EA046019B1 - COMPOSITIONS AND METHODS FOR TREATING RETINA DISORDERS - Google Patents

Description

Field of invention

The present invention relates to therapies for the treatment and/or prevention of retinal disorders, in particular cone dystrophies, cone-rod dystrophies and achromatopsia.

Prior Art

In many mammalian species, including mice and humans, the rod photoreceptors, which mediate vision in dim light, greatly outnumber the cone photoreceptors. However, in the industrialized world, in which lighting enables cone function throughout the day and night, rod-mediated vision is less important. Many patients with absent rod function are identified only incidentally from birth and, in fact, are unable to recognize their visual impairment. In contrast, when cone dysfunction occurs, patients are always symptomatic and often suffer from visual impairment that depends on the degree of their cone dysfunction.

In some conditions, only or mainly the cones are lost or do not function normally, while the rods remain relatively intact. Such conditions may be known as cone-rod dystrophy or cone-rod dystrophy (CRD). Cone or cone-rod dystrophies are inherited retinal dystrophies characterized by the initial loss of cones or sometimes the simultaneous loss of both rods and cones. Symptoms include loss of vision, sensitivity to bright light, and poor color vision. For example, achromatopsia is a severe, inherited retinal dystrophy with complete absence of cone function from birth, but presumably normal rod function. Mutations in multiple genes, including CNGA3, CNGB3 and PDE6C, are associated with this disease. Each of the disease-causing genes encodes an essential component of the cone phototransduction cascade, which converts light into an electrical signal by hyperpolarizing the photoreceptor cell. A deficiency, for example, in the CNGA3 or CNGB3 protein in cone photoreceptor cells results in the inability of these cells to hyperpolarize in response to light. As a result, the cells initially survive but do not function, and the patient suffers from poor visual acuity, lack of color vision, and photophobia from birth. Different groups have developed therapy protocols in CNGA3-deficient mice that improve survival and cone function, as well as vision.

Other examples of causative genes involved in the pathogenesis of cone dystrophies include KCNV2, PDE6H, GNAT2, and CACNA2D4. The KCNV2 gene encodes protein member 2 of subfamily V of voltage-gated potassium channel modifiers. Mutations in KCNV2 are associated with cone dystrophy with abnormal rod electroretinogram (ERG) or cone retinal dystrophy type 3B, an autosomal recessive disorder that causes lifelong vision loss in combination with an abnormal ERG response to a bright flash of light. The PDE6H gene encodes the inhibitory (gamma) subunit of the cone-specific cGMP (cyclic guanosine monophosphate) phosphodiesterase. Mutations in this gene are associated with cone retinal dystrophy type 3A (RCD3A). The GNAT2 gene encodes the cone-specific alpha subunit of transducin. Mutations in the gene can lead to infantile cone dystrophy. The CACNA2D4 gene encodes voltage-gated calcium channel alpha 2/delta subunit 4. Mutations in this gene can cause non-progressive cone dysfunction (retinal cone dystrophy 4, RCD4).

In age-related macular degeneration (AMD), vision impairment is primarily caused by degeneration of the cone-rich fovea in the central macula. Thus, patients lose central vision and acuity, but often have a relatively well-preserved peripheral macula and thus have some level of useful residual vision that is limited by cone deficiency outside the fovea.

There is a need to develop therapies that can improve cone survival and function to treat or prevent retinal disorders such as cone-rod dystrophies.

Summary of the invention

According to the invention, nucleic acids, transcriptional control units (TCU), optimized gene sequences, expression constructs and vectors for gene expression in cone photoreceptors are proposed.

The TCUs disclosed herein contain the M-opsin promoter or a fragment thereof under the control of the M/L-opsin locus control region (LCR) and are useful for driving high levels of expression in all three types of human cones .

Also proposed are expression constructs containing the human CNGA3 gene under the control of TCU, optimized for gene expression in cone photoreceptors, where TCU contains the M-opsin promoter or its fragment under the control of the M/L-opsin locus-control region.

In some embodiments, the TCU and expression constructs contain a 6 bp mutation. (par nuk

- 1 046019 leotides) immediately after the transcription initiation site in the M-opsin promoter or its fragment (M8 mutation), where this mutation can increase the effect of treatment from vectors and expression constructs containing this mutation over time.

In addition, a codon optimized sequence of the CNGA3 gene has been proposed and is provided as SEQ ID NO: 8.

Vectors, such as viral vectors, containing expression constructs disclosed herein are also provided. The expression construct is preferably delivered using a vector derived from adenovirus serotype 8 (AAV8) or an alternative strong AAV serotype.

The invention also provides methods for using nucleic acids, transcription control units (TCUs), optimized gene sequences, expression constructs and vectors for the treatment and/or prevention of retinal disorders or dystrophies, including but not limited to cone dystrophies such as achromatopsia.

