinal cells, particularly cone photoreceptors.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 397,180, filed August 11, 2022, and U.S. Provisional Application No. 63 / 400,095, filed August 23, 2022, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] 1. Background technology Blindness is the complete or near-complete loss of vision, and in most forms, it is incurable. It is estimated that 43 million people were blind in 2020 (GBD 2019 Blindness and Vision Impairment Collaborators, Lancet Glob Health, (2021), 9(2): e130-e143 (Non-Patent Document 1)). One of the most common causes of blindness is retinal disease, such as degeneration or dysfunction of retinal photoreceptor cells and the resulting loss of light sensitivity. Research has shown that in a certain subset of blind patients, cone photoreceptors remain in a dormant state (Cideciyan and Jacobson, Invest Ophthalmol. Vis. Sci. (2019), 60(5): 1680-1695 (Non-Patent Document 2)).

[0003] Photoreceptors are responsible for detecting and transmitting light signals (Osakada et al., (2008), Nat Biotechnol., 26(2):215-224 (Non-Patent Document 3)). The photoreceptor cells involved in vision are rods and cones. These cells contain chromophores (light-absorbing molecules) such as retinal bound to membrane proteins called opsins (e.g., rhodopsin). Rods primarily contribute to night vision (dark-adapted state), while cones primarily contribute to daytime vision (light-adapted state). When light strikes a photoreceptor, the chromophore undergoes a shape change, for example, retinal is converted from the 11-cis isomer, which predominates in dark conditions, to the all-trans isomer. Chromophore isomerization induces a conformational change in the opsin protein. This light activation triggers a series of events that lead to hyperpolarization of the photoreceptor cell membrane potential. Thus, unlike most sensory neurons, which depolarize upon exposure to stimuli, photoreceptors hyperpolarize in response to light stimuli. Choung et al., (2014), Nature Neuroscience, 17, 1123-1129 (Non-Patent Document 4), Carter and Lecea (2011), Trends Mol. Med., 17(4):197-206 (Non-Patent Document 5), Simon et al., (2020), BBRC, 527(2):325-330 (Non-Patent Document 6).

[0004] "Optogenetics" is a technical term referring to the process of making cells responsive to light, typically by genetically modifying cells to express light sensors. Therefore, optogenetic approaches have the potential to treat various types of blindness. For example, optogenetic approaches in patients with dormant cone photoreceptors focus on recreating normal, healthy human cone cells by genetically expressing light-activated hyperpolarizing ion pumps in the cone photoreceptors, which hyperpolarize in response to light stimuli in healthy conditions, thereby resensitizing these dysfunctional cone cells to light. (Choung et al., (2014), Nature Neuroscience, 17, 1123-1129 (Non-Patent Document 4); Carter and Lecea (2011), Trends Mol. Med., 17(4):197-206 (Non-Patent Document 5); Simon et al., (2020), BBRC, 527(2):325-330 (Non-Patent Document 6). Khabou et al., JCI Insight, (2018) 3 (2): e96029 (non-patent document 7), Nikonov et al., (2022) TVST, 11 (5): 24 (non-patent document 8).

[0005] Another optogenetic approach focuses on transducing optogenetic proteins into ganglion cells. Sahel et al. (2021), Nature Medicine, 27, 1223-1229 (Non-Patent Document 9). Ganglion cells are the final output cells of the retina. They collect information about the visual world from bipolar and amacrine cells in the form of chemical messengers sensed by receptors that trigger electrical signals. These electrical signals result in retinal computation by generating a wide variety of spikes that are transmitted through the optic nerve to trigger vision. However, transducing ganglion cells with light-sensitive optogenetic proteins converts them into artificial photoreceptor cells, with all transduced cells possessing substantially the same light-sensing capabilities. In other words, the transduced ganglion cells sense light and generate substantially the same electrical signals in response to light, thereby eliminating the retinal computation normally present in the retina, which is important for vision. Furthermore, because ganglion cells and bipolar cells are located in a ring around the fovea and are not arranged like camera pixels, optogenetic techniques targeting these cells would result in severely distorted images.

[0006] These efforts have not been successful in restoring vision without facilitation using optical devices, and few treatments have been clinically evaluated. (Sahel et al., Nature Medicine, 27, 1223-1229 (2021)) Furthermore, well-known obstacles to the effective use of optogenetics to treat blindness include the insufficient light sensitivity of optogenetic-transduced proteins. Furthermore, selectively targeting optogenetic proteins to cone cells and achieving sufficient expression levels in cone cells remains a major technical challenge. Therefore, it remains primarily an experimental approach. An additional challenge is that studies conducted in animal models often fail to translate to human cells, necessitating the development of vectors directly on the human retina. No previous studies have demonstrated the expression and function of optogenetic constructs in human cone cells using optogenetics technology. Therefore, the translation of optogenetics-based experimental approaches to humans is highly unpredictable.

[0007] There is a need for therapeutic approaches to restore vision. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] GBD 2019 Blindness and Vision Impairment Collaborators, Lancet Glob Health,(2021),9(2):e130-e143 [Non-patent document 2] Cideciyan and Jacobson,Invest Ophthalmol.Vis.Sci.(2019),60(5):1680-1695 [Non-patent document 3] Osakada et al.,(2008),Nat Biotechnol.,26(2):215-224 [Non-patent document 4] Choung et al.,(2014),Nature Neuroscience,17,1123-1129 [Non-patent document 5] Carter and Lecea(2011),Trends Mol.Med.,17(4):197-206 [Non-patent document 6] Simon et al.,(2020),BBRC,527(2):325-330 [Non-Patent Document 7] Khabou et al.,JCI Insight,(2018)3(2):e96029 [Non-patent document 8] Nikonov et al.,(2022)TVST,11(5):24 [Non-Patent Document 9] Sahel et al.,(2021),Nature Medicine,27,1223-1229 Summary of the Invention

[0009] 2. Overview The present disclosure relates to novel optogenetic constructs comprising a depolarizing optogenetic protein, preferably a light-gated ion channel polypeptide selectively expressed in human cone cells, which, when introduced into and expressed in human cone cells, can restore light sensitivity of human cone cells and retinal information processing.

[0010] As mentioned above, photoreceptor cells hyperpolarize in response to light stimulation. Based on this well-known phenomenon, it has been thought that hyperpolarization of the photoreceptor cell membrane using a hyperpolarizing protein (e.g., halorhodopsin) is essential for the success of potential optogenetic approaches. Choung et al., (2014), Nature Neuroscience, 17, 1123-1129; Carter and Lecea (2011), Trends Mol. Med., 17(4):197-206; Simon et al., (2020), BBRC, 527(2):325-330. The present inventors unexpectedly and surprisingly found that, rather than reproducing normal, healthy human cone photoreceptor cells, they could resensitize human cone cells using a depolarizing optogenetic protein that depolarizes the human cone cell membrane in response to light stimulation. Depolarizing optogenetic proteins can generate depolarizing currents that depolarize human cone cells sufficiently to cause ganglion cell spiking, which is contrary to the normal function of photoreceptors under healthy conditions. Ganglion cells are the final output cells of the retina. Their axons converge at the optic disc, where they myelinate to form the optic nerve. Activation of ganglion cells triggers vision. Furthermore, without wishing to be bound by theory, the inventors anticipated that all depolarizing optogenetic proteins would be expected to function. However, the inventors surprisingly discovered that only some depolarizing optogenetic proteins function in human cones and are therefore suitable for the optogenetic constructs described herein.

[0011] Furthermore, the inventors have surprisingly discovered that a specific combination of regulatory elements and a specific depolarizing optogenetic protein, and optionally a reporter molecule and a synthetic intron, is required to achieve both sufficient expression and restoration of light sensitivity in human cone cells.

[0012] Furthermore, the inventors have discovered that distinct subsets of patients with retinal dystrophies and geographic atrophy due to age-related macular degeneration are particularly suitable for treatment with the optogenetic constructs disclosed herein (see Janeschitz-Kriegl et al., (2022), Investigative Ophthalmology & Visual Science, 63(7):455). The inventors have found that patients with inherited retinal dystrophies and low vision are suitable treatment candidates if they have a preserved cone photoreceptor layer in the central retina. Without being bound by theory, patients who are particularly suitable treatment candidates have at least some residual cones that appear stable over time, these cone cells are likely connected to ganglion cells, and the patients have optic nerves that appear normal.

[0013] Thus, the optogenetic constructs disclosed herein include a promoter or cone-specific promoter, a depolarizing optogenetic protein, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The optogenetic construct may optionally include a reporter molecule. The optogenetic construct preferably expresses the depolarizing optogenetic protein in human cone cells, such as dormant human cone cells that normally do not respond to light stimulation. It is desirable that other cell types, including, but not limited to, rod cells, amacrine cells, or ganglion cells, do not express the depolarizing optogenetic protein when the optogenetic construct is introduced into the cells.

[0014] In particular, the present disclosure relates to an isolated nucleic acid comprising a promoter, the promoter being a hybrid promoter comprising a first nucleotide sequence comprising at least a portion of a cone-specific promoter and a second nucleotide sequence comprising at least a portion of a rod-specific promoter, wherein the first and second nucleotide sequences are operably linked and function as a single promoter for expression in cone photoreceptors, or a cone-specific promoter, or an active variant, fragment, or truncated version of either of the foregoing. The isolated nucleic acid also comprises a nucleotide sequence encoding a depolarizing optogenetic protein and, optionally, a reporter molecule, and a WPRE. The promoter, the nucleotide sequence encoding the depolarizing optogenetic protein and, optionally, a reporter molecule, and the WPRE are operably linked. A preferred promoter is Pro573.2, having SEQ ID NO: 14. Another preferred promoter is the ProA7 promoter, having SEQ ID NO: 2. Another preferred promoter is 4xProSc, having SEQ ID NO: 85.

[0015] The isolated nucleic acid may include a promoter that is a hybrid promoter, such as a promoter comprising SEQ ID NO:14, SEQ ID NO:13, SEQ ID NO:12, or SEQ ID NO:11, or an active variant, fragment, or truncation of any of the foregoing sequences.

[0016] The isolated nucleic acid may further comprise a hybrid promoter, wherein the first nucleotide sequence may comprise at least about 150 contiguous nucleotides of SEQ ID NO:2, and the second nucleotide sequence may comprise at least about 264 contiguous nucleotides of SEQ ID NO:10, or an active variant, fragment, or truncation of any of the foregoing sequences. For example, the first nucleotide sequence may be selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and a sequence comprising at least about 150 contiguous nucleotides from the 3' end of SEQ ID NO:2, and the second nucleic acid may comprise SEQ ID NO:54, or an active variant of any of the foregoing.

[0017] The isolated nucleic acid may comprise a hybrid promoter, wherein the first nucleotide sequence is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and a sequence comprising at least about 150 contiguous nucleotides from the 3' end of SEQ ID NO:2, and the second nucleic acid comprises SEQ ID NO:10, or an active variant, fragment, or truncation thereof.

[0018] The isolated nucleic acid may further comprise a nucleotide sequence encoding a polyadenylation signal (PolyA) 3' to the nucleotide sequence encoding the WPRE. The nucleotide sequence encoding PolyA and the nucleotide sequence encoding the WPRE may be operably linked.

[0019] The nucleic acid may comprise a cone-specific promoter comprising at least about 150 contiguous nucleotides of SEQ ID NO: 2, or an active variant thereof. In some examples, the cone-specific promoter is selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and a sequence comprising at least about 150 contiguous nucleotides from the 3' end of SEQ ID NO: 2, or an active variant thereof.

[0020] The nucleic acid may comprise a hybrid promoter or a cone-specific promoter comprising 1 to about 10 copies of a sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, a sequence comprising at least about 150 contiguous nucleotides from the 3' end of SEQ ID NO: 2, an active variant of any of the foregoing, and combinations thereof. The hybrid promoter may comprise 1 to about 10 copies of SEQ ID NO: 54, or an active variant thereof.

[0021] The WPRE in the nucleic acids disclosed herein may comprise a nucleotide sequence having at least 70% identity to SEQ ID NO:3 (e.g., SEQ ID NO:8). The isolated nucleic acid may further comprise a nucleotide sequence encoding a polyadenylation signal (PolyA) 3' to the nucleotide sequence encoding the WPRE. The nucleotide sequence encoding PolyA and the nucleotide sequence encoding the WPRE may be operably linked. The WPRE in the nucleic acids disclosed herein may comprise a nucleotide sequence having at least 70% identity to SEQ ID NO:86. When the nucleic acids disclosed herein comprise PolyA, the PolyA may comprise a nucleotide sequence having SEQ ID NO:87.

[0022] The isolated nucleic acid may further comprise an AAV inverted terminal repeat (ITR). The isolated nucleic acid may comprise a first AAV ITR located 5' to the promoter and a second AAV ITR located 3' to the WPRE and preferably 3' to the PolyA signal. The depolarizing optogenetic protein may be a light-responsive polypeptide. The light-responsive polypeptide may be a light-gated ion channel polypeptide. The light-gated ion channel polypeptide may be a channelrhodopsin or a functional variant thereof. The light-gated ion channel polypeptide may be a channelrhodopsin. The channelrhodopsin may be a ReaChR polypeptide or a functional variant thereof.

[0023] The isolated nucleic acid preferably comprises a nucleotide sequence encoding ReaChR or a functional variant thereof, which may be selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 73, or an amino acid sequence having at least about 70% identity to any of the foregoing.

[0024] The isolated nucleic acid may comprise a nucleic acid comprising SEQ ID NO:18, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:35, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83.

[0025] Some preferred optogenetic constructs comprise a nucleotide sequence comprising SEQ ID NO: 60, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83. A preferred optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 60.

[0026] The isolated nucleic acid may further comprise a channelrhodopsin or functional variant thereof engineered to enhance membrane localization when expressed in human cone photoreceptors. The engineered channelrhodopsin may comprise SEQ ID NO: 57 or a functional variant thereof.

[0027] In some embodiments, the isolated nucleic acid does not encode an optional molecule.

[0028] The promoter may comprise two or more first nucleotide sequences, two or more second nucleotide sequences, two or more cone-specific promoters, or two or more of them. The nucleic acid may further comprise an intron.

[0029] The nucleic acid may comprise a nucleotide sequence comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:35, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83, or a sequence with at least about 70% identity to any of the foregoing. In certain embodiments, the nucleic acid may comprise a nucleotide sequence comprising SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:32, SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:9, SEQ ID NO:15, and / or SEQ ID NO:87, or a sequence with at least about 70% identity to any of the foregoing.

[0030] The nucleic acid may further comprise an active variant having at least about 70% identity to the corresponding reference sequence. The active variant may comprise one or more of codon optimization, reduction or elimination of CpG, removal of alternative start sites, removal of repeats, removal of hairpins, removal of unnecessary splice donor and acceptor sites, deletion of ITR end dissociation sites, addition of stuffer sequences, or addition of miRNA.

[0031] The present disclosure also relates to a viral particle comprising the nucleic acid described herein. The viral particle can be an AAV particle. The present disclosure further relates to a host cell comprising the nucleic acid or viral particle described herein.

[0032] The present disclosure further relates to an AAV vector comprising the nucleic acid described herein and an AAV capsid. In the AAV vector, the AAV capsid can be an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid. A host cell can contain the AAV vector described herein.

[0033] Also provided herein are pharmaceutical compositions comprising the nucleic acids, viral particles, AAV vectors, or host cells described herein. The pharmaceutical composition can also comprise a pharmaceutically acceptable excipient.

[0034] The present disclosure also relates to a method of delivering a depolarizing optogenetic protein to a human cone cell in a subject in need thereof, the method comprising administering to the subject a nucleic acid, viral particle, AAV vector, host cell, or pharmaceutical composition described herein.

[0035] The present disclosure also relates to a method for treating a retinal disease, the method comprising administering to a subject in need thereof an effective amount of a nucleic acid, viral particle, AAV vector, host cell, or pharmaceutical composition described herein.

[0036] The present disclosure also relates to a method for treating retinal diseases, such as restoring vision or restoring the light sensitivity of human cone photoreceptor cells, or treating retinal degeneration, comprising administering to a subject in need thereof an effective amount of the nucleic acid, viral particle, AAV vector, host cell, or pharmaceutical composition disclosed herein. The nucleic acid, AAV vector, or pharmaceutical composition thereof can be administered by subretinal injection, intravitreal injection, or suprachoroidal injection. The nucleic acid, AAV vector, or pharmaceutical composition can be administered before, after, or at the onset of photoreceptor loss or dysfunction.

[0037] When a nucleic acid sequence encoding a depolarizing optogenetic protein is introduced into human cone cells, the depolarizing optogenetic protein can mediate a depolarizing current that depolarizes human cone cells when exposed to light. The depolarizing current is strong enough to be delivered to ganglion cells and generate a current that causes light-driven ganglion cell spiking. This depolarization is preferably equivalent to or stronger than the light-driven ganglion cell spiking in functional human cone cells. Light-driven ganglion cell spiking can be evaluated using any suitable assay. For example, light-driven ganglion cell spiking can be measured using a multi-electrode array.

[0038] Any retinal disease may be suitable for treatment using the compositions disclosed herein according to the methods disclosed herein. Exemplary retinal disorders that may be suitable for treatment include, but are not limited to, 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, choroideremia, post-retinal detachment, cone dysfunction, pigment epithelial retinal degeneration, retinal vein occlusion, and geographic atrophy. Treatment using the compositions disclosed herein according to the methods disclosed herein is suitable for subjects with vision-impairing disorders in which the optic nerve retains at least some function.

[0039] In some embodiments, the depolarizing optogenetic protein may be expressed in the plasma membrane of human cone cells. In some embodiments, the depolarizing optogenetic protein may be capable of mediating a depolarizing current that depolarizes human cone cells when exposed to light. In some embodiments, the depolarization of human cone cells induces light-driven ganglion cell spiking that may be equivalent to light-driven ganglion cell spiking in functional human cone cells. In some embodiments, light-driven ganglion cell spiking may be assessed using a multi-electrode array.

[0040] The nucleic acid may be capable of restoring light sensitivity when introduced into human cone cells, and the restoration of light sensitivity occurs when (i) a depolarizing optogenetic protein is expressed in the cell membrane of human cone cells, (ii) the depolarizing optogenetic protein is capable of mediating a depolarizing current that depolarizes human cone cells when exposed to light, and (iii) the depolarization of human cone cells induces light-driven ganglion cell spiking.

[0041] In particular, the present disclosure relates to an isolated nucleic acid comprising: (a) a first nucleotide sequence selected from the group consisting of a nucleotide sequence of at least 150 nucleotides having at least 70% identity to a sequence of equal length from SEQ ID NO:2, a nucleotide sequence having at least 70% identity to SEQ ID NO:22, a nucleotide sequence having at least 70% identity to SEQ ID NO:23, and a combination thereof; and a second nucleotide sequence of at least about 370 nucleotides having at least 70% identity to a sequence of equal length from SEQ ID NO:10; or (b) a promoter comprising a cone-specific promoter of at least about 150 nucleotides and not more than 499 nucleotides having at least 70% identity to a sequence of equal length from SEQ ID NO:2. The isolated nucleic acid also comprises a nucleotide sequence encoding a depolarizing optogenetics protein and optionally a reporter molecule, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The promoter, the nucleotide sequence encoding a depolarizing optogenetics protein and optionally a reporter molecule, and the WPRE are operably linked.

[0042] The isolated nucleic acid may further comprise a nucleotide sequence encoding a polyadenylation signal (PolyA) 3' to the nucleotide sequence encoding the WPRE. The nucleotide sequence encoding PolyA and the nucleotide sequence encoding the WPRE may be operably linked.

[0043] The isolated nucleic acid may further comprise a nucleotide sequence encoding an AAV inverted terminal repeat (ITR). The isolated nucleic acid may comprise a first AAV ITR located 5' to the promoter and a second AAV ITR located 3' to the WPRE and preferably 3' to the PolyA signal. The depolarizing optogenetic protein may be a light-responsive polypeptide. The light-responsive polypeptide may be a light-gated ion channel polypeptide. The light-gated ion channel polypeptide may be a channelrhodopsin or a functional variant thereof.

[0044] The light-gated ion channel polypeptide can be a channelrhodopsin. The channelrhodopsin can be a ReaChR polypeptide or a functional variant thereof.

[0045] In some embodiments, the isolated nucleic acid does not encode an optional reporter molecule.

[0046] The WPRE may comprise a nucleotide sequence having at least 70% identity to SEQ ID NO: 3. The WPRE may comprise a nucleotide sequence having at least 70% identity to SEQ ID NO:86.

[0047] The promoter may comprise two or more first nucleotide sequences, two or more second nucleotide sequences, two or more cone-specific promoters, or two or more thereof. The nucleic acid may further comprise an intron. The promoter may comprise one or more sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:45.

[0048] The present disclosure also relates to viral particles comprising the nucleic acids described herein.

[0049] The viral particle may be an AAV particle. The viral particle may be capable of transducing about 10% of degenerated human cone cells. The present disclosure further relates to a host cell comprising the nucleic acid or viral particle described herein.

[0050] The present disclosure further relates to an AAV vector comprising the nucleic acid described herein and an AAV capsid. In the AAV vector, the AAV capsid can be an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid. The promoter of the AAV vector can include SEQ ID NO: 2, the depolarizing optogenetic protein can be ReaChR, the optional reporter molecule can be absent, PolyA can be present, and the vector has SEQ ID NO: 9, and the AAV capsid can be AAV5.

[0051] The promoter of the AAV vector may comprise SEQ ID NO: 14, the depolarizing optogenetic protein may be ReaChR, the optional reporter molecule may be absent, PolyA may be present, and have SEQ ID NO: 9, and the AAV capsid may be AAV5. The promoter of the AAV vector may comprise SEQ ID NO: 12, the depolarizing optogenetic protein may be ReaChR, the optional reporter molecule may be absent, PolyA may be present, and have SEQ ID NO: 9, and the AAV capsid may be AAV5. The host cell may contain the AAV vector described herein. The promoter of the AAV may comprise SEQ ID NO: 85, the depolarizing optogenetic protein may be ReaChR, the optional reporter molecule may be absent, PolyA may be present, and have SEQ ID NO: 9, and the AAV capsid may be AAV5. The AAV promoter may comprise SEQ ID NO: 14, the depolarizing optogenetic protein may be ReaChR, the optional reporter molecule may be absent, PolyA may be present, and have SEQ ID NO: 87, and the AAV capsid may be AAV5. The AAV promoter may comprise SEQ ID NO: 12, the depolarizing optogenetic protein may be ReaChR, the optional reporter molecule may be absent, PolyA may be present, and have SEQ ID NO: 87, and the AAV capsid may be AAV5. The AAV promoter may comprise SEQ ID NO: 85, the depolarizing optogenetic protein may be ReaChR, the optional reporter molecule may be absent, PolyA may be present, and have SEQ ID NO: 87, and the AAV capsid may be AAV5.

[0052] The promoter of the AAV vector may comprise SEQ ID NO: 85, the depolarizing optogenetic protein may be ReaChR, the optional reporter molecule may be absent, PolyA may be present and has SEQ ID NO: 9, and the AAV capsid may be AAV5 and has SEQ ID NO: 84. The promoter of the AAV vector may comprise SEQ ID NO: 14, the depolarizing optogenetic protein may be ReaChR, the optional reporter molecule may be absent, PolyA may be present and has SEQ ID NO: 9, and the AAV capsid may be AAV5 and has SEQ ID NO: 84. The promoter of the AAV vector may comprise SEQ ID NO: 12, the depolarizing optogenetic protein may be ReaChR, the optional reporter molecule may be absent, PolyA may be present and has SEQ ID NO: 9, and the AAV capsid may be AAV5 and has SEQ ID NO: 84.

[0053] The promoter of the AAV vector may comprise SEQ ID NO: 12, the depolarizing optogenetic protein may be ReaChR, the optional reporter molecule may be absent, PolyA may be present and have SEQ ID NO: 87, and the AAV capsid may be AAV5 and have SEQ ID NO: 84. The promoter of the AAV vector may comprise SEQ ID NO: 85, the depolarizing optogenetic protein may be ReaChR, the optional reporter molecule may be absent, PolyA may be present and have SEQ ID NO: 87, and the AAV capsid may be AAV5 and have SEQ ID NO: 84. The promoter of the AAV vector may comprise SEQ ID NO: 14, the depolarizing optogenetic protein may be ReaChR, the optional reporter molecule may be absent, PolyA may be present and have SEQ ID NO: 87, and the AAV capsid may be AAV5 and have SEQ ID NO: 84.

[0054] Also provided herein are pharmaceutical compositions comprising the nucleic acids, viral particles, AAV vectors, or host cells described herein. The pharmaceutical composition can also comprise a pharmaceutically acceptable excipient.

[0055] The present disclosure also relates to a method of delivering a depolarizing optogenetic protein to a human cone cell in a subject in need thereof, the method comprising administering to the subject a nucleic acid, viral particle, AAV vector, host cell, or pharmaceutical composition described herein.

[0056] The present disclosure also relates to a method for treating a retinal disease, the method comprising administering to a subject in need thereof an effective amount of a nucleic acid, viral particle, AAV vector, host cell, or pharmaceutical composition described herein.

[0057] The present disclosure also relates to a method for treating retinal diseases, such as restoring vision or restoring the light sensitivity of human cone photoreceptor cells, or treating retinal degeneration, comprising administering to a subject in need thereof an effective amount of the nucleic acid, viral particle, AAV vector, host cell, or pharmaceutical composition disclosed herein. The nucleic acid, AAV vector, or pharmaceutical composition thereof can be administered by subretinal injection. The nucleic acid, AAV vector, or pharmaceutical composition can be administered before, after, or at the onset of photoreceptor loss or dysfunction.

[0058] When a nucleic acid sequence encoding a depolarizing optogenetic protein is introduced into human cone cells, the depolarizing optogenetic protein can mediate a depolarizing current that depolarizes human cone cells when exposed to light. The depolarizing current is strong enough to be delivered to ganglion cells and generate a current that causes light-driven ganglion cell spiking. This depolarization is preferably equivalent to or stronger than the light-driven ganglion cell spiking in functional human cone cells. Light-driven ganglion cell spiking can be evaluated using any suitable assay. For example, light-driven ganglion cell spiking can be measured using a multi-electrode array.

[0059] Any retinal disease may be suitable for treatment using the compositions disclosed herein according to the methods disclosed herein. Exemplary retinal disorders that may be suitable for treatment include, but are not limited to, 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, choroideremia, post-retinal detachment, cone dysfunction, pigment epithelial retinal degeneration, retinal vein occlusion, and geographic atrophy. Treatment using the compositions disclosed herein according to the methods disclosed herein is suitable for subjects with vision-impairing disorders in which the optic nerve retains at least some function.

[0060] In some embodiments, the depolarizing optogenetic protein may be expressed in the plasma membrane of human cone cells. In some embodiments, the depolarizing optogenetic protein may be capable of mediating a depolarizing current that depolarizes human cone cells when exposed to light. In some embodiments, the depolarization of human cone cells induces light-driven ganglion cell spiking that may be equivalent to light-driven ganglion cell spiking in functional human cone cells. In some embodiments, light-driven ganglion cell spiking may be assessed using a multi-electrode array.

[0061] The nucleic acid may be capable of restoring light sensitivity when introduced into human cone cells, and the restoration of light sensitivity occurs when (i) a depolarizing optogenetic protein is expressed in the cell membrane of human cone cells, (ii) the depolarizing optogenetic protein is capable of mediating a depolarizing current that depolarizes human cone cells when exposed to light, and (iii) the depolarization of human cone cells induces light-driven ganglion cell spiking. [Brief explanation of the drawings]

[0062] 3. Brief description of the drawings [Figure 1A] Figures 1A-1E are graphs showing the expression of AAV8-BP2-ProA7-ChrimsonR-tdTomato (without WPRE), AAV8-BP2-CAG-ChrimsonR-tdTomato (without WPRE), AAV8-BP2-ProA7-ChrimsonR-tdTomato-WPRE, and AAV8-BP2-ProA7-vfChrimson-EYFP-WPRE in different model systems. The percentage of cone transduction was determined based on the cone marker (cone arrestin) in mouse, human, and non-human primate (NHP) cones. When the ProA7 promoter was used, the WPRE element was not required for construct expression in mouse retina and human retinal organoids (Figure 1A, Figure 1B). However, when the WPRE was absent from the ProA7 construct, there was no cone expression in mature human retinal cones in retinal explants (Figure 1C). The nonselective CAG promoter does not require the WPRE element to express optogenetic proteins in cones. Addition of the WPRE element to the AAV8-BP2-ProA7-ChrimsonR-tdT construct resulted in expression in human cones (Figure 1C). Thus, in the human retina, the WPRE element is required for the ProA7 promoter but not for the CAG promoter, indicating that the requirement for the WPRE element is promoter-dependent (Figure 1C). The AAV8-BP2-ProA7-vfChrimson-EYFP-WPRE vector construct was also expressed in cone cells in nonhuman primate (macaque) retinas after in vivo injection (Figure 1D). ProA7 conferred primarily cone-specific expression in mouse retina, with cone specificity nearly 100% in both human and nonhuman primate cones (Figure 1E). CAG-driven expression of ChrimsonR was not cone-specific, as other cells were also targeted (Figure 1E). (All vectors in Figures 1A-1E contain the hGH polyA sequence). [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D]See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 2] Figures 2A and 2B are graphs showing the percentage of cones transduced with the AAV-ProA7-vfChrimson-EYFP-WPRE-hGH polyA construct in different AAV capsid serotypes: AAV8-BP2, AAV-PHP.B, AAV-PHP.eB, AAV-NHP26, and AAV-44.9(E531D). Figure A shows the percentage of cones transduced in human retinal organoids at a dose of 8.5E11 vector genomes (vg). Figure B shows the percentage of cones transduced in human retinal explants at a high dose (2.8E12 v.g.) and a low dose (3.8E11 v.g.) using the same vector containing different capsids. Vector genome numbers were determined by digital droplet PCR of WPRE to allow for titer matching between different constructs. [Figure 3] Figures 3A and 3B are graphs showing the percentage of cones transduced with vector constructs containing AAV8-BP2, ProA7 promoter, WPRE, hGH polyA, different transgenes, and reporter tags. The following transgenes were tested: vfChrimson, ChrimsonR, CatCh, ChrMine, ReaChR, fChrimson-C174, UBI-fChrimson, fChrimson, and Jaws. Either EYFP, Citrine, or tdTomato (tdT) reporter tags were included in the constructs. (A) shows the percentage of cones transduced in retinal organoids at a dose of 8.5E11 v.g. per organoid. (B) shows the percentage of cones transduced in macular human retinal explants at a high dose (2.8E12 v.g.) and a low dose (3.8E11 v.g.), respectively. [Figure 4]Figures 4A and 4B show the results of a combinatorial screen using AAV8-BP2, AAV-PHP.eB, and AAV-NHP26 capsids and all combinations of vfChrimson-EYFP, ChrimsonR-tdTomato, ReaChR-Citrine, and ChrMine-EYFP transgenes. (A) shows the percentage of cones transduced in human retinal organoids at a dose of 8.5E11 v.g. (B) shows the percentage of cones transduced in human retinas at a lower dose (3.8E11 v.g.). ProA7-ReaChR-Citrine-WPRE-hGH polyA and ProA7-ChrMine-EYFP-WPRE-hGH polyA resulted in very high levels of expression, even when different capsids were used in both model systems. [Figure 5] Figures 5A and 5B are graphs showing the results of capsid screening using AAV5, AAV8, or AAV9 capsids and the ProA7-ReaChR-Citrine-WPRE-hGH polyA transgene. A shows the percentage of cones transduced into human retinal organoids at a dose of 8.5E11 v.g. B shows the percentage of cones transduced into human retinas at a lower dose (3.8E11 v.g.). AAV5, AAV8, and AAV9 resulted in efficient expression of ProA7-ReaChR-Citrine-WPRE-hGH polyA in both retinal organoids and human retinal explants. [Figure 6A]Figures 6A-6D are graphs showing the light responsiveness and membrane localization of optogenetic constructs in cultured retinas. In Figures 6A-6C, all constructs tested contained the ProA7 promoter, fluorescent reporter, WPRE, and hGH polyA. The vectors tested were AAV8-BP2-ProA7-ReaChR-Citrine-WPRE-hGH polyA, AAV9-PHP.eB-ProA7-ReaChR-Citrine-WPRE-hGH polyA, AAV9-PHP.eB-ProA7-ChrMine-EYFP-WPRE-hGH polyA, AAV5-ProA7-ReaChR-Citrine-WPRE-hGH polyA, AAV8-BP2-ProA7-Jaws-EYFP-WPRE-hGH polyA, AAV8-BP2-ProA7-ChrimsonR-EYFP-WPRE-hGH polyA, and AAV-NHP26-ProA7-ReaChR-Citrine-WPRE-hGH polyA. Untreated human retinal explants were used as controls. Untreated human retinal explants lost light sensitivity within a few hours, while only optogenetically treated retinas exhibited light responses several weeks after explantation. All recordings were performed 4–8 weeks after AAV treatment. Figure 6A shows the correlation index (a measure of light responsiveness) for all constructs and the negative control. Figure 6B shows the absolute modulation index (another measure of light responsiveness) for the constructs and the negative control. Of all capsids tested, only ProA7-ReaChR-Citrine-WPRE-hGH polyA produced a light response in human retinas. Figure 6C shows the percentage of cells in which the optogenetic protein localized exclusively to the plasma membrane. Without being bound by theory, the light response observed with ReaChR may be explained by its superior membrane localization (Figure 6C). Figure 6D shows additional tests using hyperpolarizing channels (eGTACR1 and HcKCR1) in human retinas with AAV5-ProA7 constructs containing WPRE and hGH polyA. Unlike ReaChR (constructs used the ProX573.2 promoter), these hyperpolarizing optogenetic channels do not elicit a light response. [Figure 6B]See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 7] Figures 7A-7C are graphs showing light-driven activity in human retinal explants transduced with AAV vectors containing ProA7-ReaChR-WPRE-hGH polyA or ProA7-ReaChR-Citrine-WPRE-hGH polyA. (A) Multielectrode array recordings of light-driven ganglion cell spiking from human retinal explants transduced with AAV5 capsids containing ProA7-ReaChR-Citrine-WPRE-hGH polyA. (B) Various optogenetic-driven light responses of major cell types in the human retina (sustained ON cells, transient ON cells, ON / OFF cells, transient OFF cells, and sustained OFF cells) to a 2-s white light flash are shown. This diversity of responses demonstrates the reestablishment of retinal information processing through optogenetics. These responses are identical to normal human light responses as shown by Cowan C et al. (2020) Cell 182(6):1623-1640. (B) Light responsiveness as a function of photon flux for human retinal explants transduced with an AAV5 capsid containing ProA7-ReaChR-Citrine-WPRE-hGH polyA or the same construct lacking the fluorescently tagged citrine. (C) Frequency modulation index in retinas transduced with ProA7-ReaChR-Citrine-WPRE-hGH polyA, with modulation observed for frequencies up to 23.2 Hz. This suggests that optogenetically treated retinas respond to alternating stimuli at approximately 23 frames per second. [Figure 8]Figures 8A-8C show the photoresponsiveness of AAV5-ProA7-ReaChR-citrine-WPRE-hGH polyA vector-transduced and non-transduced (control) areas of explanted retinas from non-human primates (cynomolgus macaques) after subretinal injection in vivo, as well as cone transduction in transduced areas ("blebs"). Acute recordings detected light responses immediately after explantation, suggesting good tissue viability ("Control Acute" in A). The retinas were then cultured for 1-2 days to neutralize endogenous light responses. As expected, no light responses were detected in untreated (non-blebs) areas after culture (Control (non-blebs) in A). In treated (blebs) areas, optogenetic light responses were evident in transient ON cells, sustained ON cells, ON / OFF cells, and ON / OFF-inhibited and OFF-inhibited cells at the two highest dose levels (A and B). At the lowest dose (1.5e10 v.g.), no light response was observed (A). C shows the cone transduction efficiency at different dose levels (n=2 eyes per dose). All dose levels included n=2 per dose except for the 1.5e10 dose, which was used in one eye. The dots represent different images taken from different locations within the bleb. [Figure 9A]Figures 9A-9B show cone transduction efficiency and photoresponsiveness by the AAV5-ProA7-ReaChR-citrine-WPRE-hGH polyA vector in explanted retinas containing the fovea. Immunostaining for GFP was performed to detect the citrine tag in the transduced tissue. Cone transduction was highly efficient across all donors, ranging in age from 18 to 58 years. Figure 9A shows cone transduction in retinal explants from four human donors. Figure 9B presents the optogenetic light response, measured as the photoresponsiveness index (R, intertrial correlation), after transduction of explanted retinas from four additional human donors, ranging in age from 18 to 58 years, with the AAV5-ProA7-ReaChR-citrine-WPRE-hGH polyA vector. Figure 9C shows the cone transduction efficiency of the AAV5-ProA7-ReaChr-citrine-WPRE-hGH polyA vector in explanted retinas from 12 human donors, whose donor ages ranged from 18 to 80 years. Figure 9D shows the photoresponsiveness index (R, intertrial correlation) in explanted retinas from 12 donors. "Control" represents untreated cultured retinas that exhibit no light sensitivity. "Acute" represents normal light response from freshly isolated, uncultured retinal pieces. Donor A in Figures 9A and 9B corresponds to donor 5 in Figures 9C and 9D. Donor B in Figures 9A and 9B corresponds to donor 7 in Figures 9C and 9D. Donor C in Figures 9A and 9B corresponds to donor 6 in Figures 9C and 9D. Quantification of cone transduction was performed for all 12 donors using the same background threshold. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 10] 10 is a schematic diagram showing the structure of three vectors containing ReaChR-citrine, WPRE, and hGH polyA, and differing in promoters: ProA7, Pro572.2, and Pro573.2. ITR: inverted terminal repeat. [Figure 11A]Figures 11A-11E are graphs showing the transduction and light responsiveness of optogenetic constructs under the control of the promoters ProA7, Pro572.2, or Pro573.2 (Pro572.2 and Pro573.2 are also referred to as "ProX572.2" and "ProX573.2," respectively) in human retinal explants. Figures 11A and 11B are graphs showing the percentage of cone transduction in human retinal explants (Figure 11A) and function (light responsiveness) in human retinal explants (Figure 11B) for ProA7- and Pro572.2-driven optogenetic vectors. Figure 11C shows that the Pro573.2-driven construct resulted in a significantly stronger light response in human retinas compared to the ProA7-driven construct. Figure 11D shows that the Pro573.2-driven construct resulted in a higher percentage of light-responsive cells. FIG. 11E shows that the percentage of cones transduced with optogenetic vectors containing ProA7 or Pro573.2 was similar. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 11D] See legend to Figure 11A. [Figure 11E] See legend to Figure 11A. [Figure 12] 12A and 12B show the photoresponsiveness and percentage of photoresponsive cells in human retinal explants from three donors transduced with AAV5-Pro573.2-ReaChR-WPRE-hGH polyA. A is a graph showing the photoresponsiveness index. B is a graph showing the percentage of photoresponsive cells in the explants. [Figure 13]Figures 13A and 13B show that transduction of human retinal explants with the AAV5-Pro573.2-ReaChR-WPRE-hGH polyA AAV resulted in a better light response compared to the AAV5-ProA7-ReaChR-citrine-WPRE-hGH polyA. The Pro573.2-driven construct drove a significantly stronger light response (A) and was more sensitive (the curve is shifted to the left for the Pro573.2 promoter-driven construct) (B). [Figure 14] Figures 14A and 14B show the light response and percentage of light-responsive cells in control, acute, and bleb tissue samples from macaque retinas after subretinal injection of the AAV5-Pro573.2-ReaChR-WPRE-hGH polyA vector. "Acute" refers to freshly dissected retinas, from which normal light responses can be measured. "Control" and "bleb" tissue sections were cultured to eliminate normal light responses before analysis. "Control" is from an untreated area of ​​the retina and, as expected, showed little light response. "Bleb" is from a treated area of ​​the retina and showed light response. (C) shows that the full diversity of light responses (five classes of cells) in the treated bleb sample was detected after transduction with the AAV5-Pro573.2-ReaChR-WPRE-hGH polyA vector. [Figure 15] Figure 15 shows light responses from blind rd1 mice injected with AAV5-ProA7-ReaChR-citrine-WPRE-hGH polyA. The small number of light-responsive cells in controls may represent endogenously light-sensitive cells in the retina. [Figure 16]Figure 16 is a graph showing GFP expression levels in human retinal organoids after transduction with AAV5 and AAVPhP.eB vectors encoding GFP under the control of different promoters at a dose of 1E10 or 1E11 viral genomes (vg) per well. The vectors tested were 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 17] Figures 17A and 17B show cone labeling and cone specificity for constructs under the control of the ProA7 or 4xProSC promoter. A is a quantitative plot of expression in cross sections of human retinal organoids transduced with AAVPhP.eB-ProA7-EGFP-WPRE or AAVPhP.eB-4xProSC-EGFP-WPRE. Quantification of GFP+ cell density as a percentage of cone photoreceptor density is shown. B is a quantitative plot of AAV targeting specificity, shown as the percentage of major (circles) and minor (squares) cell types or classes of cells expressing GFP in organoids transduced with AAVPhP.eB-ProA7-EGFP-WPRE or AAVPhP.eB-4xProSC-EGFP-WPRE. [Figure 18]Figure 18 shows 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. Left: GFP (green). Center left, immunostaining with the cone marker CAR (magenta). Center right, immunostaining with the rod marker NRL (blue), GFP, and a cone marker (signal overlap visible in white). Right: GFP and cone marker, plus nuclear staining (Hoechst, white). DETAILED DESCRIPTION OF THE INVENTION

[0063] Detailed Description The present disclosure relates to an optogenetic approach to restoring light sensitivity in resting human cone cells. The approach disclosed herein contradicts the natural light stimulation mechanism in human cone cells, which hyperpolarize in response to light stimulation. As described and exemplified herein, an optogenetic construct has been developed that causes depolarization of cone cells in response to light stimulation and has been unexpectedly shown to restore light sensitivity in resting cone cells. Indeed, the degree of light sensitivity and depolarization of cone cells is sufficient to trigger light-modulated current spikes in downstream retinal ganglion cells (RGCs), which are well known for transmitting visual information to the brain via action potentials. Accordingly, the present disclosure relates to nucleic acid constructs and viral vectors comprising a depolarizing optogenetic construct, including a depolarizing optogenetic protein, preferably a light-gated ion channel protein. The nucleic acid contains expression control elements (e.g., promoter, cis-regulatory elements) that specifically express the depolarizing optogenetic protein in transduced human cone cells at levels sufficient to restore light sensitivity in the human cone cells. Also disclosed are preferred combinations of expression control elements, optogenetic proteins, and viral vectors, such as AAV capsids, that result in selective delivery and expression of optogenetic proteins in human cone cells.

[0064] As mentioned above, conventional wisdom in the field suggests that optogenetic approaches to restore light sensitivity in photoreceptors, such as cone cells, should focus on recapitulating the normal physiological process of light activation in cone cells, which hyperpolarize in response to light stimulation under normal, healthy conditions ( Choung et al., (2014), Nature Neuroscience, 17, 1123-1129 ; Carter and Lecea (2011), Trends Mol. Med., 17(4):197-206 ; Simon et al., (2020), BBRC, 527(2):325-330 ). The present inventors went against conventional wisdom and surprisingly found that, rather than recapitulating the light-induced hyperpolarization mechanism of normal, healthy cone cells, light sensitivity and light-induced signaling through RGCs can be achieved in human cone cells by using a light-activated depolarizing ion channel protein. We found that the responses of all types of ganglion cells present in normal human retina (Cowan C et al. (2020) Cell 182(6)1623-1640) were reproduced in retinas treated with optogenetics using depolarizing channels. Importantly and unexpectedly, depolarizing optogenetic proteins can generate depolarizing currents strong enough to generate current spiking in RGCs, which may result in the restoration of human vision. The depolarization approach described herein is distinct from and reciprocal to the hyperpolarization mechanism for light perception in healthy human photoreceptors.

[0065] As described herein, the inventors have also surprisingly discovered that combining certain regulatory elements with particular depolarizing optogenetic proteins (and optionally reporter molecules) results in improved or superior expression and light sensitivity in transduced human cones.

[0066] The present disclosure relates to a nucleic acid comprising: i) a promoter as described herein; ii) a nucleotide sequence encoding a depolarizing optogenetic protein, preferably ReaChR, and optionally a reporter molecule, preferably Citrine; iii) a viral post-transcriptional regulatory element (PRE), preferably a Woodchuck Hepatitis Virus PRE (WPRE); and iv) optionally a synthetic intron. The promoter, the nucleotide sequence encoding the depolarizing optogenetic protein and optionally the reporter molecule, and the PRE are operably linked.

[0067] Generally, the promoter, in the context of the nucleic acids disclosed herein, confers selective expression in cone photoreceptors, preferably human cone photoreceptors.

[0068] In some embodiments, the promoter can be a ProA7 promoter having SEQ ID NO:2 or a variant thereof, including a functional fragment thereof. The functional fragment can be a truncated form of ProA7 comprising about 150 to 499 nucleotides from SEQ ID NO:2, for example, about 150 to 499 nucleotides from the 3' end of SEQ ID NO:2.

[0069] In some embodiments, the promoter can be the PR1.7 promoter having SEQ ID NO: 22 or a variant thereof, including a functional fragment thereof.

[0070] In some embodiments, the promoter can be the hG1.7 promoter having SEQ ID NO: 23 or a variant thereof, including a functional fragment thereof.