Accordingly, in one aspect of the invention there is provided a transcription control unit (TCU) up to 2500 nucleotides in length comprising, in the 5' to 3' direction:

(a) a locus control region (LCR) containing:

(i) SEQ ID NO: 1 or (ii) a sequence having at least 90% sequence identity with the specified sequence (a)(i); and (b) a promoter element comprising:

(i) at least 200 nucleotides of either SEQ ID NO: 2 or SEQ ID NO: 17; or (ii) a sequence having at least 90% sequence identity with the specified sequence (b)(i);

wherein said TCU exhibits cone photoreceptor-specific promoter activity.

According to the above aspect, the promoter element (b) may optionally comprise at least the last 200 or the last 500 nucleotides of either SEQ ID NO: 2 or SEQ ID NO: 17, or a sequence having at least 90% identity sequences with the last 200 or last 500 nucleotides of either SEQ ID NO: 2 or SEQ ID NO: 17.

In any of the above aspects, the promoter element (b) may comprise at least 200 nucleotides of SEQ ID NO: 3, optionally wherein the promoter element (b) also contains a sequence of at least 10 contiguous nucleotides selected from nucleotides 1-35 of SEQ ID NO: 3, or a sequence containing at least 10 contiguous nucleotides selected from a sequence having at least 90% sequence identity to nucleotides 1-35 of SEQ ID NO: 3.

In any of the above aspects, promoter element (b) may comprise SEQ ID NO: 3 [529 bp promoter element in hG1.7] or SEQ ID NO: 5 [247 bp promoter element ., in hG1.4] or a sequence having at least 90% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 5.

In any of the above aspects, the promoter element may further comprise SEQ ID NO: 16 [M8 mutation]. For example, in accordance with any of the above aspects, nucleotides corresponding to nucleotides 1934-1939 (GGGCCG) of SEQ ID NO: 2 may be replaced by SEQ ID NO: 16.

In one aspect, TCU comprises SEQ ID NO: 4 [variant hG1.7 promoter construct missing four nucleotides], SEQ ID NO: 6 [hG1.4 construct] or SEQ ID NO: 15 [hG1.7 promoter construct in product].

The invention also provides an expression construct containing TCU as described herein, wherein the TCU is operably linked to a sequence to be expressed in a cone photoreceptor specific manner. In one embodiment, the sequence operably associated with TCU comprises a gene encoding CNGA3, CNGB3, PDE6C, PDE6H, GNAT2, KCNV2, or CACNA2D4. In some embodiments, the operably linked sequence comprises SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14, or has at least 80% sequence identity to SEQ ID NO: 7, 8, 9, 10. 11, 12, 13 or 14 and has the ability to restore cone photoreceptor function. In one embodiment, the operably linked sequence comprises SEQ ID NO: 8 [CNGA3 codon optimized sequence], or a sequence that has at least 80% sequence identity to SEQ ID NO: 8 and has the ability to restore cone photoreceptor function.

The invention also provides vectors containing any of the nucleic acids, TCUs, promoter fragments, codon optimized genes and/or expression constructs described herein. In some embodiments, the vector is a viral vector.

In some embodiments, the vector is an AAV vector and/or contains an AAV genome or

- 2 046019 its derivative. In one embodiment, the derivative is a chimeric, shuffled, or capsid-modified derivative. In one embodiment, the AAV genome is derived from a naturally occurring AAV serotype or isolate or clade. In one embodiment, the AAV genome is derived from AAV serotype 2 (AAV2), AAV serotype 4 (AAV4), or AAV serotype 8 (AAV8), and/or the AAV capsid is derived from AAV8. In a preferred embodiment, the genome is derived from AAV2 and the capsid is derived from AAV8. In one embodiment, the AAV vector carries a gene encoding CNGA3.

The invention further provides host cells containing a nucleic acid or vector disclosed herein, as well as host cells that produce a nucleic acid or viral vector as disclosed herein. In one embodiment, the host cell is a HEK293 or HEK293T cell.

Also provided are pharmaceutical compositions containing the nucleic acid or vector described herein and a pharmaceutically acceptable carrier.

The invention further provides methods of using the nucleic acids, vectors, optimized gene sequences and/or expression constructs described herein in a method for preventing or treating retinal disorders. In one embodiment, the nucleic acids, vectors, optimized gene sequences and/or expression constructs described herein are used in the manufacture of a medicament for the treatment or prevention of retinal disorders. Also provided is a method of treating or preventing retinal disorders in a patient in need thereof, comprising administering a therapeutically effective amount of a vector disclosed herein. In one embodiment, the retinal disorder is achromatopsia. In some embodiments, the vector is administered to a patient via direct retinal injection, subretinal injection, or intravitreal injection.