[0071] In some embodiments, the promoter can be a hybrid promoter containing a ProA7-derived component having SEQ ID NO:2 or a variant thereof, including a functional fragment thereof, e.g., a truncated version of SEQ ID NO:2 comprising about 150-499 nucleotides, e.g., about 150-499 nucleotides from the 3' end of SEQ ID NO:2. Hybrid promoters also include a rod-specific promoter-derived component having SEQ ID NO:10 or a variant thereof, including a functional fragment thereof. The functional fragment can comprise about 370-999 nucleotides from SEQ ID NO:10, e.g., about 370-999 nucleotides from the 3' end of SEQ ID NO:10 or about 895-999 nucleotides from the 5' end of SEQ ID NO:10. Similarly, hybrid promoters can contain other cone-specific promoters or variants thereof, including functional fragments thereof, as desired, and rod-specific promoter-derived components, e.g., variants including SEQ ID NO:10 or functional fragments thereof.

[0072] A preferred nucleic acid comprises a ProA7 promoter, a ReaChR optogenetic protein, optionally Citrine as a reporter molecule, and a WPRE. Preferably, the nucleic acid also comprises a nucleotide sequence encoding a suitable polyadenylation signal (PolyA, such as human growth hormone (hGH) polyA) 3' to the WPRE. The respective nucleotide sequences are operably linked. The nucleic acid may further comprise an inverted terminal repeat (ITR), for example, an AAV ITR. For example, the nucleic acid may comprise an AAV ITR 5' to the promoter and an AAV ITR 3' to the WPRE, or preferably 3' to the PolyA signal. If desired, the nucleic acid may further encode an AAV capsid protein.

[0073] When a nucleic acid sequence encoding a depolarizing optogenetic protein is introduced into human cone cells, it can be expressed (transcribed and translated), and the encoded protein can be localized to the plasma membrane of human cone cells. When exposed to light, the depolarizing optogenetic protein can mediate a depolarizing current that depolarizes human cone cells. Depolarization of human cone cells can induce light-driven current spikes in RGCs. This spiking can be similar or equivalent to light-driven RGC spiking in a functional human retina with healthy cone cells. Light-driven RGC spiking can be measured, for example, using a multi-electrode array.

[0074] The present disclosure further relates to methods of using nucleic acids in the treatment of diseases, conditions, and disorders associated with retinal disease and vision restoration.

[0075] Also provided herein are pharmaceutical compositions comprising nucleic acids encoding the optogenetic constructs, as well as recombinant expression vectors and host cells for producing the optogenetic constructs disclosed herein.

[0076] Certain exemplary and preferred embodiments are described in detail herein, and the embodiments herein should not be construed as limiting the scope of the present disclosure.

[0077] A. Promoter a. ProA7 and its variants, fragments, and truncations As disclosed herein, the nucleic acid can include a nucleic acid sequence encoding the ProA7 promoter, or a variant, fragment, or truncation thereof.

[0078] The ProA7 promoter is known to be useful for driving high expression of desired genes in various species, tissues, and cell types. For example, the present inventors have shown that the ProA7 promoter alone can drive expression in mouse photoreceptor cone cells and various human cell types, including preferentially driving expression in human cone cells but not in other retinal cells. See, for example, Juettner et al. Nature Neuroscience 22, 1345-1356 (2019). However, the present inventors surprisingly discovered that the ProA7 promoter alone does not drive the expression of depolarizing optogenetic proteins in human photoreceptor cone cells. The present inventors surprisingly discovered that the ProA7 promoter in combination with a posttranscriptional regulatory element (PRE), preferably the woodchuck hepatitis virus PRE (WPRE), can drive expression in human photoreceptor cone cells. It has also been found that combining ProA7 and a PRE (e.g., WPRE) with a specific depolarizing optogenetic protein affects expression levels.

[0079] The ProA7 promoter disclosed herein has specificity for human cone cells, and drives high levels of expression in cone cells, such as dormant cone cells that are no longer responsive to light.The ProA7 promoter can have promoter activity in human cone cells of retinal explants.In some cases, the ProA7 promoter does not have substantial promoter activity in other cells of the human retina, such as rod cells (for example, less than about 10% promoter activity is observed in non-cone cells).

[0080] 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: 2, and a second component (second nucleotide sequence) derived from a rod-specific promoter, such as SEQ ID NO: 10, 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: 2) from which the first component (first nucleotide sequence) is derived.

[0081] 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, a promoter can have at least about 90% selectivity for cone cells compared to rod cells, 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 human cone photoreceptors compared to other cells, such as human rod photoreceptors.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.

[0082] 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.

[0083] 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 90% selectivity for rod cells compared to cone cells, 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.

[0084] 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.

[0085] A promoter comprising the ProA7 promoter or a variant, fragment, or truncated form thereof may be operably linked to a nucleotide sequence encoding a depolarizing optogenetics protein. The ProA7 promoter or a variant, fragment, or truncated form thereof may be operably linked to a reporter molecule, if desired. The ProA7 promoter or a variant, fragment, or truncated form thereof may comprise a transcription sequence that mediates expression of a depolarizing optogenetics protein.

[0086] The ProA7 promoter is described in International Publication No. WO2017046084. Generally, the ProA7 promoter has a nucleotide sequence comprising SEQ ID NO: 2. The ProA7 promoter may comprise a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 2. The ProA7 promoter 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:2.

[0087] If desired, the promoter can be a ProA7 variant of SEQ ID NO: 2 that retains promoter activity. The variant ProA7 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 SEQ ID NO: 2.

[0088] The ProA7 variant may be a truncated version of the ProA7 promoter (e.g., containing less than 500 nt of ProA7 due to a deletion of nucleotides at the 5' end). The truncated ProA7 promoter may contain a sequence of at least about 150 nucleotides, and preferably has at least about 70% or more sequence identity with SEQ ID NO:2 over the entire length of the variant sequence. For example, a ProA7 variant can comprise a 5' truncated form of ProA7, and the sequence of the variant can be substantially identical to SEQ ID NO:2 over the entire length of the variant, or 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% identical to SEQ ID NO:2 over the entire length of the variant.

[0089] A truncated ProA7 promoter with a 5' truncation of ProA7 can contain any number of nucleotides from about 150 to 499 (i.e., one less than the length of SEQ ID NO:2). For example, the nucleotide sequence of a 5' truncated Pro7 variant can be the same as nucleotides 386 to 500 of SEQ ID NO:2, or the same as nucleotides 2 to 500 of SEQ ID NO:2.

[0090] In some embodiments, the 5' truncated ProA7 promoter is about 150 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 from SEQ ID NO:2, e.g., from the 3' end of SEQ ID NO:2. 178nt, 179nt, 180nt, 181nt, 182nt, 183nt, 184nt, 185nt, 186nt, 187nt, 188nt, 189nt, 190nt, 191nt, 192nt, 193nt, 194nt, 195 nt, about 196nt, about 197nt, about 198nt, about 199nt, about 200nt, about 201nt, about 202nt, about 203nt, about 204nt, about 205nt, about 206nt, about 207nt, about 208nt, about 209nt, about 210nt, about 211nt, about 212nt, about 21 3nt, about 214nt, about 215nt, about 216nt, about 217nt, about 218nt, about 219nt, about 220nt, about 221nt, about 222nt, about 223nt, about 224nt, about 225nt, about 226nt, about 227nt, about 228nt, about 229nt, about 230nt, about 2 31nt, approx. 232nt, approx. 233nt, approx. 234nt, approx. 235nt, approx. 236nt, approx. 237nt, approx. 238nt, approx. 239nt, approx. 240nt, approx. 249nt, about 250nt, about 251nt, about 252nt, about 253nt, about 254nt, about 255nt, about 256nt, about 257nt, about 258nt, about 259nt, about 260nt, about 261nt, about 262nt, about 263nt, about 264nt, about 265nt, about 266nt, 267nt, 268nt, 269nt, 270nt, 271nt, 272nt, 273nt, 274nt, 275nt, 276nt, 277nt, 278nt, 279nt, 280nt, 281nt, 282nt, 283nt, 284nt,about 285nt, about 286nt, about 287nt, 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 t, about 321nt, about 322nt, about 323nt, 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 6nt, about 357nt, about 358nt, about 359nt, 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 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 443nt, about 444nt, about 445nt, about 446nt, about 447nt, about 448nt, about 449nt, about 450nt, about 451nt, about 452nt, about 453nt, about 454nt, about 455nt, about 456nt, about 457nt, about 458ntapprox. 428nt, approx. 429nt, approx. 430nt, approx. 431nt, approx. 432nt, approx. 433nt, approx. 434nt, approx. 435nt, approx. 436nt, approx. 46nt, 447nt, 448nt, 449nt, 450nt, 451nt, 452nt, 453nt, 454nt, 455nt, 456nt, 457nt, 458nt, 459nt, 460nt, 461nt, 462nt, 463nt, 464 nt, about 465 nt, 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.

[0091] A truncated ProA7 promoter (eg, a 5' truncated version) may comprise a nucleic acid sequence having at least 70% or more sequence identity with SEQ ID NO: 2 over the entire length of the truncated ProA7 promoter. For example, a ProA7 variant can include a 5' truncated form, and the sequence of the variant can be substantially identical to SEQ ID NO:2 over the entire length of the variant, or 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% identical to SEQ ID NO:2 over the entire length of the variant.

[0092] The truncated ProA7 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:2.

[0093] An exemplary truncated ProA7 promoter suitable for use in the constructs of the present disclosure may comprise a sequence of about 395 nt having at least about 70% or more identity to the 3' end of SEQ ID NO: 2 over 395 nt. For example, the truncated ProA7 promoter may comprise SEQ ID NO: 24.

[0094] An exemplary truncated ProA7 promoter suitable for use in the constructs of the present disclosure can comprise a sequence of about 290 nt having at least about 70% or more identity to the 3' end of SEQ ID NO: 2 over 290 nt. For example, the truncated ProA7 promoter can comprise SEQ ID NO: 25.

[0095] An exemplary truncated ProA7 promoter suitable for use in the constructs of the present disclosure can comprise a sequence of approximately 185 nt having at least 70% or more identity to the 3' end of SEQ ID NO: 2 over 185 nt. For example, a truncated ProA7 promoter can comprise SEQ ID NO: 26. The promoter of SEQ ID NO: 26 is referred to as ProA7 5'3 or ProSC.

[0096] An exemplary truncated ProA7 promoter suitable for use in the constructs of the present disclosure can comprise an approximately 150 nt sequence having at least 70% or more identity over 150 nt to the 3' end of SEQ ID NO: 2. For example, the truncated ProA7 promoter can comprise SEQ ID NO: 27.

[0097] In some embodiments, the promoter comprises ProA7 or a truncated ProA7 comprising SEQ ID NO:2, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27. As further described herein, ProA7 promoters, fragments, or variants can be used as a single copy or multiple copies of the promoter sequence, if desired. In some embodiments, the promoter can comprise two or more ProA7-derived components, either the same or different, e.g., 1 to about 10 copies of the same or different sequences. For example, 1 to about 10 copies (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 copies) of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, or any combination thereof. In various embodiments, the cone-specific promoter contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 copies of SEQ ID NO:26.

[0098] ProA7 and truncated ProA7 promoters are suitable for inclusion in the nucleic acids of the present disclosure.

[0099] b. PR1.7, hG1.7, and variants, fragments, and truncations thereof Other suitable promoters include the PR1.7 promoter, which comprises SEQ ID NO: 22, or a sequence having at least 70% identity to SEQ ID NO: 22. The PR1.7 promoter can 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: 22.

[0100] The PR1.7 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:22.

[0101] A PR1.7 variant can be a truncated version of the PR1.7 promoter (e.g., containing less than 1724 nt of PR1.7 due to a deletion of nucleotides at the 5' end). The truncated PR1.7 promoter can contain a sequence of at least about 150 nucleotides, and preferably has at least about 70% or more sequence identity with SEQ ID NO: 22 over the entire length of the variant sequence. For example, a PR1.7 variant can comprise a truncated form of PR1.7, and the sequence of the variant can be substantially identical to SEQ ID NO:22 over the entire length of the variant, or 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% identical to SEQ ID NO:22 over the entire length of the variant.

[0102] The truncated PR1.7 promoter can contain any number of nucleotides from about 150 to 1723 (ie, one less than the length of SEQ ID NO:22).

[0103] In some embodiments, the truncated PR1.7 promoter comprises about 150 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 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 300 nt, about 310 nt, about 311 nt, about 312 nt, about 313 nt, about 314 nt, about 315 nt, about 316 nt, about 317 nt, about 318 nt, about 319 nt, about 320 nt, about 321 nt, about 322 nt, about 323 nt, about 324 nt, about 325 nt, about 326 nt, about 327 nt, about 328 nt, about 329 nt, about 330 nt, about 180nt, 181nt, 182nt, 183nt, 184nt, 185nt, 186nt, 187nt, 188nt, 189nt, 190nt, 191nt, 192nt, 193nt, 194nt, 195nt, 196nt, 197nt , about 198nt, about 199nt, about 200nt, about 201nt, about 202nt, about 203nt, about 204nt, about 205nt, about 206nt, about 207nt, about 208nt, about 209nt, about 210nt, about 211nt, about 212nt, about 213nt, about 214nt, about 215n t, about 216nt, about 217nt, about 218nt, about 219nt, about 220nt, about 221nt, about 222nt, about 223nt, about 224nt, about 225nt, about 226nt, about 227nt, about 228nt, about 229nt, about 230nt, about 231nt, about 232nt, about 23 3nt, about 234nt, about 235nt, about 236nt, about 237nt, about 238nt, about 239nt, about 240nt, about 241nt, about 242nt, about 243nt, about 244nt, about 245nt, about 246nt, about 247nt, about 248nt, about 249nt, about 250nt, about 2 51nt, approx. 252nt, approx. 253nt, approx. 254nt, approx. 255nt, approx. 256nt, approx. 257nt, approx. 258nt, approx. 259nt, approx. 260nt, approx. 269nt, about 270nt, about 271nt, about 272nt, about 273nt, about 274nt, about 275nt, about 276nt, about 277nt, about 278nt, about 279nt, about 280nt, about 281nt, about 282nt, about 283nt, about 284nt, about 285nt, about 286nt,about 287nt, 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 t, about 323nt, 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 8nt, about 359nt, 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 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 443nt, about 444nt, about 445nt, about 446nt, about 447nt, about 448nt, about 449nt, 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 t, about 466nt, about 467nt, about 468nt, about 469nt, about 470nt, 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 487nt, 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 1nt, 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 about 537nt, about 538nt, about 539nt, about 540nt, 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, 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 t, about 609nt, about 610nt, about 611nt, 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 4nt, 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 about 680nt, about 681nt, about 682nt, about 683nt, 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, 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 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 4nt, 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 t, about 819nt, about 820nt, about 821nt, about 822nt, about 823nt, about 824nt, about 825nt, about 826nt, 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, 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 t, 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 4nt, about 925nt, about 926nt, about 927nt, about 928nt, about 929nt, about 930nt, about 931nt, about 932nt, about 933nt, about 934nt, about 935nt, about 936nt, about 937nt, about 938nt, about 939nt, about 940nt, about 941nt, about 942nt, about 943nt, about 944nt, about 945nt, about 946nt, about 947nt, about 948nt, about 949nt, about 950nt, about 951nt, about 952nt, about 953nt, about 954nt, about 955nt, about 956nt, about 957nt, about 958nt, about 959nt, about 960nt, about 961nt, about 962nt, about 963nt, about 964nt, about 965nt, about 966nt, about 967nt, about 968nt, about 969nt, about 970nt, about 971nt, about 972nt, about 973nt, about 974nt, about 975nt, about 976nt, about 977nt, about 978nt, about 979nt, about 980nt, about 981nt, about 982nt, about 983nt, about 984nt, about 985nt, about 986nt, about 987nt, about 988nt, about 989nt, about 990nt, about 991nt, about 992nt, about 993nt, about 994nt, about 995nt,about 996nt, about 997nt, about 998nt, about 999nt, about 1000nt, about 1001nt, about 1002nt, about 1003nt, about 1004nt, about 1005nt, about 1006nt, about 1007nt, about 1008nt, about 1009nt, about 1010nt, about 1011nt, about 1012nt, about 1013nt, about 1014nt, about 1015nt, about 1016nt, about 1017nt, about 1018nt, about 1019nt, about 1020nt, about 1021nt, about 1022nt, about 1023nt, about 1024nt, about 1025nt, about 1026nt, about 1027nt nt, about 1028nt, about 1029nt, about 1030nt, about 1031nt, about 1032nt, about 1033nt, about 1034nt, about 1035nt, about 1036nt, about 1037nt, about 1038nt, about 1039nt, about 1040nt, about 1041nt, about 1042nt, about 1043nt, about 1044nt, about 1045nt, about 1046nt, about 1047nt, about 1048nt, about 1049nt, about 1050nt, about 1051nt, about 1052nt, about 1053nt, about 1054nt, about 1055nt, about 1056nt, about 1057nt, about 1058nt t, about 1059nt, about 1060nt, about 1061nt, about 1062nt, about 1063nt, about 1064nt, about 1065nt, about 1066nt, about 1067nt, about 1068nt, about 1069nt, about 1070nt, about 1071nt, about 1072nt, about 1073nt, about 1074nt, about 1075nt, about 1076nt, about 1077nt, about 1078nt, about 1079nt, about 1080nt, about 1081nt, about 1082nt, about 1083nt, about 1084nt, about 1085nt, about 1086nt, about 1087nt, about 1088nt, about 1089nt nt, about 1090nt, about 1091nt, about 1092nt, about 1093nt, about 1094nt, about 1095nt, about 1096nt, about 1097nt, about 1098nt, about 1099nt, about 1100nt, about 1101nt, about 1102nt, about 1103nt, about 1104nt, about 1105nt, about 1106nt, about 1107nt, about 1108nt, about 1109nt, about 1110nt, about 1111nt, about 1112nt, about 1113nt, about 1114nt, about 1115nt, about 1116nt, about 1117nt, about 1118nt, about 1119nt, about 1120nt,about 1121nt, about 1122nt, about 1123nt, about 1124nt, about 1125nt, about 1126nt, about 1127nt, about 1128nt, about 1129nt, about 1130nt, about 1131nt, about 1132nt, about 1133nt, about 1134nt, about 1135nt, about 1136nt, about 1137nt, about 1138nt, about 1139nt, about 1140nt, about 1141nt, about 1142nt, about 1143nt, about 1144nt, about 1145nt, about 1146nt, about 1147nt, about 1148nt, about 1149nt, about 1150nt, about 1151nt, about 1152nt 152nt, about 1153nt, about 1154nt, about 1155nt, about 1156nt, about 1157nt, about 1158nt, about 1159nt, about 1160nt, about 1161nt, about 1162nt, about 1163nt, about 1164nt, about 1165nt, about 1166nt, about 1167nt, about 1168nt, about 1169nt, about 1170nt, about 1171nt, about 1172nt, about 1173nt, about 1174nt, about 1175nt, about 1176nt, about 1177nt, about 1178nt, about 1179nt, about 1180nt, about 1181nt, about 1182nt, about 1183nt 3nt, about 1184nt, about 1185nt, about 1186nt, about 1187nt, about 1188nt, about 1189nt, about 1190nt, about 1191nt, about 1192nt, about 1193nt, about 1194nt, about 1195nt, about 1196nt, about 1197nt, about 1198nt, about 1199nt, about 1200nt, about 1201nt, about 1202nt, about 1203nt, about 1204nt, about 1205nt, about 1206nt, about 1207nt, about 1208nt, about 1209nt, about 1210nt, about 1211nt, about 1212nt, about 1213nt, about 1214nt t, about 1215nt, about 1216nt, about 1217nt, about 1218nt, about 1219nt, about 1220nt, about 1221nt, about 1222nt, about 1223nt, about 1224nt, about 1225nt, about 1226nt, about 1227nt, about 1228nt, about 1229nt, about 1230nt, about 1231nt, about 1232nt, about 1233nt, about 1234nt, about 1235nt, about 1236nt, about 1237nt, about 1238nt, about 1239nt, about 1240nt, about 1241nt, about 1242nt, about 1243nt, about 1244nt, about 1245nt,about 1246nt, about 1247nt, about 1248nt, about 1249nt, about 1250nt, about 1251nt, about 1252nt, about 1253nt, about 1254nt, about 1255nt, about 1256nt, about 1257nt, about 1258nt, about 1259nt, about 1260nt, about 1261nt, about 1262nt, about 1263nt, about 1264nt, about 1265nt, about 1266nt, about 1267nt, about 1268nt, about 1269nt, about 1270nt, about 1271nt, about 1272nt, about 1273nt, about 1274nt, about 1275nt, about 1276nt, about 1 277nt, about 1278nt, about 1279nt, about 1280nt, about 1281nt, about 1282nt, about 1283nt, about 1284nt, about 1285nt, about 1286nt, about 1287nt, about 1288nt, about 1289nt, about 1290nt, about 1291nt, about 1292nt, about 1293nt, about 1294nt, about 1295nt, about 1296nt, about 1297nt, about 1298nt, about 1299nt, about 1300nt, about 1301nt, about 1302nt, about 1303nt, about 1304nt, about 1305nt, about 1306nt, about 1307nt, about 1308nt 8nt, about 1309nt, about 1310nt, about 1311nt, about 1312nt, about 1313nt, about 1314nt, about 1315nt, about 1316nt, about 1317nt, about 1318nt, about 1319nt, about 1320nt, about 1321nt, about 1322nt, about 1323nt, about 1324nt, about 1325nt, about 1326nt, about 1327nt, about 1328nt, about 1329nt, about 1330nt, about 1331nt, about 1332nt, about 1333nt, about 1334nt, about 1335nt, about 1336nt, about 1337nt, about 1338nt, about 1339nt t, about 1340nt, about 1341nt, about 1342nt, about 1343nt, about 1344nt, about 1345nt, about 1346nt, about 1347nt, about 1348nt, about 1349nt, about 1350nt, about 1351nt, about 1352nt, about 1353nt, about 1354nt, about 1355nt, about 1356nt, about 1357nt, about 1358nt, about 1359nt, about 1360nt, about 1361nt, about 1362nt, about 1363nt, about 1364nt, about 1365nt, about 1366nt, about 1367nt, about 1368nt, about 1369nt, about 1370nt,about 1371nt, about 1372nt, about 1373nt, about 1374nt, about 1375nt, about 1376nt, about 1377nt, about 1378nt, about 1379nt, about 1380nt, about 1381nt, about 1382nt, about 1383nt, about 1384nt, about 1385nt, about 1386nt, about 1387nt, about 1388nt, about 1389nt, about 1390nt, about 1391nt, about 1392nt, about 1393nt, about 1394nt, about 1395nt, about 1396nt, about 1397nt, about 1398nt, about 1399nt, about 1400nt, about 1401nt, about 1402nt 402nt, about 1403nt, about 1404nt, about 1405nt, about 1406nt, about 1407nt, about 1408nt, about 1409nt, about 1410nt, about 1411nt, about 1412nt, about 1413nt, about 1414nt, about 1415nt, about 1416nt, about 1417nt, about 1418nt, about 1419nt, about 1420nt, about 1421nt, about 1422nt, about 1423nt, about 1424nt, about 1425nt, about 1426nt, about 1427nt, about 1428nt, about 1429nt, about 1430nt, about 1431nt, about 1432nt, about 1433nt 3nt, about 1434nt, about 1435nt, about 1436nt, about 1437nt, about 1438nt, about 1439nt, about 1440nt, about 1441nt, about 1442nt, about 1443nt, about 1444nt, about 1445nt, about 1446nt, about 1447nt, about 1448nt, about 1449nt, about 1450nt, about 1451nt, about 1452nt, about 1453nt, about 1454nt, about 1455nt, about 1456nt, about 1457nt, about 1458nt, about 1459nt, about 1460nt, about 1461nt, about 1462nt, about 1463nt, about 1464nt t, about 1465nt, about 1466nt, about 1467nt, about 1468nt, about 1469nt, about 1470nt, about 1471nt, about 1472nt, about 1473nt, about 1474nt, about 1475nt, about 1476nt, about 1477nt, about 1478nt, about 1479nt, about 1480nt, about 1481nt, about 1482nt, about 1483nt, about 1484nt, about 1485nt, about 1486nt, about 1487nt, about 1488nt, about 1489nt, about 1490nt, about 1491nt, about 1492nt, about 1493nt, about 1494nt, about 1495nt,about 1496nt, about 1497nt, about 1498nt, about 1499nt, about 1500nt, about 1501nt, about 1502nt, about 1503nt, about 1504nt, about 1505nt, about 1506nt, about 1507nt, about 1508nt, about 1509nt, about 1510nt, about 1511nt, about 1512nt, about 1513nt, about 1514nt, about 1515nt, about 1516nt, about 1517nt, about 1518nt, about 1519nt, about 1520nt, about 1521nt, about 1522nt, about 1523nt, about 1524nt, about 1525nt, about 1526nt, about 1527nt, about 1528nt, about 1529nt, about 1530nt, about 1531nt, about 1532nt 1532nt, about 1533nt, about 1534nt, about 1535nt, about 1536nt, about 1537nt, about 1538nt, about 1539nt, about 1540nt, about 1541nt, about 1542nt, about 1543nt, about 1544nt, about 1545nt, about 1546nt, about 1547nt, about 1548nt, about 1549nt, about 1550nt, about 1551nt, about 1552nt, about 1553nt, about 1554nt, about 1555nt, about 1556nt, about 1557nt, about 1558nt, about 1559nt, about 1560nt, about 1561nt, about 1562nt, about 1563nt 3nt, about 1564nt, about 1565nt, about 1566nt, about 1567nt, about 1568nt, about 1569nt, about 1570nt, about 1571nt, about 1572nt, about 1573nt, about 1574nt, about 1575nt, about 1576nt, about 1577nt, about 1578nt, about 1579nt, about 1580nt, about 1581nt, about 1582nt, about 1583nt, about 1584nt, about 1585nt, about 1586nt, about 1587nt, about 1588nt, about 1589nt, about 1590nt, about 1591nt, about 1592nt, about 1593nt, about 1594nt t, about 1595nt, about 1596nt, about 1597nt, about 1598nt, about 1599nt, about 1600nt, about 1601nt, about 1602nt, about 1603nt, about 1604nt, about 1605nt, about 1606nt, about 1607nt, about 1608nt, about 1609nt, about 1610nt, about 1611nt, about 1612nt, about 1613nt, about 1614nt, about 1615nt, about 1616nt, about 1617nt, about 1618nt, about 1619nt, about 1620nt, about 1621nt, about 1622nt, about 1623nt, about 1624nt, about 1625nt,About 1626nt, about 1627nt, about 1628nt, about 1629nt, about 1630nt, about 1631nt, about 1632nt, about 1633nt, about 1634nt, about 1635nt, about 1636nt, about 1637nt, about 16 38nt, about 1639nt, about 1640nt, about 1641nt, about 1642nt, about 1643nt, about 1644nt, about 1645nt, about 1646nt, about 1647nt, about 1648nt, about 1649nt, about 1650n t, about 1651nt, about 1652nt, about 1653nt, about 1654nt, about 1655nt, about 1656nt, about 1657nt, about 1658nt, about 1659nt, about 1660nt, about 1661nt, about 1662nt, about 1663nt, about 1664nt, about 1665nt, about 1666nt, about 1667nt, about 1668nt, about 1669nt, about 1670nt, about 1671nt, about 1672nt, about 1673nt, about 1674nt, about 167 5nt, about 1676nt, about 1677nt, about 1678nt, about 1679nt, about 1680nt, about 1681nt, about 1682nt, about 1683nt, about 1684nt, about 1685nt, about 1686nt, about 1687nt , about 1688nt, about 1689nt, about 1690nt, about 1691nt, about 1692nt, about 1693nt, about 1694nt, about 1695nt, about 1696nt, about 1697nt, about 1698nt, about 1699nt, about 1 700 nt, about 1701 nt, about 1702 nt, about 1703 nt, about 1704 nt, about 1705 nt, about 1706 nt, about 1707 nt, about 1708 nt, about 1709 nt, about 1710 nt, about 1711 nt, about 1712 nt, about 1713 nt, about 1714 nt, about 1715 nt, about 1716 nt, about 1717 nt, about 1718 nt, about 1719 nt, about 1720 nt, about 1721 nt, about 1722 nt, or about 1723 nt.

[0104] Another suitable promoter is the hG1.7 promoter, which comprises SEQ ID NO: 23, or a sequence having at least 70% identity to SEQ ID NO: 23. The hG1.7 promoter can 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 an equivalent length sequence from SEQ ID NO: 23.

[0105] The hG1.7 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 aligned sequence of the same length from SEQ ID NO:23.

[0106] The hG1.7 variant may be a truncated version of the hG1.7 promoter (e.g., may contain less than 1782 nt of hG1.7). The truncated hG1.7 promoter may contain a sequence of at least about 150 nucleotides, and preferably has at least about 70% or more sequence identity with SEQ ID NO: 23 over the entire length of the variant sequence. For example, an hG1.7 variant can include a truncated form of hG1.7, and the sequence of the variant can be substantially identical to SEQ ID NO:23 over the entire length of the variant, or 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% identical to SEQ ID NO:23 over the entire length of the variant.

[0107] The truncated hG1.7 promoter can contain any number of nucleotides from about 150 to 1781 (ie, one less than the length of SEQ ID NO:23).

[0108] In some embodiments, the truncated hG1.7 promoter comprises about 150 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 180nt, 181nt, 182nt, 183nt, 184nt, 185nt, 186nt, 187nt, 188nt, 189nt, 190nt, 191nt, 192nt, 193nt, 194nt, 195nt, 196nt, 197nt , about 198nt, about 199nt, about 200nt, about 201nt, about 202nt, about 203nt, about 204nt, about 205nt, about 206nt, about 207nt, about 208nt, about 209nt, about 210nt, about 211nt, about 212nt, about 213nt, about 214nt, about 215n t, about 216nt, about 217nt, about 218nt, about 219nt, about 220nt, about 221nt, about 222nt, about 223nt, about 224nt, about 225nt, about 226nt, about 227nt, about 228nt, about 229nt, about 230nt, about 231nt, about 232nt, about 23 3nt, about 234nt, about 235nt, about 236nt, about 237nt, about 238nt, about 239nt, about 240nt, about 241nt, about 242nt, about 243nt, about 244nt, about 245nt, about 246nt, about 247nt, about 248nt, about 249nt, about 250nt, about 2 51nt, approx. 252nt, approx. 253nt, approx. 254nt, approx. 255nt, approx. 256nt, approx. 257nt, approx. 258nt, approx. 259nt, approx. 260nt, approx. 269nt, about 270nt, about 271nt, about 272nt, about 273nt, about 274nt, about 275nt, about 276nt, about 277nt, about 278nt, about 279nt, about 280nt, about 281nt, about 282nt, about 283nt, about 284nt, about 285nt, about 286nt,about 287nt, 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 t, about 323nt, 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 8nt, about 359nt, 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 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 443nt, about 444nt, about 445nt, about 446nt, about 447nt, about 448nt, about 449nt, 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 t, about 466nt, about 467nt, about 468nt, about 469nt, about 470nt, 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 487nt, 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 1nt, 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 about 537nt, about 538nt, about 539nt, about 540nt, 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, 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 t, about 609nt, about 610nt, about 611nt, 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 4nt, 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 about 680nt, about 681nt, about 682nt, about 683nt, 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, 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 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 4nt, 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 t, about 819nt, about 820nt, about 821nt, about 822nt, about 823nt, about 824nt, about 825nt, about 826nt, 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, 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 t, 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 4nt, about 925nt, about 926nt, about 927nt, about 928nt, about 929nt, about 930nt, about 931nt, about 932nt, about 933nt, about 934nt, about 935nt, about 936nt, about 937nt, about 938nt, about 939nt, about 940nt, about 941nt, about 942nt, about 943nt, about 944nt, about 945nt, about 946nt, about 947nt, about 948nt, about 949nt, about 950nt, about 951nt, about 952nt, about 953nt, about 954nt, about 955nt, about 956nt, about 957nt, about 958nt, about 959nt, about 960nt, about 961nt, about 962nt, about 963nt, about 964nt, about 965nt, about 966nt, about 967nt, about 968nt, about 969nt, about 970nt, about 971nt, about 972nt, about 973nt, about 974nt, about 975nt, about 976nt, about 977nt, about 978nt, about 979nt, about 980nt, about 981nt, about 982nt, about 983nt, about 984nt, about 985nt, about 986nt, about 987nt, about 988nt, about 989nt, about 990nt, about 991nt, about 992nt, about 993nt, about 994nt, about 995nt,about 996nt, about 997nt, about 998nt, about 999nt, about 1000nt, about 1001nt, about 1002nt, about 1003nt, about 1004nt, about 1005nt, about 1006nt, about 1007nt, about 1008nt, about 1009nt, about 1010nt, about 1011nt, about 1012nt, about 1013nt, about 1014nt, about 1015nt, about 1016nt, about 1017nt, about 1018nt, about 1019nt, about 1020nt, about 1021nt, about 1022nt, about 1023nt, about 1024nt, about 1025nt, about 1026nt, about 1027nt nt, about 1028nt, about 1029nt, about 1030nt, about 1031nt, about 1032nt, about 1033nt, about 1034nt, about 1035nt, about 1036nt, about 1037nt, about 1038nt, about 1039nt, about 1040nt, about 1041nt, about 1042nt, about 1043nt, about 1044nt, about 1045nt, about 1046nt, about 1047nt, about 1048nt, about 1049nt, about 1050nt, about 1051nt, about 1052nt, about 1053nt, about 1054nt, about 1055nt, about 1056nt, about 1057nt, about 1058nt t, about 1059nt, about 1060nt, about 1061nt, about 1062nt, about 1063nt, about 1064nt, about 1065nt, about 1066nt, about 1067nt, about 1068nt, about 1069nt, about 1070nt, about 1071nt, about 1072nt, about 1073nt, about 1074nt, about 1075nt, about 1076nt, about 1077nt, about 1078nt, about 1079nt, about 1080nt, about 1081nt, about 1082nt, about 1083nt, about 1084nt, about 1085nt, about 1086nt, about 1087nt, about 1088nt, about 1089nt nt, about 1090nt, about 1091nt, about 1092nt, about 1093nt, about 1094nt, about 1095nt, about 1096nt, about 1097nt, about 1098nt, about 1099nt, about 1100nt, about 1101nt, about 1102nt, about 1103nt, about 1104nt, about 1105nt, about 1106nt, about 1107nt, about 1108nt, about 1109nt, about 1110nt, about 1111nt, about 1112nt, about 1113nt, about 1114nt, about 1115nt, about 1116nt, about 1117nt, about 1118nt, about 1119nt, about 1120nt,about 1121nt, about 1122nt, about 1123nt, about 1124nt, about 1125nt, about 1126nt, about 1127nt, about 1128nt, about 1129nt, about 1130nt, about 1131nt, about 1132nt, about 1133nt, about 1134nt, about 1135nt, about 1136nt, about 1137nt, about 1138nt, about 1139nt, about 1140nt, about 1141nt, about 1142nt, about 1143nt, about 1144nt, about 1145nt, about 1146nt, about 1147nt, about 1148nt, about 1149nt, about 1150nt, about 1151nt, about 1152nt 152nt, about 1153nt, about 1154nt, about 1155nt, about 1156nt, about 1157nt, about 1158nt, about 1159nt, about 1160nt, about 1161nt, about 1162nt, about 1163nt, about 1164nt, about 1165nt, about 1166nt, about 1167nt, about 1168nt, about 1169nt, about 1170nt, about 1171nt, about 1172nt, about 1173nt, about 1174nt, about 1175nt, about 1176nt, about 1177nt, about 1178nt, about 1179nt, about 1180nt, about 1181nt, about 1182nt, about 1183nt 3nt, about 1184nt, about 1185nt, about 1186nt, about 1187nt, about 1188nt, about 1189nt, about 1190nt, about 1191nt, about 1192nt, about 1193nt, about 1194nt, about 1195nt, about 1196nt, about 1197nt, about 1198nt, about 1199nt, about 1200nt, about 1201nt, about 1202nt, about 1203nt, about 1204nt, about 1205nt, about 1206nt, about 1207nt, about 1208nt, about 1209nt, about 1210nt, about 1211nt, about 1212nt, about 1213nt, about 1214nt t, about 1215nt, about 1216nt, about 1217nt, about 1218nt, about 1219nt, about 1220nt, about 1221nt, about 1222nt, about 1223nt, about 1224nt, about 1225nt, about 1226nt, about 1227nt, about 1228nt, about 1229nt, about 1230nt, about 1231nt, about 1232nt, about 1233nt, about 1234nt, about 1235nt, about 1236nt, about 1237nt, about 1238nt, about 1239nt, about 1240nt, about 1241nt, about 1242nt, about 1243nt, about 1244nt, about 1245nt,about 1246nt, about 1247nt, about 1248nt, about 1249nt, about 1250nt, about 1251nt, about 1252nt, about 1253nt, about 1254nt, about 1255nt, about 1256nt, about 1257nt, about 1258nt, about 1259nt, about 1260nt, about 1261nt, about 1262nt, about 1263nt, about 1264nt, about 1265nt, about 1266nt, about 1267nt, about 1268nt, about 1269nt, about 1270nt, about 1271nt, about 1272nt, about 1273nt, about 1274nt, about 1275nt, about 1276nt, about 1 277nt, about 1278nt, about 1279nt, about 1280nt, about 1281nt, about 1282nt, about 1283nt, about 1284nt, about 1285nt, about 1286nt, about 1287nt, about 1288nt, about 1289nt, about 1290nt, about 1291nt, about 1292nt, about 1293nt, about 1294nt, about 1295nt, about 1296nt, about 1297nt, about 1298nt, about 1299nt, about 1300nt, about 1301nt, about 1302nt, about 1303nt, about 1304nt, about 1305nt, about 1306nt, about 1307nt, about 1308nt 8nt, about 1309nt, about 1310nt, about 1311nt, about 1312nt, about 1313nt, about 1314nt, about 1315nt, about 1316nt, about 1317nt, about 1318nt, about 1319nt, about 1320nt, about 1321nt, about 1322nt, about 1323nt, about 1324nt, about 1325nt, about 1326nt, about 1327nt, about 1328nt, about 1329nt, about 1330nt, about 1331nt, about 1332nt, about 1333nt, about 1334nt, about 1335nt, about 1336nt, about 1337nt, about 1338nt, about 1339nt t, about 1340nt, about 1341nt, about 1342nt, about 1343nt, about 1344nt, about 1345nt, about 1346nt, about 1347nt, about 1348nt, about 1349nt, about 1350nt, about 1351nt, about 1352nt, about 1353nt, about 1354nt, about 1355nt, about 1356nt, about 1357nt, about 1358nt, about 1359nt, about 1360nt, about 1361nt, about 1362nt, about 1363nt, about 1364nt, about 1365nt, about 1366nt, about 1367nt, about 1368nt, about 1369nt, about 1370nt,about 1371nt, about 1372nt, about 1373nt, about 1374nt, about 1375nt, about 1376nt, about 1377nt, about 1378nt, about 1379nt, about 1380nt, about 1381nt, about 1382nt, about 1383nt, about 1384nt, about 1385nt, about 1386nt, about 1387nt, about 1388nt, about 1389nt, about 1390nt, about 1391nt, about 1392nt, about 1393nt, about 1394nt, about 1395nt, about 1396nt, about 1397nt, about 1398nt, about 1399nt, about 1400nt, about 1401nt, about 1402nt 402nt, about 1403nt, about 1404nt, about 1405nt, about 1406nt, about 1407nt, about 1408nt, about 1409nt, about 1410nt, about 1411nt, about 1412nt, about 1413nt, about 1414nt, about 1415nt, about 1416nt, about 1417nt, about 1418nt, about 1419nt, about 1420nt, about 1421nt, about 1422nt, about 1423nt, about 1424nt, about 1425nt, about 1426nt, about 1427nt, about 1428nt, about 1429nt, about 1430nt, about 1431nt, about 1432nt, about 1433nt 3nt, about 1434nt, about 1435nt, about 1436nt, about 1437nt, about 1438nt, about 1439nt, about 1440nt, about 1441nt, about 1442nt, about 1443nt, about 1444nt, about 1445nt, about 1446nt, about 1447nt, about 1448nt, about 1449nt, about 1450nt, about 1451nt, about 1452nt, about 1453nt, about 1454nt, about 1455nt, about 1456nt, about 1457nt, about 1458nt, about 1459nt, about 1460nt, about 1461nt, about 1462nt, about 1463nt, about 1464nt t, about 1465nt, about 1466nt, about 1467nt, about 1468nt, about 1469nt, about 1470nt, about 1471nt, about 1472nt, about 1473nt, about 1474nt, about 1475nt, about 1476nt, about 1477nt, about 1478nt, about 1479nt, about 1480nt, about 1481nt, about 1482nt, about 1483nt, about 1484nt, about 1485nt, about 1486nt, about 1487nt, about 1488nt, about 1489nt, about 1490nt, about 1491nt, about 1492nt, about 1493nt, about 1494nt, about 1495nt,about 1496nt, about 1497nt, about 1498nt, about 1499nt, about 1500nt, about 1501nt, about 1502nt, about 1503nt, about 1504nt, about 1505nt, about 1506nt, about 1507nt, about 1508nt, about 1509nt, about 1510nt, about 1511nt, about 1512nt, about 1513nt, about 1514nt, about 1515nt, about 1516nt, about 1517nt, about 1518nt, about 1519nt, about 1520nt, about 1521nt, about 1522nt, about 1523nt, about 1524nt, about 1525nt, about 1526nt, about 1527nt, about 1528nt, about 1529nt, about 1530nt, about 1531nt, about 1532nt 1532nt, about 1533nt, about 1534nt, about 1535nt, about 1536nt, about 1537nt, about 1538nt, about 1539nt, about 1540nt, about 1541nt, about 1542nt, about 1543nt, about 1544nt, about 1545nt, about 1546nt, about 1547nt, about 1548nt, about 1549nt, about 1550nt, about 1551nt, about 1552nt, about 1553nt, about 1554nt, about 1555nt, about 1556nt, about 1557nt, about 1558nt, about 1559nt, about 1560nt, about 1561nt, about 1562nt, about 1563nt 3nt, about 1564nt, about 1565nt, about 1566nt, about 1567nt, about 1568nt, about 1569nt, about 1570nt, about 1571nt, about 1572nt, about 1573nt, about 1574nt, about 1575nt, about 1576nt, about 1577nt, about 1578nt, about 1579nt, about 1580nt, about 1581nt, about 1582nt, about 1583nt, about 1584nt, about 1585nt, about 1586nt, about 1587nt, about 1588nt, about 1589nt, about 1590nt, about 1591nt, about 1592nt, about 1593nt, about 1594nt t, about 1595nt, about 1596nt, about 1597nt, about 1598nt, about 1599nt, about 1600nt, about 1601nt, about 1602nt, about 1603nt, about 1604nt, about 1605nt, about 1606nt, about 1607nt, about 1608nt, about 1609nt, about 1610nt, about 1611nt, about 1612nt, about 1613nt, about 1614nt, about 1615nt, about 1616nt, about 1617nt, about 1618nt, about 1619nt, about 1620nt, about 1621nt, about 1622nt, about 1623nt, about 1624nt, about 1625nt,about 1626nt, about 1627nt, about 1628nt, about 1629nt, about 1630nt, about 1631nt, about 1632nt, about 1633nt, about 1634nt, about 1635nt, about 1636nt, about 1637nt, about 1638nt, about 1639nt, about 1640nt, about 1641nt, about 1642nt, about 1643nt, about 1644nt, about 1645nt, about 1646nt, about 1647nt, about 1648nt, about 1649nt, about 1650nt, about 1651nt, about 1652nt, about 1653nt, about 1654nt, about 1655nt, about 1656nt, about 1 657nt, about 1658nt, about 1659nt, about 1660nt, about 1661nt, about 1662nt, about 1663nt, about 1664nt, about 1665nt, about 1666nt, about 1667nt, about 1668nt, about 1669nt, about 1670nt, about 1671nt, about 1672nt, about 1673nt, about 1674nt, about 1675nt, about 1676nt, about 1677nt, about 1678nt, about 1679nt, about 1680nt, about 1681nt, about 1682nt, about 1683nt, about 1684nt, about 1685nt, about 1686nt, about 1687nt, about 1688nt 8nt, about 1689nt, about 1690nt, about 1691nt, about 1692nt, about 1693nt, about 1694nt, about 1695nt, about 1696nt, about 1697nt, about 1698nt, about 1699nt, about 1700nt, about 1701nt, about 1702nt, about 1703nt, about 1704nt, about 1705nt, about 1706nt, about 1707nt, about 1708nt, about 1709nt, about 1710nt, about 1711nt, about 1712nt, about 1713nt, about 1714nt, about 1715nt, about 1716nt, about 1717nt, about 1718nt, about 1719nt t, about 1720nt, about 1721nt, about 1722nt, about 1723nt, about 1724nt, about 1725nt, about 1726nt, about 1727nt, about 1728nt, about 1729nt, about 1730nt, about 1731nt, about 1732nt, about 1733nt, about 1734nt, about 1735nt, about 1736nt, about 1737nt, about 1738nt, about 1739nt, about 1740nt, about 1741nt, about 1742nt, about 1743nt, about 1744nt, about 1745nt, about 1746nt, about 1747nt, about 1748nt, about 1749nt, about 1750nt,It may contain about 1751 nt, about 1752 nt, about 1753 nt, about 1754 nt, about 1755 nt, about 1756 nt, about 1757 nt, about 1758 nt, about 1759 nt, about 1760 nt, about 1761 nt, about 1762 nt, about 1763 nt, about 1764 nt, about 1765 nt, about 1766 nt, about 1767 nt, about 1768 nt, about 1769 nt, about 1770 nt, about 1771 nt, about 1772 nt, about 1773 nt, about 1774 nt, about 1775 nt, about 1776 nt, about 1777 nt, about 1778 nt, about 1779 nt, about 1780 nt, or about 1781 nt.

[0109] As described herein, if desired, the RP1.7 promoter, hG1.7 promoter, and any fragment or variant may be used in single or multiple copies, for example, from 1 copy to about 10 copies (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 copies).