Brief description of graphic materials

In fig. Figure 1 shows the results of an in vivo reporter gene expression study in transgenic mice to evaluate the effect of locus control region (LCR) position on the green (M) opsin core promoter. Top left: Row of normal human red and green pigment genes, showing positions of LCRs, transcription units, and exons. Bottom left: Rows of modified visual pigment genes, shown at 10x magnification. The initiation sites and direction of transcription are indicated by arrows. P _red is the promoter of the human red pigment gene. _{G reen} is _the promoter of the human green pigment gene. AR (from the English alkaline phosphatase), human placental alkaline phosphatase. lacZ, E. coli β-galactosidase. Right: Pie charts showing the proportion of transgene-expressing cells that express AP only (red), lacZ only (dark green), and AP and lacZ (yellow) or lacZAP (light green) in chimeric or germline-transmitted mice lines for the designs shown on the left. The number of cells from different mice derived from the same ES cell line were pooled to produce one single pie chart. Cell numbers for those mouse lines from which the PGK-neo marker was removed by crossing with cre germline mice are shown immediately to the right of the pie chart for the corresponding parental line.

In fig. Figure 2 shows a schematic representation of the chromosomal rearrangement of the red (L-) and green (M-) opsin promoters (boxed, top). Schematic representations of previously designed transcription units (pR2.1 and PR1.7), as well as the designed transcription control units disclosed in this study. The LCR is a locus control region.

In fig. Figure 3 illustrates the transduction profile of cone cells transduced with the AAVssh10 vector expressing green fluorescent protein (GFP) under the control of TCU hG1.4. Cryosections of human retina derived from an embryonic stem cell transduced with AAVshh10-hG1.4(M8)-GFP are shown. The transduction profile of cone cells was visualized by GFP imaging (A). Co-localization of blue opsin (S-opsin; B) with GFP (B') and red/green opsin (L/M-opsin; C) with GFP (C) is shown after staining with antibodies that bind either blue opsin ( S-opsin), or with red/green opsins (L/M opsin).

In fig. Figure 4 illustrates that inclusion of the M8 sequence in TCU enhances the restoration of photopic responses in CNGA3 knockout mice. Shown are the photopic electroretinogram (ERG) responses of Cnga3 knockout mice treated with AAV2/8 vectors carrying codon-optimized CNGA3 (coCNGA3) constructs driven by different TCUs with or without the M8 sequence. Shown are ERG responses at 1 month (left columns) and 2 months (right columns) after injection. All animals were treated at 1 month of age, with the exception of the far right group; this group was treated for 2 weeks.

In fig. Figure 5 illustrates that codon-optimized CNGA3 restores photopic responses in CNGA3 knockout mice more effectively than wild-type CNGA3. Photopic ERG responses in Cnga3 knockout mice treated with AAV2/8 vectors carrying codon-optimized CNGA3 (co) and wild-type CNGA3 (non-co) constructs.

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Fig. 6 illustrates that the AAV2/8 vector expressing CNGA3 under the control of TCU hG1.4 is effective in restoring cone function in CNGA3 knockout mice. (A) Photopic ERG traces of AAV8-treated and untreated Cnga3 knockout mice. A- and B-waves are annotated. The Y-axis represents µV. Light brightness settings: 10 cd/ ^m2 . (B) Photopic ERG responses of Cnga3 knockout mice treated with either AAV2/8-hG1.4(M8).coCNGA3 or AAV2/5-hG1.4(M8).coCNGA3. Light brightness settings: 10 cd/ ^m2 .

Fig. 7 illustrates long-term in vivo restoration of retinal sensitivity by AAV2/8 vectors expressing CNGA3 under the control of two different optimized TCUs up to 6 months post-treatment. Cnga3-deficient mice at 2 weeks of age were injected subretinal with either AAV2/8-hG1.4(M8).coCNGA3 (n = 14) or AAV2/8-hG1.7(M8).coCNGA3 (n = 13) (titer 1x 10 ¹² mg/ml in both cases). Unprocessed (n equals 3). Light brightness settings: 10 cd/ ^m2 .

Fig. 8 illustrates increased cone survival in vivo 3-4 months after treatment with AAV2/8 vectors expressing CNGA3 under the control of TCU hG1.7. Single-plane confocal images of flat mount retinas from a 3- to 4-month-old C57BL/6J mouse (A), or an age-matched Cnga3-deficient mouse that was uninjected (B) or injected (C) with AAV2/8-hG1.7 (M8).coCNGA3 at 2 weeks of age. Retinas were stained with cone arrestin and cleared. Scale bar: 5 µm.

Fig. 9 illustrates long-term increased cone survival in vivo 13 months after treatment with an AAV2/8 vector expressing CNGA3 under the control of TCU hG1.7. Z-projection confocal images (A,B) or single-plane confocal images (C,D) of a flat mount retina from a 14-month-old Cnga3-deficient mouse injected with AAV2/8hG1.7(M8).coCNGA3 at age 2 weeks. Untreated mice do not show positive PNA staining at this age. Retinal flat mounts were stained with PNA (A, C) and cone arrestin (B, D) and cleared. Scale bar: 10 µm (A, B), 5 µm (C, D).

Fig. 10 illustrates quantification of improvement in synaptic integrity between cone cells and neuronal maintenance (bipolar cells) in vivo 3-4 months after treatment with AAV2/8 vector expressing CNGA3 under the control of TCU hG1.7. Assessment was performed using the signal intensity of the synapse marker Gpr179. Signal intensity analysis of Gpr179 staining was performed on single-plane confocal images of retinal flat mounts from a 3-4 month old C57BL/6J mouse or an age-matched Cnga3-deficient mouse that was not injected or injected with AAV2/8-hG1.7(M8) .coCNGA3 at 2 weeks of age. Retinas were stained with Gpr179 and PNA and then cleared. Gpr179 staining was monitored by freehand line drawing for several Gpr179 stains associated with the cone stalk (PNA staining was used to confirm cone stalks) and more than 10 Gpr179 stains associated with the rod spherule (A). Signal intensity was calculated (B; white line: Gpr179, red line: PNA). The signal intensity peaks from each source were averaged, and the ratio of GPr179 staining associated with cone stalks to GPr179 staining associated with rod spherules (CP/RS) was calculated. CP/RS from four different positions was used for statistical analysis (Bonferroni multiple comparison test (ns): p greater than 0.05, **: p less than or equal to 0.01, *: p less or equal to 0.05)). The error bar shows SEM (Standard Error of the Mean). (WITH).

Fig. 11 illustrates that expression of CNGA3 under the control of TCU hG1.4 in AAV8 vectors results in improved ERG responses in CNGA3-deficient mice compared to AAV Anc80L65, AAV44.9 or AAV5 vectors, respectively. (A). Comparison of Anc80L65 and AAV8. AAV-Anc80L65 or AAV8 carrying the hG1.4(M8).coCNGA3 expression cassette was delivered to Cnga3-deficient mice at 2 weeks of age. **: p less than or equal to 0.01, *: p less than or equal to 0.05. Error bars indicate SEM. (IN). Comparison of AAV8 and AAV44.9 for delivery of CNGA3 in Cnga3-deficient mice at 4 weeks of age. Error bars indicate SEM. (C) Comparison of AAV5 and AAV8. AAV5 or AAV8 carrying the hG1.4(M8).coCNGA3 expression cassette was delivered to Cnga3-deficient mice at 2 weeks of age. **: p less than or equal to 0.01, *: p less than or equal to 0.05. Error bars indicate SEM.

Fig. 12 illustrates the improved expression levels of TCU hG1.4 and hG1.7 carrying the M8 mutation compared to the expression levels observed for known cone promoters. (A) 17- to 19-week-old hEBs were transduced with AAVShH10 expressing eGFP under two different green opsin promoters (hG1.4 and hG1.7) and harvested 2 weeks later ( n = 6–8 for each promoter). After dissociation, cells were analyzed with respect to the relative median fluorescence intensity (MFI) in GFP-positive cells (relative MFI in hEB transduced with AAVShH10-eGFP, analyzed on the same day of the experiment, was calculated as a ratio to MFI in EVs transduced with AAV ShH10-1.7L-eGFP) by flow cytometry. An asterisk indicates a significant difference (p less than or equal to 0.01). Error bars indicate SEM. (B) Photopic ERG responses of treated Cnga3 knockout mice treated with either AAV2/8-CAR-CNGA3 or left untreated. The Y-axis represents µV. Setting the light brightness 4 046019: 10 cd/m ² .

Brief description of sequences

SEQ ID NO: 1 shows the DNA sequence of a 1.2 kb fragment of the human M/L opsin locus control region. (one thousand base pairs)

SEQ ID NO: 2 shows the DNA sequence of a 2.0 kb fragment of the human opsin promoter.

SEQ ID NO: 3 shows the DNA sequence of a 500 bp fragment of the human opsin promoter M.

SEQ ID NO: 4 shows the DNA sequence of the hG1.7(M8) variant construct, which consists of a 1.2 kb human opsin M/L locus control region fragment followed by a human opsin promoter M fragment 500 bp in size, and the specified fragment of the opsin promoter includes the M8 mutation.

SEQ ID NO: 5 shows the DNA sequence of a 200 bp fragment of the human opsin promoter M.

SEQ ID NO: 6 shows the cDNA sequence of the hG1.4(M8) construct, which consists of a 1.2 kb human opsin M/L locus control region fragment followed by a human opsin promoter M fragment of 200 bp, and the specified fragment of the opsin promoter includes the M8 mutation.

SEQ ID NO: 7 shows the cDC sequence of the human CNGA3 gene.

SEQ ID NO: 8 shows the codon optimized cDNA sequence of the human CNGA3 gene.

SEQ ID NO: 9 shows the cDC sequence of the human PDE6C gene.

SEQ ID NO: 10 shows the cDNA sequence of the human PDE6H gene.

SEQ ID NO: 11 shows the cDNA sequence of the human GNAT2 gene.

SEQ ID NO: 12 shows the cDNA sequence of the human KCNV2 gene.

SEQ ID NO: 13 shows the cDNA sequence of the human CACNA2D4 gene.

SEQ ID NO: 14 shows the cDNA sequence of the human CNGB3 gene.

SEQ ID NO: 15 shows the DNA sequence of the hG1.7(M8) construct, which contains a 1.2 kb human opsin M/L locus control region fragment followed by a GATC sequence and an M opsin promoter fragment human 500 bp in size, and the specified fragment of the opsin promoter includes the M8 mutation.