[0110] c. Hybrid promoters The promoter may be a synthetic hybrid promoter. A preferred hybrid promoter herein refers to a promoter comprising a ProA7-derived component having SEQ ID NO: 2 or a variant thereof, including a functional fragment thereof, and a rod-specific promoter-derived component having SEQ ID NO: 10 or a variant thereof, including a functional fragment thereof, wherein the two components are operably linked to function as a single promoter.

[0111] The inventors surprisingly discovered that such synthetic promoters are unexpectedly cone-specific, having enhanced promoter activity in human cone cells compared to the cone-specific promoter (e.g., SEQ ID NO: 2) from which the ProA7 component is derived.

[0112] ProA7-derived components, including variants, fragments, and truncations thereof, are described elsewhere herein.

[0113] The rod-specific promoter element may have a nucleotide sequence comprising SEQ ID NO: 10. The rod-specific promoter element may comprise a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:10. A rod-specific promoter component can 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:10.

[0114] If desired, the promoter can comprise a rod-specific promoter element variant of SEQ ID NO: 10 that retains promoter activity. The variant rod-specific promoter element 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 SEQ ID NO: 10.

[0115] A functional fragment can comprise about 370 to 999 nucleotides from SEQ ID NO:10, for example, about 370 to 999 nucleotides from the 3' end of SEQ ID NO:10 or about 895 to 999 nucleotides from the 5' end of SEQ ID NO:10.

[0116] Preferably, the rod-specific promoter element comprises at least about 370 nucleotides and has at least 70% identity to a sequence of equal length from SEQ ID NO: 10. In this specification, the promoter consisting of SEQ ID NO: 10 is sometimes referred to as ProA330. For example, a rod-specific promoter element 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: 10 over the entire length of the rod-specific promoter element. Preferably, the rod-specific promoter element may have at least 90% identity to SEQ ID NO: 10 over the entire length of the rod-specific promoter element.

[0117] These embodiments include, but are not limited to, those in which the ProA7-derived component has at least 90% identity to a sequence equal in length from the 3' end of SEQ ID NO:2.

[0118] Exemplary rod-specific promoter elements that can be used in the promoters of the present disclosure include SEQ ID NO: 10 and nucleotide sequences having at least about 90% identity to SEQ ID NO: 10 over the entire length of the rod-specific promoter element.

[0119] A rod-specific promoter element 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:10.

[0120] A rod-specific promoter element may comprise a sequence having at least 70% identity to a sequence of equal length from the 3' end of SEQ ID NO: 10. A rod-specific promoter element according to this paragraph may be considered a 5' truncated version of SEQ ID NO: 10, since nucleotides have been removed from the 5' end of SEQ ID NO: 10 to form a rod-specific promoter element.

[0121] A rod-specific promoter element may comprise a sequence having at least 70% identity to a sequence of equal length from the 5' end of SEQ ID NO: 10. A rod-specific promoter element according to this paragraph may be considered a 3' truncated version of SEQ ID NO: 10, since nucleotides have been removed from the 3' end of SEQ ID NO: 10 to form a rod-specific promoter element.

[0122] The rod-specific promoter element may be a 5'-truncated version, a 3'-truncated version, or both, of SEQ ID NO: 10 and may comprise less than 1000 nt of SEQ ID NO: 10 due to deletion of nucleotides at the 5' and / or 3' ends. For example, the rod-specific promoter element may be identical to nucleotides 106 to 1000 of SEQ ID NO: 10, or may be identical to nucleotides 1 to 895 of SEQ ID NO: 10. Preferably, the rod-specific promoter element may comprise or consist of nucleotides 631 to 895 of SEQ ID NO: 10, which is believed to be the minimum portion of SEQ ID NO: 10 required for rod-specific promoter activity.

[0123] The rod-specific promoter element can contain any number of nucleotides between 370 and 1000. For example, the rod-specific promoter element 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, about 40 1nt, approx. 402nt, approx. 403nt, approx. 404nt, approx. 405nt, approx. 406nt, approx. 407nt, approx. 408nt, approx. 409nt, approx. 410nt, approx. 8nt, approx. 419nt, approx. 420nt, approx. 421nt, approx. 422nt, approx. 423nt, approx. 424nt, approx. 425nt, approx. 426nt, approx. 427nt, approx. nt, approx. 436nt, approx. 437nt, approx. 438nt, approx. 439nt, approx. 440nt, approx. 441nt, approx. 442nt, approx. 443nt, approx. 444nt, approx. 445nt, approx. 446nt, approx. nt, 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, about 467nt, about 468nt, about 469n t, about 470nt, 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 487nt, 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 t, about 540nt, 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 5nt, about 576nt, 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 about 611nt, 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 t, about 683nt, 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 8nt, about 719nt, 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 about 754nt, 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 t, about 826nt, 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 1nt, about 862nt, 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, about 934nt, about 935nt, about 936nt, about 937nt, about 938nt, about 939nt, about 940nt, about 941nt, about 942nt, about 943nt, about 944nt, about 945nt, about 946nt, about 947nt, about 948nt, about 949nt, about 9 50nt, about 951nt, about 952nt, about 953nt, about 954nt, about 955nt, about 956nt, about 957nt, about 958nt, about 959nt, about 960nt, about 961nt, about 962nt, about 963nt, about 964nt, about 965nt, about 966nt, about 967 nt, about 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.

[0124] Some preferred rod-specific promoter components for use in the promoters of the present disclosure have a nucleotide sequence comprising from about 370 nucleotides to about 1000 nucleotides from the 3' end of SEQ ID NO:10.

[0125] Some preferred rod-specific promoter components for use in the promoters of the present disclosure have a nucleotide sequence comprising from about 895 nucleotides to about 1000 nucleotides from the 5' end of SEQ ID NO:10.

[0126] Some preferred rod-specific promoter components for use in the promoters of the present disclosure have a nucleotide sequence comprising or consisting of nucleotides 631 to 895 of SEQ ID NO:10.

[0127] The rod-specific promoter element may comprise a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 10 over the entire length of the rod-specific promoter element. A rod-specific promoter element 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: 10 over the entire length of the rod-specific promoter element. Preferably, the rod-specific promoter element may have at least 90% identity to SEQ ID NO: 10 over the entire length of the rod-specific promoter element.

[0128] A rod-specific promoter element 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:10.

[0129] An exemplary rod-specific promoter component may comprise an approximately 895 nt sequence having at least 70% identity to the 3' end of SEQ ID NO: 10. For example, a rod-specific promoter component may comprise SEQ ID NO: 36. An exemplary rod-specific promoter component may comprise an approximately 790 nt sequence having at least 70% identity to the 3' end of SEQ ID NO: 10. For example, a rod-specific promoter component may comprise SEQ ID NO: 37. An exemplary rod-specific promoter component may comprise an approximately 685 nt sequence having at least 70% identity to the 3' end of SEQ ID NO: 10. For example, a rod-specific promoter component may comprise SEQ ID NO: 38. An exemplary rod-specific promoter component may comprise an approximately 580 nt sequence having at least 70% identity to the 3' end of SEQ ID NO: 10. For example, a rod-specific promoter component may comprise SEQ ID NO: 39. An exemplary rod-specific promoter component may comprise an approximately 475 nt sequence having at least 70% identity to the 3' end of SEQ ID NO: 10. For example, a rod-specific promoter component may comprise SEQ ID NO: 40. An exemplary rod-specific promoter component may comprise an approximately 390 nt sequence having at least 70% identity to the 3' end of SEQ ID NO: 10. For example, a rod-specific promoter component may comprise SEQ ID NO: 41. An exemplary rod-specific promoter component may comprise an approximately 895 nt sequence having at least 70% identity to the 5' end of SEQ ID NO: 10. For example, a rod-specific promoter component may comprise SEQ ID NO: 45.

[0130] The rod-specific promoter component can include any number of nucleotides from about 260 to about 369 nucleotides of SEQ ID NO: 10. For example, the rod-specific promoter component can include any number of nucleotides from about 261 nt, 262 nt, 263 nt, 264 nt, 265 nt, 266 nt, 267 nt, 268 nt, 269 nt, 270 nt, 271 nt, 272 nt, 272 nt, 274 nt, 275 nt, 276 nt, 277 nt, 278 nt, 279 nt, 280 nt, 281 nt, 282 nt, 283 nt, 284 nt of SEQ ID NO: 10. , 285nt, 286nt, 287nt, 288nt, 289nt, 290nt, 291nt, 292nt, 293nt, 294nt, 295nt, 296nt, 297nt, 298nt, 2 99nt, 300nt, 301nt, 302nt, 303nt, 304nt, 305nt, 306nt, 307nt, 308nt, 309nt, 310nt, 311nt, 312nt, 313n t, 314nt, 315nt, 316nt, 317nt, 318nt, 319nt, 320nt, 321nt, 322nt, 323nt, 324nt, 325nt, 326nt, 327nt, 328nt, 329nt, 330nt, 331nt, 332nt, 333nt, 334nt, 335nt, 336nt, 337nt, 338nt, 339nt, 340nt, 341nt, 342 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:54 or a sequence having at least about 70% identity to SEQ ID NO:54.

[0131] In some embodiments, the rod-specific promoter components comprise SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:45. In some embodiments, the rod-specific promoter components comprise or consist of nucleotides 631-895 of SEQ ID NO:10. Another preferred specific such promoter is designated min330 (SEQ ID NO:54). In some embodiments, in addition to the ProA7-derived components and the rod-specific promoter-derived components, the hybrid promoter can further comprise one or more other rod-specific or cone-specific promoter sequences. In some embodiments, the hybrid promoter can further comprise nucleotide sequences derived from one or more other cone-specific promoters or cone-specific promoters, such as hG1.7 (SEQ ID NO:23) or PR1.7 (SEQ ID NO:22), or variants, fragments, or truncations thereof.

[0132] The ProA7-derived component, the rod-specific promoter-derived component, and any other promoter sequences (if included) can each be included in a hybrid promoter as a single copy or multiple copies. In some embodiments, a promoter can include two or more ProA7-derived components, two or more rod-specific promoter-derived components, or both. When two or more ProA7-derived components are included in a promoter, the two ProA7-derived components need not be identical, as long as each individually meets the criteria described herein for ProA7 or its variants, fragments, or truncations. Similarly, when two or more rod-specific promoter-derived components are included in a promoter, the two rod-specific promoter-derived components need not be identical, as long as each individually meets the criteria described herein for rod-specific promoter-derived components.

[0133] In the hybrid promoters of the present disclosure, the ProA7-derived element(s) and the rod-specific promoter-derived element(s) can be positioned in any desired order, with or without other sequences between them. As exemplified herein, hybrid promoters containing the same ProA7-derived element and the same rod-specific promoter-derived element in different orders are effective in driving expression of a reporter gene in human cone photoreceptors.

[0134] The promoter may comprise, from 5' to 3', a ProA7-derived component linked to a rod-specific promoter-derived component, with no sequence between them. The promoter may comprise, from 5' to 3', a rod-specific promoter-derived component linked to a ProA7-derived component, with no sequence between them. In another example, the promoter may comprise two ProA7-derived components (component A and component B) and one rod-specific promoter-derived component, arranged 5' to 3' as follows: component A / rod-specific promoter-derived component / component B.

[0135] In some embodiments, a promoter may comprise one or more promoter units, each of which comprises a ProA7-derived component and a rod-specific promoter-derived component, in any order. The promoter units as separate molecules have cone-specific promoter activity. For example, a promoter may comprise one to about four promoter units.

[0136] Some particular promoters may contain, from 5' to 3', a ProA7-derived element comprising SEQ ID NO: 2 and a rod-specific promoter-derived element comprising SEQ ID NO: 10. A particular such promoter, designated Pro572, has SEQ ID NO: 11.

[0137] Some particular promoters may comprise, 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO: 10 and a ProA7-derived component comprising SEQ ID NO: 2. A particular such promoter, designated Pro573, has SEQ ID NO: 13. Some particular promoters may comprise, 5' to 3', a ProA7-derived component comprising SEQ ID NO: 24 and a rod-specific promoter-derived component comprising SEQ ID NO: 10. Some particular promoters may comprise, 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO: 10 and a ProA7-derived component comprising SEQ ID NO: 24. Some particular promoters may comprise, 5' to 3', a ProA7-derived component comprising SEQ ID NO: 25 and a rod-specific promoter-derived component comprising SEQ ID NO: 10. Some particular promoters may comprise, 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO: 10 and a ProA7-derived component comprising SEQ ID NO: 25.

[0138] Some particular promoters may contain, from 5' to 3', a ProA7-derived element comprising SEQ ID NO: 26 and a rod-specific promoter-derived element comprising SEQ ID NO: 10. One such particular promoter, which is a preferred promoter, is designated Pro572.2 (SEQ ID NO: 12).

[0139] In some embodiments, the promoter may comprise, from 5' to 3', a rod-specific promoter-derived element comprising SEQ ID NO: 10 and a ProA7-derived element comprising SEQ ID NO: 26. A particular such promoter that is also preferred is designated Pro573.2 (SEQ ID NO: 14).

[0140] Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:27 and a rod-specific promoter-derived component comprising SEQ ID NO:10. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:10 and a ProA7-derived component comprising SEQ ID NO:27. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:2 and a rod-specific promoter-derived component comprising SEQ ID NO:36. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:36 and a ProA7-derived component comprising SEQ ID NO:2. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:2 and a rod-specific promoter-derived component comprising SEQ ID NO:37. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:37 and a ProA7-derived component comprising SEQ ID NO:2. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:2 and a rod-specific promoter-derived component comprising SEQ ID NO:38. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:38 and a ProA7-derived component comprising SEQ ID NO:2. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:2 and a rod-specific promoter-derived component comprising SEQ ID NO:39. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:39 and a ProA7-derived component comprising SEQ ID NO:2. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:2 and a rod-specific promoter-derived component comprising SEQ ID NO:40. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:40 and a ProA7-derived component comprising SEQ ID NO:2. Some particular promoters may contain, from 5' to 3', a ProA7-derived element comprising SEQ ID NO:2 and a rod-specific promoter-derived element comprising SEQ ID NO:41.Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:41 and a ProA7-derived component comprising SEQ ID NO:2. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:2 and a rod-specific promoter-derived component comprising SEQ ID NO:45. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:45 and a ProA7-derived component comprising SEQ ID NO:24. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:2 and a rod-specific promoter-derived component comprising SEQ ID NO:36. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:36 and a ProA7-derived component comprising SEQ ID NO:24. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:24 and a rod-specific promoter-derived component comprising SEQ ID NO:37. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:37 and a ProA7-derived component comprising SEQ ID NO:24. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:24 and a rod-specific promoter-derived component comprising SEQ ID NO:38. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:38 and a ProA7-derived component comprising SEQ ID NO:24. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:24 and a rod-specific promoter-derived component comprising SEQ ID NO:39. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:39 and a ProA7-derived component comprising SEQ ID NO:24. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:24 and a rod-specific promoter-derived component comprising SEQ ID NO:40. Some particular promoters may contain, from 5' to 3', a rod-specific promoter-derived element comprising SEQ ID NO:40 and a ProA7-derived element comprising SEQ ID NO:24.Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:24 and a rod-specific promoter-derived component comprising SEQ ID NO:41. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:41 and a ProA7-derived component comprising SEQ ID NO:24. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:24 and a rod-specific promoter-derived component comprising SEQ ID NO:45. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:45 and a ProA7-derived component comprising SEQ ID NO:24. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:25 and a rod-specific promoter-derived component comprising SEQ ID NO:36. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:36 and a ProA7-derived component comprising SEQ ID NO:25. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:25 and a rod-specific promoter-derived component comprising SEQ ID NO:37. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:37 and a ProA7-derived component comprising SEQ ID NO:25. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:25 and a rod-specific promoter-derived component comprising SEQ ID NO:38. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:38 and a ProA7-derived component comprising SEQ ID NO:25. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:25 and a rod-specific promoter-derived component comprising SEQ ID NO:39. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:39 and a ProA7-derived component comprising SEQ ID NO:25. Some particular promoters may contain, from 5' to 3', a ProA7-derived element comprising SEQ ID NO:25 and a rod-specific promoter-derived element comprising SEQ ID NO:40.Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:40 and a ProA7-derived component comprising SEQ ID NO:25. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:25 and a rod-specific promoter-derived component comprising SEQ ID NO:41. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:41 and a ProA7-derived component comprising SEQ ID NO:25. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:25 and a rod-specific promoter-derived component comprising SEQ ID NO:45. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:45 and a ProA7-derived component comprising SEQ ID NO:25. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:26 and a rod-specific promoter-derived component comprising SEQ ID NO:36. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:36 and a ProA7-derived component comprising SEQ ID NO:26. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:26 and a rod-specific promoter-derived component comprising SEQ ID NO:37. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:37 and a ProA7-derived component comprising SEQ ID NO:26. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:26 and a rod-specific promoter-derived component comprising SEQ ID NO:38. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:38 and a ProA7-derived component comprising SEQ ID NO:26. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:26 and a rod-specific promoter-derived component comprising SEQ ID NO:39. Some particular promoters may contain, from 5' to 3', a rod-specific promoter-derived element comprising SEQ ID NO:39 and a ProA7-derived element comprising SEQ ID NO:26.Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:26 and a rod-specific promoter-derived component comprising SEQ ID NO:40. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:40 and a ProA7-derived component comprising SEQ ID NO:26. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:26 and a rod-specific promoter-derived component comprising SEQ ID NO:41. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:41 and a ProA7-derived component comprising SEQ ID NO:26. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:26 and a rod-specific promoter-derived component comprising SEQ ID NO:45. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:45 and a ProA7-derived component comprising SEQ ID NO:26. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO: 27 and a rod-specific promoter-derived component comprising SEQ ID NO: 36. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO: 36 and a ProA7-derived component comprising SEQ ID NO: 27. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO: 27 and a rod-specific promoter-derived component comprising SEQ ID NO: 37. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO: 37 and a ProA7-derived component comprising SEQ ID NO: 27. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:27 and a rod-specific promoter-derived component comprising SEQ ID NO:38. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:38 and a ProA7-derived component comprising SEQ ID NO:27. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:27 and a rod-specific promoter-derived component comprising SEQ ID NO:39. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:39 and a ProA7-derived component comprising SEQ ID NO:27. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:27 and a rod-specific promoter-derived component comprising SEQ ID NO:40. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:40 and a ProA7-derived component comprising SEQ ID NO:27. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:27 and a rod-specific promoter-derived component comprising SEQ ID NO:41. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:41 and a ProA7-derived component comprising SEQ ID NO:27. Some particular promoters may comprise, from 5' to 3', a ProA7-derived component comprising SEQ ID NO:27 and a rod-specific promoter-derived component comprising SEQ ID NO:45. Some particular promoters may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO:45 and a ProA7-derived component comprising SEQ ID NO:27.

[0141] The hybrid promoter may comprise, from 5' to 3', a rod-specific promoter-derived component comprising SEQ ID NO: 54 or a functional variant thereof, and a ProSC-derived component comprising SEQ ID NO: 26 or a functional variant thereof. Alternatively, the hybrid promoter may comprise, from 5' to 3', a ProSC-derived component comprising SEQ ID NO: 26 or a functional variant thereof, and a rod-specific promoter-derived component comprising SEQ ID NO: 54 or a functional variant thereof.

[0142] In certain preferred embodiments, the hybrid promoter contains about 700 or fewer nucleotides, about 600 or fewer nucleotides, or more preferably, about 500 or fewer nucleotides.

[0143] B. Optogenetic Proteins As disclosed herein, the nucleic acid comprises a nucleotide sequence encoding a depolarizing optogenetics protein. The depolarizing optogenetics protein can be expressed in human cone cells transduced with the nucleic acid and localized to the cell membrane of the human cone cells. When exposed to light, the depolarizing optogenetics protein can mediate a depolarizing current that can depolarize human cone cells. The depolarization of human cone cells can induce light-driven current spikes in RGCs.

[0144] Depolarizing optogenetic proteins can be activated or excited by any desired type of light, such as green light, red light, blue light, violet light, or yellow light. The light can have a wavelength of about 400 nm to about 700 nm. For example, the light can have a wavelength of about 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, or 700 nm.

[0145] Human cone cells containing the depolarizing optogenetic proteins described herein can have a resting potential of about -30 mV to -35 mV. In response to activation (e.g., light stimulation), the depolarizing optogenetic proteins can generate a voltage (i.e., an action potential) of at least about -40 mV to about -55 mV or more across the plasma membrane of human cone cells. Human cone cells can be depolarized by at least about 1 mV to about 20 mV or more. For example, human pyramidal cells can be depolarized by at least about 1 mV to about 2 mV, about 1 mV to about 3 mV, about 1 mV to about 4 mV, about 1 mV to about 5 mV, about 1 mV to about 6 mV, about 1 mV to about 7 mV, about 1 mV to about 8 mV, about 1 mV to about 9 mV, about 1 mV to about 10 mV, about 1 mV to about 11 mV, about 1 mV to about 12 mV, about 1 mV to about 13 mV, about 1 mV to about 14 mV, about 1 mV to about 19 mV, or about 1 mV to about 20 mV.

[0146] Preferably, the depolarizing optogenetic protein is capable of generating a voltage across the plasma membrane of human pyramidal cells that is sufficient to induce the firing of action potentials that can induce light-driven ganglion cell spiking.

[0147] Depolarizing optogenetic proteins can mediate depolarizing currents that are about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, or greater than other light-activated ion-gated channel proteins.

[0148] The depolarizing optogenetic protein is preferably a light-gated ion channel protein.Suitable light-gated ion channel proteins include, but are not limited to, channelrhodopsin (e.g., channelrhodopsin-1 (ChR1), channelrhodopsin 2 (ChR2)), its variant, or a combination thereof.Channelrhodopsin can be ChrMine polypeptide, Chrimson polypeptide, ReaChR polypeptide, their variant, or a combination thereof.

[0149] Examples of depolarizing optogenetic proteins that may be suitable for the optogenetic constructs disclosed herein include ChrimsonR, ReaChR, ChrMine, fChrimson, and vfChrimson. A preferred depolarizing optogenetic protein is ReaChR.

[0150] The amino acid sequence of ReaChR is provided as SEQ ID NO: 32. Nucleotide sequences encoding ReaChR are provided in SEQ ID NO: 16, SEQ ID NO: 33, and SEQ ID NO: 34. SEQ ID NO: 33 and SEQ ID NO: 34 are examples of nucleic acids encoding ReaChR that have been codon-optimized for expression in human cells.

[0151] Other depolarizing optogenetic proteins that may be compatible with the optogenetic constructs disclosed herein include, but are not limited to, ChR2, ChETA, SFO, VChR1, Chronos, PsChR2, CoChR, CsChR, CheRiff, C1C2, and C1V1.

[0152] The depolarizing optogenetic protein can be a functional variant of a light-gated ion channel protein, for example, the depolarizing optogenetic protein can be a functional variant of ChrimsonR, ReaChR, ChrMine, fChrimson, or vfChrimson.