SEQ ID NO: 16 shows the sequence of the M8 mutation.

SEQ ID NO: 17 shows the DNA sequence of a 2.0 kb M fragment of the human opsin promoter containing the M8 mutation.

Detailed Description of the Invention

It should be understood that various uses of the disclosed polynucleotide sequences may be tailored to specific needs in the art. It should also be understood that the terminology used herein is offered for the purpose of describing specific embodiments of the invention only and is not intended to be limiting.

Further, as used in this specification and the accompanying claims, terms in the singular include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a polynucleotide includes polynucleotides, a reference to a promoter includes promoters, a reference to a vector includes two or more such vectors, and the like. M/L opsin and L/M opsin are used interchangeably to refer to green and red opsin.

All publications, patents and patent applications cited herein, above or below, are incorporated herein by reference in their entirety.

Transcription control units (TCU)

In one aspect, the invention provides a TCU optimized for gene expression in cone photoreceptor cells. In one embodiment, the disclosure provides a TCU that contains an M/L opsin locus control region (LCR) fragment. In a preferred embodiment, the TCU contains a fragment of the human M/L opsin locus control region (LCR).

In another embodiment, the disclosure provides a TCU that contains a promoter region, such as the M opsin promoter or a fragment thereof. In a preferred embodiment, the TCU disclosed herein comprises the human M opsin promoter or a fragment thereof.

In some embodiments, the TCU comprises fragments and/or variants of the human M/L opsin locus control region (LCR) and the human M opsin promoter or fragment thereof, wherein the TCU has cone photoreceptor-specific promoter activity.

In one embodiment, the TCU contains an LCR that contains a nucleotide sequence, typically contiguous nucleotides, from SEQ ID NO: 1 that provides cone photoreceptor-specific expression of an operably linked polynucleotide sequence. In addition, an LCR is contemplated that contains a sequence that has at least

- 5 046019 at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or according at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the LCR comprises a deletion or insertion of one or more nucleotides, where the deletion or insertion does not abolish cone photoreceptor-specific expression of a gene operably associated with the modified LCR.

In one embodiment, the TCU comprises an M opsin promoter or fragment thereof, wherein the M opsin promoter or fragment thereof comprises a nucleotide sequence, typically contiguous nucleotides, from SEQ ID NO: 2 or SEQ ID NO: 17 that provides cone photoreceptor-specific expression of an operably linked polynucleotide sequence. The M opsin promoter or fragment thereof may, for example, contain up to 1200 nucleotides of SEQ ID NO: 2 or SEQ ID NO: 17, and preferably no more than 1100, no more than 1000, no more than 900, no more than 800, no more than 700, no more than 600, no more than 500, no more than 400, no more than 300, or no more than 200 nucleotides of SEQ ID NO: 2 or SEQ ID NO: 17. In some embodiments, the M opsin promoter fragment contains of at least 200, 300, 400 or 500 nucleotides of SEQ ID NO: 2 or SEQ ID NO: 17. In addition, a fragment of the M opsin promoter is contemplated having at least 75%, at least 80%, at least at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with at least 200, at least 300, at least 400 or at least 500 contiguous nucleotides of SEQ ID NO: 2 or SEQ ID NO: 17. In some embodiments, the M-opsin promoter or fragment thereof comprises a deletion or insertion of one or more nucleotides, where the deletion or insertion does not eliminate a specific in relation to the cone photoreceptor, the expression of a gene operably linked to a modified M-opsin promoter or fragment. In some embodiments, the M-opsin promoter or fragment thereof consists essentially of SEQ ID NO: 2 or SEQ ID NO: 17. In some embodiments, the M-opsin promoter or fragment thereof consists of SEQ ID NO: 2 or SEQ ID NO: 17.

Preferably, the TCU contains an M opsin promoter fragment that contains SEQ ID NO: 3 or a sequence that is substantially identical to SEQ ID NO: 3. Also contemplated is a TCU containing an M opsin promoter fragment that contains at least 200, at least 300, at least 400 or at least 500 nucleotides of SEQ ID NO: 3 or sequence having at least 75%, at least 80%, at least 85%, at least 90% -th, at least 95%, at least 98%, or at least 99% sequence identity to at least 200, at least 300, at least 400, or at least 500 nucleotides of SEQ ID NO: 3. In some embodiments, the M opsin promoter fragment consists essentially of SEQ ID NO: 3. In some embodiments, the M opsin promoter fragment consists of SEQ ID NO: 3.

In some embodiments, the TCU contains at least 200 nucleotides of SEQ ID NO: 3 or a sequence having at least 90% sequence identity to at least 200 nucleotides of SEQ ID NO: 3 and a sequence containing at least 10 of at least at least 15, at least 20, at least 25, at least 30, or at least 35 contiguous nucleotides 1-35 SEQ ID NO: 3. In some embodiments, the TCU contains at least 200 SEQ ID NO: 3 nucleotides or sequences having at least 90% sequence identity to at least 200 nucleotides of SEQ ID NO: 3 and a sequence having at least 90% sequence identity to at least 10, at least 15, at least 20, less than 25, at least 30, or at least 35 contiguous nucleotides corresponding to nucleotides 1-35 of SEQ ID NO: 3.