[0153] A functional variant may differ from a light-gated ion channel protein by one or several amino acids (including substitutions, deletions, insertions, or any combination thereof) and substantially retain the ability to mediate a depolarizing current that depolarizes human cone cells upon exposure to light. A functional variant may contain at least one or more amino acid substitutions, deletions, or insertions compared to a light-gated ion channel polypeptide. A functional variant may 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 amino acid changes compared to a light-gated ion channel protein. The amino acid substitutions may be conservative or non-conservative, but are preferably conservative. "Conservative" amino acid substitutions, as used herein, generally refer to the substitution of one amino acid residue with another amino acid residue within a recognized group, which may alter the structure of the peptide but substantially retain the biological activity of the peptide. Conservative amino acid substitutions are known to those skilled in the art. Conservative amino acid substitutions may include, but are not limited to, substitutions between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. For example, one skilled in the art would reasonably expect that a single substitution of leucine with isoleucine or valine, a single substitution of aspartic acid with glutamic acid, a single substitution of threonine with serine, or a similar substitution of an amino acid with a structurally related amino acid, would not significantly affect the biological activity of the resulting molecule.

[0154] The nucleotide sequence of coding depolarizing optogenetics protein can be modified to optimize codon usage according to the organism, cell or organoid that wants to express depolarizing optogenetics protein by any suitable method.Some methods and algorithms for codon optimization are conventional and well known to those skilled in the art.The codon optimization of the nucleotide sequence of coding depolarizing optogenetics protein can increase the translation rate of depolarizing optogenetics protein. Depolarizing optogenetic proteins can be engineered to include one or more membrane-localization sequence(s), such as those of other membrane-associated proteins (e.g., other optogenetic proteins). Without wishing to be bound by any particular mechanism or theory, it is believed that such engineered depolarizing optogenetic proteins can enhance membrane localization. As an illustrative and non-limiting example, the transmembrane domain of ReaChR can be inserted into another depolarizing optogenetic protein, for example, replacing the transmembrane domain of the other depolarizing optogenetic protein to provide the other depolarizing optogenetic protein with an additional membrane-localization sequence. In another example, the membrane-localization sequence of ReaChR (SEQ ID NO: 57) can be added to a depolarizing optogenetic protein that does not contain it, or can replace the membrane-localization sequence of the desired depolarizing optogenetic protein. For example, ChRMine, Chrimson, Jaws, HcKCR1, or eGTACR1 can be engineered to improve membrane localization by replacing the N-terminus with the corresponding membrane localization sequence (SEQ ID NO: 57) of ReaChr. In particular, optogenetic proteins such as depolarizing channelrhodopsin can be engineered to improve membrane localization by replacing the N-terminus of channelrhodopsin (e.g., ChR1) with (SEQ ID NO: 57). (See, for example, Lin et al., Biophysical Journal, 96: 1803-1814 (2009); Lin et al., Nat. Neurosci. 16 (10): 1499-1508 (2013)).

[0155] C. Reporter Molecules The nucleic acid can include a nucleotide sequence encoding a reporter molecule, if desired. If present, the optional reporter molecule may be operably linked to the nucleotide sequence encoding the depolarizing optogenetics protein. The reporter molecule may be detectable in human cone cells. The depolarizing optogenetics protein and the reporter molecule may be components of a fusion protein, with the reporter molecule optionally located at the C-terminus or N-terminus of the depolarizing optogenetics protein. The depolarizing optogenetics protein and the reporter molecule may be fused directly to each other, or indirectly, for example, via a suitable linker sequence. For example, the reporter may be fused directly to the C-terminus or N-terminus of the depolarizing optogenetics protein, or indirectly via a suitable linker peptide. Without being bound by theory, the reporter molecule may enhance membrane localization of the optogenetics construct. Suitable reporter molecules include, for example, tdTomato, enhanced yellow fluorescent protein (EYFP), Citrine, green fluorescent protein (GFP), cyan fluorescent protein, red fluorescent protein, or functional variants thereof. A preferred reporter molecule is Citrine.

[0156] Without wishing to be bound by any particular theoretical mechanism, it is believed that the inclusion of a reporter molecule operably linked to the depolarizing optogenetic protein may enhance membrane localization of the depolarizing optogenetic protein, even though the depolarizing optogenetic protein itself is naturally membrane-associated.

[0157] D. PRE As disclosed herein, the nucleic acid comprises a nucleotide sequence encoding a PRE (preferably a WPRE). A PRE is a nucleic acid sequence that contributes to regulating the expression of the DNS sequence in which it resides. A PRE may optionally comprise three components (alpha, beta, and gamma). The activity of a PRE may depend on the number of components present.

[0158] As disclosed and exemplified herein, a promoter such as the ProA7 promoter, a variant, fragment, or truncated version thereof, or a hybrid promoter comprising ProA7-derived components and rod-specific promoter-derived components, and a WPRE element in combination with a depolarizing optogenetic protein can result in high levels of expression of the depolarizing optogenetic protein in human cone cells.

[0159] As disclosed above, the present inventors have surprisingly discovered that the ProA7 promoter in combination with a post-transcriptional regulatory element (PRE), preferably the woodchuck hepatitis virus PRE (WPRE), can drive high expression levels of depolarizing optogenetic proteins in human photoreceptor cone cells.

[0160] The WPRE may be operably linked to nucleotides encoding a depolarizing optogenetic protein and other expression control elements, such as a promoter and PolyA signal as described herein.

[0161] Any suitable WPRE can be used, such as a naturally occurring WPRE or a WPRE that contains 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.

[0162] Suitable WPREs are described in U.S. Publication No. US2021 / 0032656. Generally, a WPRE may have a nucleotide sequence comprising SEQ ID NO:3. A WPRE may comprise a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:3. A WPRE may 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:3.

[0163] 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.

[0164] The WPRE may have a nucleotide sequence comprising SEQ ID NO: 8. The WPRE may comprise a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 3. The WPRE may 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: 8.

[0165] 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.

[0166] Preferably, the WPRE may have a nucleotide sequence comprising SEQ ID NO: 3. The WPRE may comprise a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 3. The WPRE may 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:86.

[0167] 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.

[0168] E. Polyadenylation Signal and Inverted Terminal Repeats The nucleic acid may also contain a nucleotide sequence encoding a suitable PolyA 3' to the WPRE. 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. The human growth hormone (hGH) PolyA signal is a preferred PolyA.

[0169] The PolyA can be in any suitable position, but preferably the PolyA is 3' to the WPRE.

[0170] 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.

[0171] PolyA can have a nucleotide sequence comprising SEQ ID NO: 87. PolyA can comprise a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 87. PolyA 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: 87.

[0172] 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.

[0173] The nucleic acid may further comprise an AAV inverted terminal repeat (ITR). For example, an AAV ITR 5' to the promoter and an AAV ITR 3' to the WPRE or 3' to PolyA. Specific examples of AAV ITRs are SEQ ID NO: 15 and SEQ ID NO: 31. The ITRs can be independently selected from wild-type ITRs and, optionally, self-complementary (scAAV) ITRs. Other sequences functionally equivalent to the AAV 5'ITR and / or AAV 3'ITR (e.g., parvovirus terminal repeats) can also be used.

[0174] F. Adeno-Associated Virus Vector Capsid The present disclosure also relates to a viral vector comprising the nucleic acid disclosed herein.The viral vector can be the AAV vector comprising AAV capsid.Without being bound by theory, AAV capsid may improve the selective delivery of nucleic acid to cone cells, and may also improve the expression of optogenetic constructs (i.e., nucleic acid).

[0175] 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 and variant capsids are also well known in the art.

[0176] Exemplary AAV capsids suitable for the optogenetic constructs disclosed herein 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.

[0177] 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.

[0178] 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 natural or non-natural 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.

[0179] G. Combining Elements As disclosed herein, the inventors have discovered that a preferred combination of an expression control element, a specific depolarizing optogenetic protein, and optionally a reporter molecule results in high-level and selective expression of the depolarizing optogenetic protein in human cone cells, sufficient to restore light sensitivity to human cone cells, particularly those that are unresponsive to light. A preferred expression control element for the nucleic acid disclosed herein encoding a depolarizing optogenetic protein is a promoter comprising ProA7, or a variant, fragment, or truncation thereof, or a hybrid promoter comprising ProA7-derived components and rod-specific promoter-derived components, and a WPRE. The optogenetic constructs disclosed herein typically further comprise a suitable polyadenylation signal (PolyA) 3' to the WPRE. The optogenetic construct may further comprise one or more AAV inverted terminal repeats (ITRs). The ITRs may be 5' to the promoter and / or 3' to the WPRE or PolyA (if PolyA is present). Modified ITRs, such as self-complementary ITRs, or other sequences that are functionally equivalent to the AAV 5'ITR and / or AAV 3'ITR (e.g., parvovirus terminal repeats), can also be used in the optogenetic constructs.

[0180] The optogenetic construct may contain a single copy or multiple copies of a promoter or any of its elements in a hybrid promoter. For example, the construct may contain multiple copies of ProA7 or a variant, fragment, or truncated form thereof, multiple copies of ProA330 or a variant, fragment, or truncated form thereof, or a hybrid promoter containing Pro-A7-derived components and rod-specific promoter-derived components (e.g., ProA330-derived components), and a WPRE. In one embodiment, the construct may contain, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the ProA7 promoter or a variant, fragment, or truncated form thereof. In another embodiment, the construct may contain, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the ProA330 promoter or a variant, fragment, or truncated form thereof. In some embodiments, the construct may contain, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of a hybrid promoter comprising a Pro-A7-derived component and a cone- or rod-specific promoter-derived component. Each hybrid promoter comprising a Pro-A7-derived component, a cone- or rod-specific promoter-derived component, and optionally any other rod- or cone-specific promoter sequence may be included as a single copy or multiple copies. When a promoter comprising ProA7, or a variant, fragment, or truncated form thereof, ProA330, or a variant, fragment, or truncated form thereof, or a hybrid promoter comprising a Pro-A7-derived component and a rod-specific promoter-derived component is included, the two or more promoter sequences do not need to be identical. The multiple promoter copies may also be located in any position. Multiple copies of a promoter comprising ProA7, or a variant, fragment, or truncation thereof, Pro330, or a variant, fragment, or truncation thereof, or a hybrid promoter comprising Pro-A7-derived elements and rod-specific promoter-derived elements may be included in any order.Certain combinations of depolarizing optogenetic proteins and optional reporter molecules can improve expression and function in human cone cells, particularly in human cone cells that do not respond to light. For example, the depolarizing optogenetic protein can be ChrimsonR and the reporter molecule can be tdTomato. For example, the depolarizing optogenetic protein can be ChrimsonR and the reporter molecule can be EYFP. For example, the depolarizing optogenetic protein can be ReaChR and the reporter molecule can be Citrine. For example, the depolarizing optogenetic protein can be ChrMine and the reporter molecule can be EYFP. For example, the depolarizing optogenetic protein can be fChrimson and the reporter molecule can be TdTomato. For example, the depolarizing optogenetic protein can be vfChrimson and the reporter molecule can be TdTomato.

[0181] A preferred depolarizing optogenetic protein is ReaChr, and if present, a preferred reporter molecule is Citrine.

[0182] The optogenetic construct can be an AAV vector comprising a nucleic acid disclosed herein and an AAV capsid.

[0183] Exemplary optogenetic constructs (i.e., AAV vectors) comprising a depolarizing optogenetic protein, an optional and optional reporter molecule, and an AAV capsid protein are described in Table 1 below.

[0184] [Table 1]

[0185] The optogenetic construct (i.e., AAV vector) may comprise a nucleic acid comprising a promoter, ChrimsonR as a depolarizing optogenetic protein, a WPRE, and a PolyA signal. The nucleic acid may further comprise tdTomato as a reporter molecule. The nucleic acid may further comprise an AAV ITR 5' to the promoter and an AAV ITR 3' to the PolyA signal. The optogenetic construct may further comprise an AAV capsid, if desired. The nucleic acid constituting the ChrimsonR depolarizing optogenetic protein may comprise a promoter having a nucleotide sequence having SEQ ID NO:2. The nucleic acid constituting the ChrimsonR depolarizing optogenetic construct may comprise a WPRE having a nucleotide sequence having SEQ ID NO:3.

[0186] The optogenetic construct (i.e., AAV vector) may comprise a nucleic acid comprising a promoter, ChrimsonR as a depolarizing optogenetic protein, a WPRE, and a PolyA signal. The nucleic acid may further comprise tdTomato as a reporter molecule. The nucleic acid may further comprise an AAV ITR 5' to the promoter and an AAV ITR 3' to the PolyA signal. The optogenetic construct may further comprise an AAV capsid, if desired. The nucleic acid constituting the ChrimsonR depolarizing optogenetic protein may comprise a promoter having a nucleotide sequence having SEQ ID NO:2. The nucleic acid constituting the ChrimsonR depolarizing optogenetic construct may comprise a WPRE having a nucleotide sequence having SEQ ID NO:86.

[0187] The optogenetic constructs disclosed herein may comprise a nucleic acid sequence having at least 70% identity to SEQ ID NO:4, and may comprise an AAV capsid protein selected from AAV-BP2 capsid protein, AAV-PHP.B capsid protein, AAV-PHP.eB capsid protein, or AAV-NH26 capsid protein. For example, the nucleic acid may comprise nucleotides having at least 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 more sequence identity to SEQ ID NO:4.

[0188] The optogenetic construct (i.e., AAV) may comprise a nucleic acid comprising a ProA7 promoter, vfChrimsonR as a depolarizing optogenetic protein, a WPRE, and a PolyA signal. The optogenetic construct may further comprise EYFP as a reporter molecule. The nucleic acid may further comprise an AAV ITR 5' to the ProA7 promoter and an AAV ITR 3' to the PolyA signal. The optogenetic construct may further comprise an AAV capsid, if desired. An optogenetic construct comprising a vfChrimsonR depolarizing optogenetic construct may comprise a ProA7 promoter having a nucleotide sequence of SEQ ID NO:2. An optogenetic construct comprising a vfChrimsonR depolarizing optogenetic protein may comprise a WPRE having a nucleotide sequence of SEQ ID NO:3. An optogenetic construct comprising a vfChrimsonR depolarizing optogenetic protein may comprise a WPRE having a nucleotide sequence of SEQ ID NO:86.

[0189] The optogenetic constructs disclosed herein may comprise a nucleic acid sequence having at least 70% identity to SEQ ID NO: 5 and may comprise an AAV capsid protein. For example, the nucleic acid may comprise nucleotides having at least 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 more sequence identity to SEQ ID NO: 5.

[0190] The optogenetic construct (i.e., AAV) may comprise a nucleic acid comprising a ProA7 promoter, ChrMine as a depolarizing optogenetic protein, a WPRE, and a PolyA signal. The optogenetic construct may further comprise EYFP as a reporter molecule. The nucleic acid may further comprise an AAV ITR 5' to the ProA7 promoter and an AAV ITR 3' to the PolyA signal. The optogenetic construct may further comprise an AAV capsid protein, if desired. An optogenetic construct comprising a ChrMine depolarizing optogenetic protein may comprise a ProA7 promoter having a nucleotide sequence of SEQ ID NO:2. An optogenetic construct comprising a ChrMine depolarizing optogenetic construct may comprise a WPRE having a nucleotide sequence of SEQ ID NO:3. An optogenetic construct comprising a ChrMine depolarizing optogenetic construct may comprise a WPRE having a nucleotide sequence of SEQ ID NO:86.

[0191] The optogenetic constructs disclosed herein may comprise a nucleic acid sequence having at least 70% identity to SEQ ID NO:6, and may comprise an AAV capsid protein selected from AAV8-BP2 capsid protein, AAV-PHP.B capsid protein, AAV-PHP.eB capsid protein, or AAV-NH26 capsid protein. For example, the nucleic acid may comprise nucleotides having at least 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 more sequence identity to SEQ ID NO:6.

[0192] Specific optogenetic constructs include constructs containing a ProA7 promoter or a variant, fragment, or truncation thereof, or a hybrid promoter containing ProA7-derived components and rod-specific promoter-derived components, ReaChR as a depolarizing optogenetic protein, EYFP or mCitrine as a reporter molecule, and a nucleic acid containing WPRE. The optogenetic vector may further contain an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid (Byrne et al., JCI Insight. 2020;5(10):e135112. https: / / doi.org / 10.1172 / jci.insight.135112), an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid. Any of these capsids can be further optimized by sequence modification and screening, if desired.

[0193] A preferred optogenetic construct (i.e., AAV) comprises a nucleic acid comprising a ProA7 promoter, ReaChR as a depolarizing optogenetic protein, a WPRE, and an hGH PolyA signal. The optogenetic construct may further comprise Citrine as a reporter molecule. The nucleic acid may further comprise an AAV ITR 5' to the ProA7 promoter and an AAV ITR 3' to the PolyA signal. The optogenetic construct may further comprise an AAV capsid protein, if desired. An optogenetic construct comprising a ReaChr depolarizing optogenetic protein may comprise a ProA7 promoter having a nucleotide sequence of SEQ ID NO:2. An optogenetic construct comprising a ReaChr depolarizing optogenetic construct may comprise a WPRE having a nucleotide sequence of SEQ ID NO:3. An optogenetic construct comprising a ReaChr depolarizing optogenetic construct may comprise a WPRE having a nucleotide sequence of SEQ ID NO:86.

[0194] The optogenetic constructs disclosed herein may comprise a nucleic acid sequence having at least 70% identity to SEQ ID NO: 7. For example, the nucleic acid may comprise nucleotides having at least 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 more sequence identity to SEQ ID NO: 7. An optogenetic construct comprising a nucleic acid sequence having at least 70% identity to SEQ ID NO: 7 may further comprise an AAV capsid protein selected from AAV-PHP.eB capsid protein, AAV8-BP2 capsid protein, or AAV5 capsid protein. The optogenetic constructs disclosed herein may comprise a nucleic acid sequence having at least 70% identity to SEQ ID NO: 7 and may comprise an AAV capsid protein selected from AAV-PHP.eB capsid protein. The optogenetic constructs disclosed herein may comprise a nucleic acid sequence having at least 70% identity to SEQ ID NO: 7 and may comprise an AAV capsid protein selected from the AAV8-BP2 capsid protein. The optogenetic constructs disclosed herein may comprise a nucleic acid sequence having at least 70% identity to SEQ ID NO: 7 and may comprise an AAV capsid protein selected from the AAV5 capsid protein.

[0195] Certain optogenetic constructs include a nucleic acid described herein, wherein the promoter comprises SEQ ID NO: 2, the depolarizing optogenetic protein is ReaChR, the optional reporter molecule is absent, PolyA is present, and having SEQ ID NO: 9. The nucleic acid may comprise SEQ ID NO: 17. The AAV vector may comprise SEQ ID NO: 17 and a suitable capsid, such as an AAV5 capsid.

[0196] Certain optogenetic constructs include a nucleic acid described herein, wherein the promoter comprises SEQ ID NO: 14, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO: 16 or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO: 16, the optional reporter molecule is absent, PolyA is present, and having SEQ ID NO: 9. The nucleic acid may comprise SEQ ID NO: 18. The AAV vector may comprise SEQ ID NO: 18 and a suitable capsid, such as an AAV5 capsid.

[0197] Certain optogenetic constructs include a nucleic acid described herein, wherein the promoter comprises SEQ ID NO: 12, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO: 16 or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO: 16, the optional reporter molecule is absent, PolyA is present, and has SEQ ID NO: 9. The nucleic acid may comprise SEQ ID NO: 19. The AAV vector may comprise SEQ ID NO: 19 and a suitable capsid, such as an AAV5 capsid.

[0198] Particular optogenetic constructs include a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:22, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16 or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, the optional reporter molecule is absent, PolyA is present, and has SEQ ID NO:9. The nucleic acid may comprise SEQ ID NO:20. The AAV vector may comprise SEQ ID NO:20 and a suitable capsid, such as an AAV5 capsid.

[0199] Certain optogenetic constructs are described herein and include a nucleic acid wherein the promoter comprises SEQ ID NO:23, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16 or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, the optional reporter molecule is absent, PolyA is present, and having SEQ ID NO:9. The nucleic acid may comprise SEQ ID NO:21. The AAV vector may comprise SEQ ID NO:21 and a suitable capsid, such as an AAV5 capsid.

[0200] Particular optogenetic constructs include a nucleic acid described herein, wherein the promoter comprises SEQ ID NO: 2, the depolarizing optogenetic protein is ReaChR, the optional reporter molecule is absent, PolyA is present, and having SEQ ID NO: 87. The nucleic acid may comprise SEQ ID NO: 17. The AAV vector may comprise SEQ ID NO: 17 and a suitable capsid, such as an AAV5 capsid.

[0201] Particular optogenetic constructs include a nucleic acid described herein, wherein the promoter comprises SEQ ID NO: 14, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO: 16 or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO: 16, the optional reporter molecule is absent, PolyA is present, and having SEQ ID NO: 87. The nucleic acid may comprise SEQ ID NO: 18. The AAV vector may comprise SEQ ID NO: 18 and a suitable capsid, such as an AAV5 capsid.

[0202] Particular optogenetic constructs include a nucleic acid described herein, wherein the promoter comprises SEQ ID NO: 12, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO: 16 or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO: 16, the optional reporter molecule is absent, PolyA is present, and having SEQ ID NO: 87. The nucleic acid may comprise SEQ ID NO: 19. The AAV vector may comprise SEQ ID NO: 19 and a suitable capsid, such as an AAV5 capsid.

[0203] Particular optogenetic constructs include a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:22, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16 or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, the optional reporter molecule is absent, PolyA is present, and has SEQ ID NO:87. The nucleic acid may comprise SEQ ID NO:20. The AAV vector may comprise SEQ ID NO:20 and a suitable capsid, such as an AAV5 capsid.

[0204] Certain optogenetic constructs include a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:23, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16 or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, the optional reporter molecule is absent, PolyA is present, and has SEQ ID NO:87. The nucleic acid may comprise SEQ ID NO:21. The AAV vector may comprise SEQ ID NO:21 and a suitable capsid, such as an AAV5 capsid.