Preferably, the TCU contains an M opsin promoter fragment that contains SEQ ID NO: 5 or a sequence that is substantially identical to SEQ ID NO: 5. In some embodiments, the M opsin promoter fragment consists essentially of SEQ ID NO: 5. In some embodiments, the M fragment opsin promoter consists of SEQ ID NO: 5.

Additional promoters and fragments thereof considered for use in TCU are promoters or promoter fragments that differ in sequence from the sequences above but retain cone photoreceptor-specific promoter activity. Such sequences have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the contiguous nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 17, as defined above. The percentage sequence identity of variants is preferably measured over the entire length of the corresponding portion of SEQ ID NO: 2 or SEQ ID NO: 17 or over a region of 500, 600, 700, 800, 900, 1000, 1100 or 1200 nucleotides of SEQ ID NO: 2 or SEQ ID NO: 17, aligned to a sequence variant. Also contemplated are promoters and fragments thereof that contain a sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%%, at least 98%% or

- 6 046019 at least 99% sequence identity to SEQ ID NO: 3 and/or to SEQ ID NO: 5.

Sequence identities may be calculated using any suitable algorithm. For example, the PILEUP and BLAST algorithms can be used to calculate sequence identity or alignment (such as identifying equivalent or corresponding sequences (usually at their default settings), for example, as described in Altschul S. F. (1993) J Mol Evol 36:290-300 Altschul, S, F et al (1990) J Mol Biol 215:403-10). Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or meet some positive scoring threshold T when aligned with a word of the same length. in the database sequence. T is called the word similarity threshold (Altschul et al., supra). These initial matches of adjacent words act as seeds to initiate searches to find HSPs containing them. Word matches are expanded in both directions along each sequence as much as the cumulative alignment score can be increased. Extensions for word matches in each direction stop when: the cumulative alignment score decreases by an amount X from its maximum achieved value; the cumulative score drops to zero or below due to the accumulation of one or more balance alignments with a negative score; or the end of each sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of alignment. The BLAST program uses a default word length (W) of 11, BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) 50, expectation (E) 10 , M=5, N=4 and comparison of both circuits.

The BLAST algorithm performs a statistical analysis of the similarity of two sequences; see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One quantitative measure of similarity provided by the BLAST algorithm is the lowest cumulative probability (P(N)), which provides a measure of the probability with which a match between two polynucleotide or amino acid sequences will occur by chance. For example, a sequence is considered to be similar to another sequence if the smallest cumulative probability, when comparing the first sequence with the second sequence, is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. As an alternative, the UWGCG package offers a program called BESTFIT that can be used to calculate identity (eg, used with its default settings) (Devereux et al. (1984) Nucleic Acids Research 12, 387-395).

In some embodiments, the TCU contains an M-opsin promoter or fragment thereof that contains a mutant M8 sequence TCTAGA (SEQ ID NO: 16). In one embodiment, the TCU contains an M-opsin promoter or fragment thereof that contains one, two, three, four, five or six nucleotides of SEQ ID NO: 16. For example, nucleotides corresponding to nucleotides 1934 - 1939 (GGGCCG) SEQ ID NO: 2 , may be replaced by SEQ ID NO: 16.

In some embodiments, TCU includes additional nucleotide sequences not naturally found in the M/L opsin LCR and/or M-opsin promoter regions. Additional nucleotide sequences may be 5' or 3' of either the LCR or the M-opsin promoter region. In some embodiments, the additional sequence is located between the LCR and the M-opsin promoter region. In one embodiment, the GATC sequence is located between the LCR region and the M-opsin region.

In one embodiment, the TCU contains SEQ ID NO: 4. In one embodiment, the TCU contains SEQ ID NO: 6. In one embodiment, the TCU contains SEQ ID NO: 15.

In addition, a TCU containing a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, is contemplated. at least 98% or at least 99% sequence identity to SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 15.

In one embodiment, the TCU consists essentially of SEQ ID NO: 4. In one embodiment, the TCU consists essentially of SEQ ID NO: 6. In one embodiment, the TCU consists essentially of SEQ ID NO: 15.

In one embodiment, the TCU consists of SEQ ID NO: 4. In one embodiment, the TCU consists of SEQ ID NO: 6. In one embodiment, the TCU consists of SEQ ID NO: 15.

The TCU may additionally be placed elsewhere within the larger sequence, as long as the cone photoreceptor-specific promoter activity is retained. In embodiments, the TCUs described herein are located proximal to or immediately 5' to the gene to be expressed (eg, load) in a cone photoreceptor specific manner as described herein.

TCU can also be used in tandem with other regulatory elements, such as one or more additional promoters, enhancers and/or LCRs.

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TCU may be provided as an isolated nucleic acid molecule.