[0205] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO: 2, WPRE, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO: 16, SEQ ID NO: 33, or SEQ ID NO: 34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO: 16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO: 9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0206] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:2, WPRE operably linked to SEQ ID NO:10, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0207] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:2, WPRE operably linked to SEQ ID NO:36, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0208] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:2, WPRE operably linked to SEQ ID NO:37, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0209] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:2, WPRE operably linked to SEQ ID NO:38, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0210] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:2, WPRE operably linked to SEQ ID NO:39, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0211] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:2, WPRE operably linked to SEQ ID NO:40, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0212] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:2, WPRE operably linked to SEQ ID NO:41, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0213] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:2, WPRE operably linked to SEQ ID NO:45, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0214] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:24, WPRE, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9, and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0215] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:24, WPRE operably linked to SEQ ID NO:10, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0216] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:24, WPRE operably linked to SEQ ID NO:36, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0217] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:24, WPRE operably linked to SEQ ID NO:37, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0218] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:24, WPRE operably linked to SEQ ID NO:38, the depolarizing optogenetic protein is ReaChR encoded by a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0219] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:24, WPRE operably linked to SEQ ID NO:39, the depolarizing optogenetic protein is ReaChR encoded by a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0220] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:24, WPRE operably linked to SEQ ID NO:40, the depolarizing optogenetic protein is ReaChR encoded by a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0221] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:24, WPRE operably linked to SEQ ID NO:41, the depolarizing optogenetic protein is ReaChR encoded by a nucleotide sequence encoding SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0222] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:24, WPRE operably linked to SEQ ID NO:45, the depolarizing optogenetic protein is ReaChR encoded by a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0223] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO: 25, WPRE, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO: 16, SEQ ID NO: 33, or SEQ ID NO: 34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO: 16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO: 9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0224] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:25, WPRE operably linked to SEQ ID NO:10, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0225] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:25, WPRE operably linked to SEQ ID NO:36, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0226] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:25, WPRE operably linked to SEQ ID NO:37, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0227] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:25, WPRE operably linked to SEQ ID NO:38, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0228] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:25, WPRE operably linked to SEQ ID NO:39, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0229] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:25, WPRE operably linked to SEQ ID NO:40, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0230] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:25, WPRE operably linked to SEQ ID NO:41, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0231] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:25, WPRE operably linked to SEQ ID NO:45, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0232] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO: 26, WPRE, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO: 16, SEQ ID NO: 33, or SEQ ID NO: 34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO: 16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO: 9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0233] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:26, WPRE operably linked to SEQ ID NO:10, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0234] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:26, WPRE operably linked to SEQ ID NO:36, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0235] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:26, WPRE operably linked to SEQ ID NO:37, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0236] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:26, WPRE operably linked to SEQ ID NO:38, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0237] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:26, WPRE operably linked to SEQ ID NO:39, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0238] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:26, WPRE operably linked to SEQ ID NO:40, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0239] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:26, WPRE operably linked to SEQ ID NO:41, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0240] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:26, WPRE operably linked to SEQ ID NO:45, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0241] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:27, WPRE, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9, and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0242] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:27, WPRE operably linked to SEQ ID NO:10, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0243] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:27, WPRE operably linked to SEQ ID NO:36, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0244] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:27, WPRE operably linked to SEQ ID NO:37, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0245] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:27, WPRE operably linked to SEQ ID NO:38, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0246] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:27, WPRE operably linked to SEQ ID NO:39, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0247] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:27, WPRE operably linked to SEQ ID NO:40, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0248] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:27, WPRE operably linked to SEQ ID NO:41, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0249] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:27, WPRE operably linked to SEQ ID NO:45, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0250] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:2, WPRE operably linked to SEQ ID NO:45, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0251] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:11, WPRE operably linked to SEQ ID NO:45, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0252] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO: 12, WPRE operably linked to SEQ ID NO: 45, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO: 16, SEQ ID NO: 33, or SEQ ID NO: 34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO: 16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO: 9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0253] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO: 13, WPRE operably linked to SEQ ID NO: 45, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO: 16, SEQ ID NO: 33, or SEQ ID NO: 34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO: 16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO: 9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0254] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO: 14, WPRE operably linked to SEQ ID NO: 45, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO: 16, SEQ ID NO: 33, or SEQ ID NO: 34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO: 16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO: 9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0255] A particular optogenetic construct comprises a nucleic acid described herein, wherein the promoter comprises SEQ ID NO:26, WPRE operably linked to SEQ ID NO:45, the depolarizing optogenetic protein is ReaChR encoded by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:34, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or an optogenetic protein engineered to contain a ReaChR transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and has SEQ ID NO:9; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0256] Certain optogenetic constructs include nucleic acids described herein, wherein the promoter comprises SEQ ID NO:27, WPRE, and the depolarizing optogenetic protein is SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:73, or ReaChR encoded by a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or Re The invention comprises an optogenetic protein engineered to contain an aChR transmembrane domain, a nucleic acid in which the reporter molecule is absent, PolyA is present, and the nucleic acid has SEQ ID NO: 9, and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0257] Certain optogenetic constructs include nucleic acids described herein, wherein the promoter comprises SEQ ID NO:2, WPRE, and the depolarizing optogenetic protein is SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:73, or ReaChR encoded by a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or Re The invention comprises an optogenetic protein engineered to contain an aChR transmembrane domain, a nucleic acid in which the reporter molecule is absent, PolyA is present, and the nucleic acid has SEQ ID NO: 9, and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0258] Certain optogenetic constructs include nucleic acids described herein, wherein the promoter comprises SEQ ID NO:11, WPRE, and the depolarizing optogenetic protein is SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:73, or ReaChR encoded by a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or Re The invention comprises an optogenetic protein engineered to contain an aChR transmembrane domain, a nucleic acid in which the reporter molecule is absent, PolyA is present, and the nucleic acid has SEQ ID NO: 9, and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0259] Certain optogenetic constructs include nucleic acids described herein, wherein the promoter comprises SEQ ID NO:12, WPRE, and the depolarizing optogenetic protein is SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:73, or ReaChR encoded by a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or Re The invention comprises an optogenetic protein engineered to contain an aChR transmembrane domain, a nucleic acid in which the reporter molecule is absent, PolyA is present, and the nucleic acid has SEQ ID NO: 9, and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0260] Certain optogenetic constructs include nucleic acids described herein, wherein the promoter comprises SEQ ID NO:13, WPRE, and the depolarizing optogenetic protein is SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:73, or ReaChR encoded by a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or Re The invention comprises an optogenetic protein engineered to contain an aChR transmembrane domain, a nucleic acid in which the reporter molecule is absent, PolyA is present, and the nucleic acid has SEQ ID NO: 9, and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0261] Certain optogenetic constructs include nucleic acids described herein, wherein the promoter comprises SEQ ID NO:14, WPRE, and the depolarizing optogenetic protein is SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:73, or ReaChR encoded by a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or Re The invention comprises an optogenetic protein engineered to contain an aChR transmembrane domain, a nucleic acid in which the reporter molecule is absent, PolyA is present, and the nucleic acid has SEQ ID NO: 9, and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0262] Certain optogenetic constructs include nucleic acids described herein, wherein the promoter comprises SEQ ID NO:26, WPRE, and the depolarizing optogenetic protein is SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:73, or ReaChR encoded by a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16, or Re The invention comprises an optogenetic protein engineered to contain an aChR transmembrane domain, a nucleic acid in which the reporter molecule is absent, PolyA is present, and the nucleic acid has SEQ ID NO: 9, and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid.

[0263] Certain optogenetic constructs include nucleic acids described herein, wherein the promoter comprises SEQ ID NO:2, WPRE, and the depolarizing optogenetic protein is ReaChR or ReaChR transmembrane encoded by SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:73, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16. and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid having SEQ ID NO: 84.

[0264] Certain optogenetic constructs include nucleic acids described herein, wherein the promoter comprises SEQ ID NO:11, WPRE, and the depolarizing optogenetic protein is ReaChR or ReaChR membrane protein encoded by SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:73, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16. The present invention also includes an optogenetic protein engineered to contain a transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and the nucleic acid has SEQ ID NO: 87; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid having SEQ ID NO: 84.

[0265] Certain optogenetic constructs include nucleic acids described herein, wherein the promoter comprises SEQ ID NO:12, WPRE, and the depolarizing optogenetic protein is ReaChR or ReaChR membrane protein encoded by SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:73, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16. The present invention also includes an optogenetic protein engineered to contain a transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and the nucleic acid has SEQ ID NO: 87; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid having SEQ ID NO: 84.

[0266] Certain optogenetic constructs include nucleic acids described herein, wherein the promoter comprises SEQ ID NO:13, WPRE, and the depolarizing optogenetic protein is ReaChR or ReaChR membrane protein encoded by SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:73, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16. The present invention also includes an optogenetic protein engineered to contain a transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and the nucleic acid has SEQ ID NO: 87; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid having SEQ ID NO: 84.

[0267] Certain optogenetic constructs include nucleic acids described herein, wherein the promoter comprises SEQ ID NO:14, WPRE, and the depolarizing optogenetic protein is ReaChR or ReaChR membrane encoded by SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:73, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16. The present invention also includes an optogenetic protein engineered to contain a transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and the nucleic acid has SEQ ID NO: 87; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid having SEQ ID NO: 84.

[0268] Certain optogenetic constructs include nucleic acids described herein, wherein the promoter comprises SEQ ID NO:26, WPRE, and the depolarizing optogenetic protein is ReaChR or ReaChR membrane protein encoded by SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:73, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16. The present invention also includes an optogenetic protein engineered to contain a transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and the nucleic acid has SEQ ID NO: 87; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid having SEQ ID NO: 84.

[0269] Certain optogenetic constructs include nucleic acids described herein, wherein the promoter comprises SEQ ID NO:85, WPRE, and the depolarizing optogenetic protein is ReaChR or ReaChR membrane protein encoded by SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:73, or a nucleotide sequence encoding the same polypeptide encoded by SEQ ID NO:16. The present invention also includes an optogenetic protein engineered to contain a transmembrane domain, wherein the reporter molecule is absent, PolyA is present, and the nucleic acid has SEQ ID NO: 87; and an AAV vector comprising an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid, preferably an AAV5 capsid, a PHP.eB capsid, or an NHP26 capsid, more preferably an AAV5 capsid having SEQ ID NO: 84.

[0270] A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 4. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 5. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 6. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 7. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 17. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 18. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 19. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 20. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 21. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 35. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 55. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 56. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 60. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 67. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 68. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 71. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 72. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 75. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 76. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 77. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 80. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 81. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 82. A particular optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 83.

[0271] Some preferred optogenetic constructs comprise a nucleotide sequence comprising SEQ ID NO: 60, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83. A preferred optogenetic construct comprises a nucleotide sequence comprising SEQ ID NO: 60.

[0272] H. Polynucleotides and Vectors The present disclosure further relates to a recombinant vector comprising the nucleic acid disclosed herein or a host cell comprising the vector. AAV vectors may be based on a viral genome 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 comprises a promoter, a nucleotide sequence encoding a depolarizing optogenetics protein and optionally a reporter molecule, a WPRE, and typically a suitable polyA signal. Each of the nucleotide sequences is operably linked.

[0273] Furthermore, a vector may contain additional elements for expression of a nucleic acid, for example, a vector may include one or more ITRs, ribosome binding elements, terminators, enhancers, selectable markers, introns, polyA signals, and / or origins of replication.

[0274] 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.

[0275] 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.

[0276] 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.

[0277] Nucleic acids encoding the depolarizing optogenetic proteins disclosed herein can be packaged into viral capsids to produce viral particles, preferably AAV particles.

[0278] The viral capsid can be any functional AAV capsid.In one embodiment, the capsid is provided by a single AAV source.Alternatively, the AAV capsid can be derived from multiple sources.Any serotype of AAV known in the art, such as serotype AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, modified AAV, AAVPhP.B, or those yet to be discovered, or recombinant AAV based thereon, can be used as the source of AAV capsid.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] The nucleic acids described herein, or any components thereof (e.g., promoters, sequences encoding depolarizing optogenetic proteins), can be optimized by sequence modification using well-known methods, for example, to achieve a desired level of expression, reduce immunogenicity, or for other purposes. The optogenetic constructs disclosed herein typically further comprise a suitable polyadenylation signal (PolyA) 3' to the WPRE. The optogenetic constructs may further comprise one or more AAV inverted terminal repeats (ITRs). Suitable methods for optimizing nucleic acid constructs by sequence modification, including, for example, 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 components 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 miRNA, siRNA, shRNA, dsRNA, or gRNA sequences (see, e.g., Domenger and Grimm, Human Molecular Genetics, 2019, 28:R1-R12), modified to include inducible control systems (e.g., Tet on / off systems) (see, e.g., Gossen et al., Science, 268:1766-1769 (1995); Harvey et al., Bioinformatics-Trends and Methodologies, 449-472 (2011)), modified to include inducible control systems (e.g., Tet on / off systems) (see, e.g., Gossen et al., Science, 268:1766-1769 (1995)); 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). Methods for optimizing the nucleic acids disclosed herein are conventional and well known to those of skill in the art.

[0284] I. Therapeutic Applications Also provided herein are methods and uses for treating diseases, disorders, or conditions associated with vision loss, comprising administering a nucleic acid described herein to a subject in need thereof. 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.

[0285] The methods and compositions 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, choroideremia, post-retinal detachment, cone dysfunction, pigment epithelial retinal degeneration, retinal vein occlusion, and geographic atrophy. Treatment using the methods and compositions disclosed herein is suitable for subjects with vision-impairing disorders in which the optic nerve retains at least some function.

[0286] While any retinal disease may be suitable for therapy, the inventors have discovered a subset of patients with retinal dystrophies that are particularly suitable for therapy with the optogenetic constructs disclosed herein. The inventors have found that patients with inherited retinal dystrophies and low vision are particularly good candidates for treatment if they have a preserved cone photoreceptor layer in the central retina.

[0287] Additionally, the nucleic acid molecules of the present invention can be used to manufacture pharmaceuticals and / or to treat patients with diseases, disorders, or conditions associated with vision loss.

[0288] The subject may be a human, dog, cat, horse, or any animal in which restoration of vision is desired.

[0289] The nucleic acids disclosed herein can be administered to a subject in an amount sufficient to at least partially restore vision.

[0290] The nucleic acids provided herein can be administered to a subject by any suitable route, including, but not limited to, intraocular (e.g., subretinal injection, intravitreal injection, 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.

[0291] 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.

[0292] 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.

[0293] The methods disclosed herein can further include administering to the subject at least one additional therapeutic agent, which can be, in particular, a corticosteroid, an antibiotic, an analgesic, an immunosuppressant, or a nutritional factor, or any combination thereof.

[0294] J. Pharmaceutical Compositions The present disclosure also relates to pharmaceutical compositions comprising the nucleic acids, vectors, and components thereof. 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 comprising the nucleic acids are suitable for administration to a subject. The pharmaceutical compositions can be supplied as a solution, suspension, emulsion, or as a solid form suitable for dissolving or suspending in a liquid before use.

[0295] Pharmaceutical compositions may contain pharmaceutically acceptable excipients. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not impair the effectiveness of the biological activity of the component 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.

[0296] 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 can 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. The carrier is suitable for direct delivery to the eye, and 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.

[0297] 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.

[0298] 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.

[0299] 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.

[0300] 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.

[0301] 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.

[0302] 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.

[0303] 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.

[0304] 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.

[0305] K. Kit Also disclosed herein are kits comprising the nucleic acids, viral particles containing the nucleic acids, host cells, or pharmaceutical compositions thereof disclosed herein.

[0306] 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.

[0307] 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.

[0308] L. Definition 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, they include embodiments using any specific value within that range. Furthermore, reference to values ​​stated in a range 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 to be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise. The use of "or" means "and / or" unless the particular context in which it is used dictates otherwise.

[0309] 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.

[0310] 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.

[0311] 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.

[0312] As used herein, the term "promoter" refers to any cis-regulatory element, generally located upstream (5' region) that directs transcription of an operably linked nucleic acid.

[0313] As used herein, the term "operably linked" with respect to nucleic acid sequences refers to the positioning of nucleotide sequences on a single nucleic acid molecule that permits the components (i.e., nucleic acid sequences) to function in their intended manner. For example, a promoter is operably linked to a nucleic acid sequence encoding a depolarizing optogenetics protein if it is capable of affecting expression of the depolarizing optogenetics sequence, i.e., if the optogenetics sequence is under the transcriptional control of the promoter.

[0314] As used herein, the term "nucleic acid" or "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. A "polynucleotide" can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, or hybrid molecules containing DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. Furthermore, a polynucleotide can be composed of triple-stranded regions containing RNA or DNA, or both RNA and DNA. The backbone of a polynucleotide can comprise sugars and phosphate groups (such as those typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide can comprise a polymer of synthetic subunits, such as phosphoramidates, and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiester oligomer.

[0315] As used herein, the term "host cell" refers to a microorganism, prokaryotic cell, eukaryotic cell, or cell line cultured as a unicellular organism that can be or has been used as a recipient for the transfer of a recombinant vector or other polynucleotide, and includes the progeny of the original transfected cell. The progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent due to natural, accidental, or deliberate mutation.

[0316] As used herein, the term "therapeutically effective amount" refers to an amount of a compound (i.e., a nucleic acid) described herein that is sufficient to achieve a desired pharmacological or physiological effect under the conditions of administration. For example, a "therapeutically effective amount" may be an amount sufficient to reduce the signs or symptoms of a disease or condition (e.g., visual impairment or blindness). Those skilled in the art will understand that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject. The therapeutically effective amount of a pharmaceutical composition may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the pharmaceutical composition to elicit a desired response in the individual. A clinician of ordinary skill can determine the appropriate amount to be administered to achieve the desired therapeutic effect based on these and other considerations.

[0317] As used herein, the terms "treat," "treatment," or "treating," and grammatically related terms, refer to the amelioration of signs, symptoms, or consequences of a disease, such as prolonging survival, reducing morbidity, and / or mitigating side effects. As is readily understood in the art, complete eradication of a disease, although preferred, is not a requirement for treatment.

[0318] As used herein, the term "subject" refers to any animal, such as any mammal, including, but not limited to, a human, a non-human primate, a rodent, etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0319] Additional description of the methods and guidance for practicing the methods is provided herein.

[0320] 4. 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]

[0321] 5. Working Example Example 1. The cone-specific promoter (ProA7) requires 3' regulatory elements for expression in human mature cones. ChrimsonR, an optogenetic effector, is a light-activated channel that induces light responses in light-insensitive cells (Klapoetke NC et al. (2014), 11(3):338-346). The ChrimsonR-tdTomato (ChrimsonR-tdT) transgene was recently shown to be safe and effective after intravitreal injection in clinical trials (see Reference 1, Sahel et al.), so we performed preliminary studies on this. In this vector, the ChrimsonR-tdT sensor is under the control of a ubiquitous CAG promoter, which allows nonspecific expression, but targets ganglion cells (GCs) primarily after intravitreal injection (see References 1 and 2). To achieve cone-specific expression, we replaced the CAG promoter in the ChrimsonR-tdT vector with the cone-specific ProA7 promoter (see Reference 3) (synPVI or Gnat2_500, SEQ ID NO: 2).

[0322] AAV8-BP2-ProA7-ChrimsonR-tdT-hGHpolyA was subretinal injected into wild-type C57BL / 6 mice. Efficient expression in mouse cones was observed (Figure 1A). Next, AAV8-BP2-ProA7-ChrimsonR-tdT-hGHpolyA was tested in 30-week-old three-dimensional human retinal organoids. Similar efficient expression was observed (Figure 1B).

[0323] These constructs were then placed on human retinal explants derived from multiple organ donors for 2 days, followed by 5 weeks of culture. Surprisingly, no cones expressed the ChrimsonR-tdT transgene, in stark contrast to data obtained with mice and retinal organoids (Figure 1C).

[0324] It was hypothesized that one or more of the following factors contributed to this finding: (1) ChrimsonR-tdT may not have been expressed in human cones, and / or (2) the absence of 3′ regulatory elements in the construct may have reduced nuclear export of intronless transcripts.

[0325] When the AAV8-BP2-CAG-ChrimsonR-tdT-hGHpolyA (without WPRE) vector (SEQ ID NO: 1; see, e.g., WO2017187272A1 and WO2012 / 145601 for AAV2-7m8 viral vectors containing CAG-ChrimsonR-tdTomato) was tested in human retina, it resulted in nonspecific expression of the transgene in cones and rods (Figures 1C and 1E). This suggests that ChrimsonR-tdT can be expressed in cones, but nonspecific expression is undesirable for therapeutic purposes because it leads to abnormalities in intraretinal information processing.

[0326] Next, a modified woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was cloned downstream of ChrimsonR-tdT to create a new vector (SEQ ID NOs: 3 and 4). A weak but specific fluorescence was observed in some cones with AAV8-BP2-ProA7-ChrimsonR-tdT-hGHpolyA (Figure 1C and 1D).

[0327] Finally, changing ChrimsonR-tdT to vfChrimson-EYFP (SEQ ID NO: 5) resulted in more efficient and specific expression in human peripheral retinal cones (Figures 1C and 1D) and in non-human primate (NHP) cones after in vivo subretinal injection (Figure 1E).

[0328] These results collectively suggest that the WPRE element is required for ProA7-mediated (but not CAG-mediated) expression and that the optogenetic sensor is important for achieving high-level expression. ProA7 linked to the WPRE element results in efficient and specific expression of optogenetic constructs in human cones.

[0329] Example 2. Systematic screening of AAV capsids and optogenetic sensors in human cones Having identified a combination of vector elements (ProA7+WPRE) that jointly resulted in efficient cone-specific expression, we systematically investigated the influence of the AAV capsid and optogenetic sensors on expression using retinal organoids and human retinal explants as model systems (Figures 2 and 3). To allow for side-by-side comparisons between different vectors, samples were transduced with equal genome copies of AAV using ddPCR of WPRE as a titration method.

[0330] Organoids and macular human retinal explants were incubated for 5 weeks and stained with antibodies against fluorophores and a cone marker (cone arrestin). Cone transduction rates were determined by manual counting (organoids, 3 investigators) or by automated scripting (human retina, positive cutoff was 4 SD above the background mean). In the first screening, the ProA7-vfChrimson-EYFP-WPRE-hGH-polyA vector was packaged into the AAV8-BP2, AAV-PHP.B, AAV-PHP.eB, AAV-NHP26, and AAV44.9(E531D) capsid serotypes. All vectors transduced cones into retinal organoids (Figure 2A).

[0331] In human retina, the highest transduction rates were observed with AAV-NHP26, AAV-PHP.B, and AAV-PHP.eB. Interestingly, AAV8-BP2 was effective only at high doses, resulting in only 13.2% ± 5.0% (mean ± sd) cone transduction in central (macular) retinal explants (Figure 2B). AAV44.9(E531D) did not result in cone transduction.

[0332] In the second screen, we focused on comparing optogenetic sensors. We packaged several different sensors into AAV8-BP2 capsids: ChrimsonR-tdT, CatCH-GFP, ChrMine-tdT, ReaChR-citrine, ChrimsonR-EYFP, fChrimson-C174-EYFP (containing the CatCH-GFP mutation), ubiquitinated fChrimson-EYFP, fChrimson-EYFP, ChrMine-EYFP, and Jaws-EYFP (Figure 3). All sensors were driven by the ProA7 promoter, followed by the WPRE element and the hGH-polyA sequence.

[0333] Several sensors were expressed in organoids (Figure 3A) and human retina (Figure 3B), although overall expression was lower in human retina. Highest expression was observed with ReaChR-EYFP and Jaws-EYFP, but expression was also observed with ChrimsonR and ChrMine, regardless of whether the EYFP or tdT tag was used.

[0334] Finally, we combined three capsids (AAV-BP2, AAV-PHP.eB, and AAV-NHP26) and four sensors (vfChrimson-EYFP, ChrimsonR-tdT, ReaChR-citrine, and ChrMine-EYFP) and performed combinatorial screening in retinal organoids and human retina (Figure 4).