The TCU provided by this disclosure can be used to control gene expression (load) in a cone photoreceptor in a cone photoreceptor-specific manner. Cone photoreceptor-specific expression can be defined as expression that occurs only in the cone photoreceptor and not significantly in other cell types. Cone photoreceptor-specific expression can be defined as expression that is greater than about 10-fold higher, 20-fold higher, 50-fold higher, or 100-fold or more higher in a cone photoreceptor than in other cell types, especially rod cells. photoreceptor cells. Expression in cone photoreceptors and other cell types can be measured by any suitable standard technique known to one of ordinary skill in the art. For example, RNA expression levels can be measured by real-time quantitative PCR (polymerase chain reaction). Protein expression can be measured by Western blotting or immunohistochemistry. The TCUs proposed herein mediate functionally linked gene expression in all cone photoreceptor subtypes.

The TCU proposed by this disclosure can be used to drive significantly increased gene expression in a cone photoreceptor, compared to a reference TCU or promoter. Significantly increased expression can be defined as gene expression in a cone photoreceptor increased by more than about 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, or 300-fold, compared to expression driven by the reference TCU or promoter, including the parent Mopsin promoter, but not limited to it. Expression in cone photoreceptors and other cell types can be measured by any suitable standard technique known to one of ordinary skill in the art. For example, DNA expression levels can be measured by quantitative real-time PCR. Protein expression can be measured by Western blotting or immunohistochemistry.

The TCU provided by this disclosure can be used to control the expression of a protein-coding nucleotide sequence in a cone photoreceptor, including nucleotide sequences expressing proteins that are not normally expressed in a cone photoreceptor, such as GFP.

For example, the TCUs provided in this disclosure are useful for the expression of genes in cone photoreceptors required for normal cone photoreceptor function, including, but not limited to, the guanine nucleotide binding protein G(t) alpha 2 subunit (GNAT2), a cyclic nucleotide-gated cation channel alpha -3 (CNGA3), cyclic nucleotide gated cation channel beta 3 (CNGB3), alpha subunit of cone cGMP-specific 3',5'-cyclic phosphodiesterase (PDE6C), subunit of gamma cGMP-specific 3',5' cyclic phosphodiesterase, retinal cone rhodopsin-sensitive (PDE6H), voltage-gated potassium channel subfamily V member 2 (KCNV2), and voltage-gated calcium channel alpha 2/delta 4 subunit (CACNA2D4), which are essential proteins for normal cone function. In this regard, the present invention provides TCUs and methods for expressing, for example, the genes GNAT2, CNGA3, CNGB3, PDE6C, PDE6H, KCNV2 and CACNA2D4 in cone photoreceptors. The genes PDE6C, GNAT2 CNGA3 and CNGB3 are four of the genes contributing to achromatopsia. PDE6C is the alpha subunit of cone cGMP-specific 3',5'-cyclic phosphodiesterase. GNAT2 is a component of cone transducin, an essential element of the phototransduction cascade in cones. CNGA3 is a subunit of the cyclic nucleotide-gated cone ion channel that closes in response to light, thereby hyperpolarizing the cone cell.

Expressive designs

The invention also provides expression constructs comprising the TCU disclosed herein operably linked to a sequence, such as a gene sequence, to be expressed in a cone photoreceptor specific manner.

The term operably coupled refers to the immediate proximity in which the described components are in a relationship that allows them to function properly. A control sequence operably linked to the coding sequence is ligated such that expression of the coding sequence is achieved under conditions comparable to the control sequences. Multiple copies of the same or different polynucleotide can be introduced into an expression construct. An expression construct can be defined as a polynucleotide sequence capable of directing the expression of a protein from a polynucleotide sequence comprising a coding sequence.

Thus, the expression construct may, for example, contain a PDE6H, PDE6C, GNAT2, KCNV2, CACNA2D4, CNGA3 or CNGB3 coding sequence, for example, a polynucleotide selected from SEQ ID NO: 7-14, or a variant of SEQ ID NO: 7-14, which retains functionality

- 8 046019 protein translated from a sequence selected from SEQ ID NO: 7-14.

A polynucleotide variant selected from the group consisting of SEQ ID NO: 7-14 can be defined as any variant of the sequence of SEQ ID NO: 7-14, including naturally occurring variants in the nucleic acid sequence. A variant may be defined as having at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs 7 - 14, where the polypeptide translated from the sequence variant retains its functionality. A variant may be defined as having at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NO: 7-14, where the polypeptide translated from the sequence variant has the ability to restore cone photoreceptor function. In embodiments, this variant is a codon optimized version of the coding sequence.

The expression constructs contemplated by the disclosure can restore cone photoreceptor function. Restoration of cone photoreceptor function can be defined as recovery of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of cone photoreceptor function. Cone photoreceptor function can be analyzed by any suitable standard technique known to one skilled in the art, for example by analyzing retinal responses based on electroretinography.