[0335] As in previous experiments, the differences between the different constructs were greater in the human retina. At the low AAV dose used (3.8e11 v.g.), robust expression was observed with ReaChR-citrine and ChrMine-EYFP, while only faint expression was observed with vfChrimson-EYFP and ChrimsonR-tdT (Figure 4). When AAV5, AAV8, and AAV9 were tested with ReaChR, expression was observed with all capsids, with AAV5 being the most effective (Figure 5). Collectively, these experiments suggest that the main determinants of effective expression are the vector design and the applied sensor. Various capsids can confer efficient expression in human cones.

[0336] Example 3. ReaChR-citrine restores light sensitivity to human retinal explants. Having identified AAV capsid-optogenetic sensor combinations that resulted in efficient cone transduction and expression, we investigated whether these combinations could also drive functional light responses in cultured human retinas (Figures 6 and 7). Cultured retinas lose intrinsic photoreceptor light sensitivity within hours of explantation. This is due to the loss of the fragile outer segments and the lack of support from the RPE, which is isolated for cell culture.

[0337] Taking advantage of this feature, we investigated whether light sensitivity could be restored in these cultured retinas after AAV transduction. Indeed, if a light response was observed in the cultured explants after AAV transduction, it could only be attributed to the tested optogenetic protein.

[0338] Light responses in AAV-transduced human retinal explants from four different donors after 5–9 weeks in culture were investigated using microelectrode array recording. Briefly, explants were placed on a 256-microelectrode array with the ganglion cell layer facing the electrodes, and the activity of several ganglion cells in response to light stimuli was simultaneously recorded. Spike sorting was then performed to isolate single-cell activity. To screen light responses, a 2-second white light flash was presented, repeated 10 times with a 5-second interstimulus interval, at an intensity of 1.38E+16 photon flux / cm. 2 This intensity was previously shown to reliably activate optogenetic effectors in NHP explants (Ref. 2) and to be within the typical range of sensitivity of these microbial opsins (Ref. 4).

[0339] Non-transduced control retinas showed no light response (Figures 6A-6B). Light responses were observed in retinas treated with an AAV vector carrying the ProA7-ReaChR-citrine-WPRE-hGH transgene. The expression findings reported here suggest that capsid components were not important, as light responses were observed with all capsids tested (AAV8-BP2, AAV5, AAV9-PHP.eB, or AAV-NHP26; Figures 6A-6C). However, all other light sensors tested (ChrMine-EYFP, ChrimsonR-EYFP, and Jaws-EYFP) failed to elicit a light response (Figures 6A-6C). Further testing of additional hyperpolarizing optogenetic effectors (HcKCR1 and eGTACR1) also failed to elicit a light response (Figure 6D).

[0340] Although all tested sensors were expressed in human retina (Figures 3 and 4), ReaChR showed very robust membrane localization (Figure 6C). This suggests that excellent membrane trafficking of ReaChR is important for obtaining a light response. The importance of membrane localization is not surprising, as optogenetic sensors are generally understood to function by translocating ions across the cell membrane. ChrMine-EYFP, ChrimsonR-EYFP, and Jaws-EYFP were not efficient at membrane localization (Figure 6C), which may explain the lack of light response in human retina.

[0341] Example 4. Characterization of light responses using ReaChR-citrine Next, we further characterized the light response of the ReaChR-citrine construct (SEQ ID NO: 7) in human retinal explants (Figures 7A-7C). Normal human retinas exhibit five distinct ganglion cell responses to light flashes (see Reference 5). The first class of cells exhibits a spiking response during the 2-second light flash (sustained ON response). The second class of cells exhibits a response that fires only at the onset of the light flash (transient ON response). The third class responds at the onset and offset of the stimulus (ON / OFF response). The fourth class responds at the offset of the stimulus (transient OFF response), and the fifth class reduces its firing for the entire duration of the response (sustained OFF response).

[0342] Importantly, ReaChR-citrine-transduced human retinas produced the same five distinct types of responses, suggesting that this construct restored normal human intraretinal information processing ( Fig. 7<em>A ).

[0343] We also characterized the sensitivity of the response using ReaChR-citrine. The light response was approximately 1x10 14 ~1x10 16 photon / cm 2 / sec, which is consistent with previous reports obtained in mice using a different sensor (see reference 4).

[0344] Finally, the citrine tag was removed and retinal cultures were transduced with AAV5-ProA7-ReaChR-WPRE-hGH (SEQ ID NO: 17). Light response and higher sensitivity were observed. The sensitivity of AAV5-ProA7-ReaChR-WPRE-hGH was approximately 5x10 13 ~1x10 16 photon / cm 2 / sec, which is a half-log higher sensitivity than AAV5-ProA7-ReaChR-citrine WPRE-hGH (SEQ ID NO: 7) (Figure 7B). Figure 7C further demonstrates that retinas transduced with an optogenetic vector driven by the cone-specific ProA7 promoter can respond to frequency stimuli up to 23.2 Hz.

[0345] These experiments suggest that optogenetic stimulation of cone photoreceptors can restore light sensitivity and physiological retinal information processing within a certain range of light intensities. Furthermore, removal of the citrine tag does not inhibit light responses but results in higher sensitivity.

[0346] Example 5. Non-human primate studies A. ReaChRs are expressed in the fovea after in vivo injection into NHPs Next, AAV5-ProA7-ReaChR-citrine-WPRE-hGH (SEQ ID NO: 7) was tested in NHPs after in vivo subretinal injection. Three cynomolgus monkeys were administered three different doses of AAV5-ProA7-ReaChR-citrine-WPRE-hGH (SEQ ID NO: 7) vector (1.5x10 per eye). 10 , 1.5x10 11 , 3x10 11 The vector genome (vg) was injected bilaterally. Three months after injection, the animals were sacrificed and the retinas were explanted. The retinal explants were cultured for 1–2 days to abolish endogenous light responses.

[0347] No photoresponsive cells were observed in the non-transduced areas ("non-blebs") after this short incubation period, whereas 1.5x10 photoresponsive cells were observed in the AAV-transduced areas ("blebs"). 11 , 3x10 11 A robust light response was observed at a dose of 1.5x10 vg, confirming the in vivo functionality of the optogenetic effector (Figure 8A). 10 At the lowest dose of 1.5x10, no light response was observed (Figure 8A). Light-responsive cells exhibited sustained ON, transient ON, ON / OFF, or sustained OFF behavior, suggesting recovery of intraretinal information processing (Figure 8B). After MEA recording, tissue slices were fixed and transduction efficiency was calculated by counterstaining cones with cone arrestin (Figure 8B). 10 , 1.5x10 11 , 3x10 11 At the vg dose, mean cone transduction of 39.54%, 58.97%, and 93.94%, respectively, was observed.

[0348] Collectively, these experiments demonstrate that the AAV5-ProA7-ReaChR-citrine-WPRE-hGH vector (SEQ ID NO: 7) efficiently transduces cone photoreceptors in the macaque retina after subretinal injection and is functional in vivo.

[0349] B. AAV vectors carrying the optogenetic effector ReaChR under the control of a hybrid promoter are functional in non-human primates. To investigate and compare light responsiveness in healthy primate retinas after transduction with the AAV5-Pro573.2-ReaChR-WPRE-hgH polyA vector (SEQ ID NO: 18), cynomolgus monkeys were injected subretinal with 1.5E11vg of the AAV vector, and light responsiveness in healthy non-human primate (NHP) retinas was characterized (Figures 14A-14C).

[0350] AAV5-Pro573.2-ReaChR-WPRE-hgH polyA (SEQ ID NO: 18) was injected into three cynomolgus macaques at a dose of 1.5e11 v.g. (n=6). The control vector, AAV5-ProA7-ReaChR-citrine-WRPE-hGH (SEQ ID NO: 7), was intranasally injected into two eyes of two different macaques. To assess retinal morphology after injection, animals were followed using optical coherence tomography (OCT). After 3-4 months, animals were euthanized, and eyes were examined for gross lesions. Gene expression and photoresponsiveness were then tested.

[0351] Injected ("bleb") and non-injected ("non-bleb") retinal tissues were cultured for 1-2 days to reduce endogenous light responses, and ontogenetically driven light responses were measured using multielectrode array (MEA) recordings. "Acute" refers to freshly dissected retinas, where light responses can be measured in healthy, untreated areas of the retina. Both "control" and "bleb" samples were cultured to eliminate normal light responses. "Control" is from an untreated area of ​​the retina. AAV-transduced areas ("blebs") showed enhanced and robust light responses compared to freshly dissected retinas ("acute"), where normal light responses can be measured (Figures 14A and 14B). No light-responsive cells were observed in the control samples.

[0352] NHP retinas transduced with AAV-Pro573.2-ReaChr-WPRE-hgH polyA showed complete diversity of light responses for five different types of responses, demonstrating normal-like light responsiveness and intraretinal information processing (Figure 14C).

[0353] These experiments demonstrate that optogenetic stimulation of NHP cone photoreceptors can result in photosensitivity and physiological intraretinal information processing within a certain light intensity range. Furthermore, removal of the citrine tag does not inhibit photoresponsiveness, as photoresponsiveness was observed with the citrine-depleted AAV-Pro573.2-ReaChr-WPRE-hgH polyA construct. Importantly, these results were observed in a preclinical animal model that most closely resembles humans and is presumed to be the best model for technology transfer testing of human therapeutics. Furthermore, the efficacy of vector administration in these NHP experiments was demonstrated using the same method used to administer AAV vectors to human patients in clinical trials.

[0354] Example 6. Efficacy of AAV5-ProA7-ReaChR-citrine-WPRE-hGH in four different human donors Next, we analyzed whether the AAV5-ProA7-ReaChR-citrine-WPRE-hGH vector (SEQ ID NO: 7) transduces cone photoreceptors in four additional independent human donors. Human retinas were cultured for 4 to 6 weeks and then analyzed. Robust light responses were observed in all cases (Figure 9B). Efficient cone transduction was detected in all four donors (Figure 9A; mean efficiencies were found to be 49.06-68.03%). In donor 4, the vector was tested in the fovea. 66.92% of cones were transduced with the AAV5-ProA7-ReaChR-citrine-WPRE-hGH (SEQ ID NO: 7) vector.

[0355] Human explants from 12 human donors (8 males and 4 females) aged 18 to 80 years were also tested. The results are compared in Figures 9C and 9D. Figure 9C shows efficient cone transduction (average efficiency of 40 to 80%), and Figure 9D demonstrates robust light responses in donor retinal samples transduced with the AAV5-ProA7-ReaChr-citrine-WPRE-hGH polyA vector. Donor A in Figures 9A and 9B corresponds to donor 5 in Figures 9C and 9D. Donor B in Figures 9A and 9B corresponds to donor 7 in Figures 9C and 9D. Donor C in Figures 9A and 9B corresponds to donor 6 in Figures 9C and 9D.

[0356] These results suggest that the desirable characteristics of the AAV5-ProA7-ReaChR-citrine-WPRE-hGH polyA optogenetic vector, namely high transduction efficiency and effective light responsiveness, are reproducible in transduced human retinal explants from individuals of different ages and genders.

[0357] Example 7. Hybrid promoters containing the 185 base pair sequence of ProA7 are also functional The experiments described herein confirmed the functionality of the AAV5-ProA7-ReaChR-citrine-WPRE-hGH vector (SEQ ID NO: 7, FIG. 10) in retinal organoids, human retinal explants, and in vivo in macaque retina. The functionality of a hybrid promoter containing a 185 base pair region of the ProA7 promoter and a rod-specific promoter having SEQ ID NO: 10 was further analyzed (FIG. 10). These promoters were named ProX572.2 (SEQ ID NO: 12) and ProX573.2 (SEQ ID NO: 14) according to the order of the 185 base pair ProA7 fragment and the rod-specific promoter (FIG. 10). ProX572.2 is also known as Pro572.2, and ProX573.2 is also known as Pro573.2.

[0358] Expression and function were tested using the AAV5-ProX572.2-ReaChR-citrine-WPRE hGH polyA in human retinal explants. Expression levels were similar between the AAV5-ProA7-ReaChR-citrine-WPRE-hGH pol...

Claims

1. (i) a promoter comprising a first nucleotide sequence comprising at least a portion of a cone-specific promoter and a second nucleotide sequence comprising at least a portion of a rod-specific promoter, wherein the first and second nucleotide sequences are operably linked and function as a single promoter for expression in cone photoreceptors, or the promoter is a hybrid promoter, or a cone-specific promoter, or an active variant, fragment, or truncation of any of the foregoing; (ii) a nucleotide sequence encoding a depolarizing optogenetic protein and, optionally, a reporter molecule; (iii) woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); Including, the promoter, the nucleotide sequence encoding a depolarizing optogenetic protein and optionally a reporter molecule, and the WPRE are operably linked; Isolated nucleic acid.

2. 2. The isolated nucleic acid of claim 1, wherein the promoter is a hybrid promoter comprising SEQ ID NO: 14, SEQ ID NO: 13, SEQ ID NO: 12, or SEQ ID NO: 11, or an active variant, fragment, or truncated form of any of the foregoing.

3. the promoter is a hybrid promoter, and the first nucleotide sequence comprises at least about 150 contiguous nucleotides of SEQ ID NO:2; and the second nucleotide sequence comprises at least about 264 contiguous nucleotides of SEQ ID NO: 10, or an active variant, fragment, or truncation of any of the foregoing; The isolated nucleic acid of claim 1.

4. the first nucleotide sequence SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and A sequence comprising at least about 150 consecutive nucleotides from the 3' end of SEQ ID NO:2 is selected from the group consisting of the second nucleic acid comprises SEQ ID NO: 54, or an active variant of any of the foregoing; The isolated nucleic acid of claim 3.

5. 5. The isolated nucleic acid of claim 4, wherein the second nucleotide comprises SEQ ID NO: 10 or an active variant, fragment, or truncation thereof.

6. 2. The isolated nucleic acid of claim 1, wherein the promoter is a cone-specific promoter comprising at least about 150 consecutive nucleotides of SEQ ID NO: 2 or an active variant thereof.

7. The promoter is SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and A sequence comprising at least about 150 consecutive nucleotides from the 3' end of SEQ ID NO: 2, or an active variant thereof.

7. The isolated nucleic acid of claim 6, selected from the group consisting of:

8. the hybrid promoter or the cone-specific promoter is SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, a sequence comprising at least about 150 consecutive nucleotides from the 3' end of SEQ ID NO:2; Active variants of any of the foregoing, and combinations thereof comprising 1 to about 10 copies of a sequence selected from the group consisting of:

10. An isolated nucleic acid according to any one of the preceding claims.

9. 10. The isolated nucleic acid of any one of the preceding claims, wherein the hybrid promoter comprises 1 to about 10 copies of SEQ ID NO: 54, or an active variant thereof.

10. An isolated nucleic acid described in any one of the preceding claims, wherein the WPRE is encoded by a nucleotide sequence having at least 70% identity to SEQ ID NO: 3 (e.g., SEQ ID NO: 8), or a nucleotide sequence having at least 70% identity to SEQ ID NO:

86.

11. further comprising a nucleotide sequence encoding a polyadenylation signal (PolyA) 3' to the nucleotide sequence encoding the WPRE; the nucleotide sequence encoding the PolyA and the nucleotide sequence encoding the WPRE are operably linked; 10. An isolated nucleic acid according to any one of the preceding claims.

12. 10. The isolated nucleic acid of any one of the preceding claims, further comprising a nucleotide sequence encoding an AAV inverted terminal repeat (ITR).

13. 13. The isolated nucleic acid of claim 12, comprising a first AAV ITR 5' to the promoter and a second AAV ITR 3' to the WPRE and preferably 3' to the PolyA signal.

14. 10. The isolated nucleic acid of any one of the preceding claims, wherein the depolarizing optogenetic protein is a light-responsive polypeptide.

15. 15. The isolated nucleic acid of claim 14, wherein the light-responsive polypeptide is a light-gated ion channel polypeptide.

16. 16. The isolated nucleic acid of claim 15, wherein the light-gated ion channel polypeptide is a channelrhodopsin or a functional variant thereof.

17. 17. The isolated nucleic acid of claim 16, wherein the channelrhodopsin is a ReaChR polypeptide or a functional variant thereof.

18. 18. The isolated nucleic acid of claim 17, wherein the isolated nucleic acid comprises a nucleotide sequence encoding ReaCHR or a functional variant thereof.

19. 19. The isolated nucleic acid of claim 18, wherein the nucleotide sequence encoding ReaChR or a functional variant thereof is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 73, or an amino acid sequence having at least about 70% identity to any of the foregoing.

20. 17. The isolated nucleic acid of claim 16, wherein the channelrhodopsin or functional variant thereof is engineered to enhance membrane localization when expressed in human cone photoreceptors.

21. 21. The isolated nucleic acid of claim 20, wherein the engineered channelrhodopsin comprises SEQ ID NO: 57 or a functional variant thereof.

22. 10. The isolated nucleic acid of any one of the preceding claims, wherein the isolated nucleic acid does not encode the optional reporter molecule.

23. The promoter is two or more first nucleotide sequences, two or more second nucleotide sequences, two or more cone-specific promoters, or Two or more of them The isolated nucleic acid according to any one of claims 1 to 9, comprising:

24. 10. A nucleic acid according to any one of the preceding claims, further comprising an intron.

25. 10. The nucleic acid of any one of the preceding claims, comprising a nucleotide sequence comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:35, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83, or a sequence having at least about 70% identity to any of the foregoing.

26. 25. The nucleic acid of any one of claims 1 to 24, comprising a nucleotide sequence comprising SEQ ID NO:60, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:

83.

27. 10. The nucleic acid of any one of the preceding claims, wherein the active variant has at least about 70% identity with the corresponding reference sequence.

28. 28. The nucleic acid of claim 27, wherein the active variant comprises one or more of codon optimization, CpG reduction, removal of alternative start sites, removal of repeats, removal of hairpins, removal of unnecessary splice donor and acceptor sites, deletion of ITR end release sites, addition of stuffer sequences, or addition of miRNA.

29. A viral particle comprising a nucleic acid according to any one of the preceding claims.

30. A host cell comprising a nucleic acid according to any one of claims 1 to 28 or a viral particle according to claim 28.

31. (i) a nucleic acid according to any one of claims 1 to 28; (ii) AAV capsid; An AAV vector comprising:

32. The AAV vector of claim 31, wherein the AAV capsid is an AAV5 capsid, a PHP.eB capsid, an NHP26 capsid, an AAV8 capsid, an AAV8-BP2 capsid, an AAV9 capsid, or a PHP.B capsid.

33. 33. The AAV vector of claim 32, wherein the AAV capsid is an AAV5 capsid, and an AAV8 capsid or an AAV9 capsid.

34. A host cell comprising the AAV vector of any one of claims 31 to 33.

35. (i) a nucleic acid according to any one of claims 1 to 28, a viral particle according to claim 29, an AAV vector according to any one of claims 31 to 33, or a host cell according to claim 30 or 34, and (ii) a pharmaceutically acceptable excipient A pharmaceutical composition comprising:

36. 10. A method for delivering a depolarizing optogenetic protein to a human cone cell in a subject in need thereof, the method comprising administering to the subject a nucleic acid of any one of claims 1 to 28, a viral particle of claim 29, an AAV vector of any one of claims 31 to 33, a host cell of claim 30 or 34, or a pharmaceutical composition of claim 35.

37. 19. A method for treating a retinal disease, comprising administering to a subject in need thereof an effective amount of the nucleic acid of any one of claims 1 to 28, the viral particle of claim 29, the AAV vector of any one of claims 31 to 33, the host cell of claim 30 or 34, or the pharmaceutical composition of claim 35.

38. 19. A method for restoring vision, comprising administering to a subject in need thereof an effective amount of the nucleic acid of any one of claims 1 to 28, the viral particle of claim 29, the AAV vector of any one of claims 31 to 33, the host cell of claim 30 or 34, or the pharmaceutical composition of claim 35.

39. 19. A method for restoring the light sensitivity of human cone photoreceptor cells, the method comprising administering to said human cone photoreceptor cells in need thereof a nucleic acid according to any one of claims 1 to 28, a viral particle according to claim 29, an AAV vector according to any one of claims 31 to 33, a host cell according to claim 30 or 34, or a pharmaceutical composition according to claim 35.

40. 19. A method for treating retinal degeneration in a subject, the method comprising administering to a subject in need thereof an effective amount of the nucleic acid of any one of claims 1 to 28, the viral particle of claim 29, the AAV vector of any one of claims 31 to 33, the host cell of claim 30 or 34, or the pharmaceutical composition of claim 35.

41. 41. The method of any one of claims 36-40, wherein the subject has or is at risk of developing 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, choroideremia, pigment epithelial retinal degeneration, retinal vein occlusion, or geographic atrophy.

42. 42. The method of claim 41, wherein the subject has or is at risk of developing retinitis pigmentosa.

43. 42. The method of claim 41, wherein the subject has or is at risk of developing geographic atrophy.

44. 44. The method of any one of claims 36 to 43, wherein the nucleic acid, the viral particle, the AAV vector, the host cell, or the pharmaceutical composition is administered by subretinal injection.

45. 44. The method of any one of claims 36 to 43, wherein the nucleic acid, the viral particle, the AAV vector, the host cell, or the pharmaceutical composition is administered before or after the onset of photoreceptor loss.

46. 36. The nucleic acid of any one of claims 1 to 28, the viral particle of claim 29, the AAV vector of any one of claims 31 to 33, the host cell of claim 30 or 34, or the pharmaceutical composition of claim 35, for use in treating a retinal disease, restoring vision, restoring light sensitivity of human cone photoreceptor cells, or treating retinal degeneration in a subject in need thereof.

47. 47. The use of claim 46, wherein the subject in need thereof has or is at risk of developing retinitis pigmentosa, rod-cone dystrophy, Leber 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, choroideremia, pigment epithelial retinal degeneration, retinal vein occlusion, or geographic atrophy.

48. 48. The use of claim 47, wherein the subject has or is at risk of developing retinitis pigmentosa.

49. 48. The use of claim 47, wherein the subject has or is at risk of developing geographic atrophy.

50. 50. The method or use of any one of claims 36 to 49, wherein the depolarizing optogenetic protein is expressed in the plasma membrane of human cone cells.

51. 51. The method or use of any one of claims 36 to 50, wherein the depolarizing optogenetic protein is capable of mediating a depolarizing current that depolarizes human cone cells when exposed to light.

52. 52. The method or use of claim 51 , wherein depolarization of human pyramidal cells induces light-driven ganglion cell spiking that is equivalent to light-driven ganglion cell spiking in functional human pyramidal cells.

53. 53. The method or use of claim 52, wherein the light-driven ganglion cell spiking is assessed using a multi-electrode array.

54. 54. The method or use of any one of claims 36 to 53, wherein the nucleic acid is capable of restoring light sensitivity when introduced into a human cone cell, and wherein restoration of light sensitivity occurs when: (i) the depolarizing optogenetic protein is expressed in the cell membrane of the human cone cell; (ii) the depolarizing optogenetic protein is capable of mediating a depolarizing current that depolarizes the human cone cell when exposed to light; and (iii) depolarization of the human cone cell induces light-driven ganglion cell spiking.

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