Restoration of cone photoreceptor function can also be defined as prolongation of cone survival. Prolongation of cone survival can be defined as an increase in the period of time over which a cone photoreceptor is functional by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or greater than 100%, compared to a cone photoreceptor affected by cone dystrophy. Cone photoreceptor function can be analyzed by any suitable standard technique known to one skilled in the art, for example, by analyzing retinal responses based on electroretinography. Examples of prolonging cone survival also include improving ERG activity or slowing loss of ERG activity, improving retinal sensitivity, or slowing/reversing vision loss.

The expression construct of the invention may comprise a TCU operably linked to the CNGA3 gene. In some embodiments, the CNGA3 gene sequence comprises SEQ ID NO: 7. In some embodiments, the CNGA3 gene comprises a codon optimized sequence. The phrase codon optimization refers to a method of altering a naturally occurring polynucleotide sequence to enhance expression in a target organism, such as a human. In one embodiment of the present invention, the human CNGA3 gene, SEQ ID NO: 7, has been optimized to create SEQ ID NO: 8. In the cDNA of this optimized CNGA3 SEQ ID NO: 8, the few codons have been replaced with codons that are more commonly found and/or codons which are often found in human genes expressed at high levels.

In one embodiment, the expression construct comprises a TCU operably linked to SEQ ID NO: 8.

Vectors

The invention provides vectors containing nucleic acids, TCUs, promoters and fragments thereof, optimized genes and expression constructs disclosed herein. The vector can be of any type, for example it can be a plasmid vector or minicircular DNA.

The effectiveness of therapy generally depends on the proper and efficient delivery of donor DNA. This method is usually mediated by viral vectors. In this regard, the invention provides viral vectors which can be based, for example, on herpes simplex virus, adenovirus or lentivirus. The viral vector may be an adeno-associated virus (AAV)-based vector or a derivative thereof. AAV is a particularly attractive vector because it is generally nonpathogenic; most people have been infected with this virus during their lifetime without adverse effects. Immune privilege of ocular tissue, the result of anatomical barriers and immunomodulatory factors, renders the eye largely free from deleterious immunological reactions.

In one embodiment, the viral vector contains an AAV genome from a naturally occurring serotype, an AAV isolate or clade, or a derivative thereof.

The term AAV genome is a polynucleotide sequence that encodes the functions necessary to produce the AAV viral particle. These functions include functions operating in the AAV replication and packaging cycle in the host cell, including encapsidation of the AAV genome into the AAV viral particle. Naturally occurring AAV viruses are replication defective and rely on the presence of helper functions in trans to complete the replication and packaging cycle. Accordingly, and with the additional removal of the AAV rep and cap genes, the AAV vector genome of the invention is replication defective.

- 9 046019

The AAV genome may be present as a single strand, either sense or antisense, or alternatively as two strands. The use of a double-stranded form allows one to bypass the stage of DNA replication in the target cell and thus can accelerate the expression of the transgene.

The AAV genome can be derived from any naturally occurring AAV serotype or isolate or clade. As is known to one skilled in the art, AAV viruses occurring in nature can be classified according to different biological systems.

Typically, AAV viruses are referred to by their serotype. The term serotype corresponds to an AAV subtype variant that, due to its capsid surface antigen expression profile, has distinctive reactivity that can be used to distinguish it from other subtype variants. Typically, a virus of a particular AAV serotype does not effectively cross-react with neutralizing antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11, as well as recombinant serotypes such as Rec2 and Rec3 recently identified from primate brain. In the vectors of the invention, the genome can be derived from any AAV serotype. The capsid may also be derived from any AAV serotype. The genome and capsid may be from the same serotype or different serotypes.

In one embodiment, the genome of the vector disclosed herein is derived from AAV serotype 2 (AAV2), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), or AAV serotype 8 (AAV8). Other AAV vectors that can be used include those derived from AAV44.9 and AAVAnc80. Most preferably, the genome is derived from AAV2, which binds to target cells via the heparin sulfate proteoglycan receptor, but other serotypes of particular interest for use in the invention include AAV4, AAV5 and AAV8, which effectively transduce tissue in the eye, such as pigment epithelium retina.

AAV genome sequences or elements of AAV genomes, including ITR sequences, rep or cap genes for use in the invention, can be derived from the following accession numbers for AAV complete genome sequences: adeno-associated virus 1 NC_002077, AF063497; adeno-associated virus 2 NC_001401; adeno-associated virus 3 NC_001729; adeno-associated virus 3B NC_001863; adeno-associated virus 4 NC_001829; adeno-associated virus 5 Y18065, AF085716; adeno-associated virus 6 NC_001862; avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; avian AAV strain DA-3 NC_006263, AY629583; bovine AAV NC_005889, AY388617.

AAV viruses may also be referred to as clades or clones. It refers to the phylogenetic relationship of naturally occurring AAV viruses and typically refers to a phylogenetic group of AAV viruses that can be traced back to a common ancestor and includes all of its descendants. In addition, AAV viruses may be referred to by referring to a specific isolate, namely a genetic isolate of a particular AAV virus found in nature. The term genetic isolate describes a population of AAV viruses that has undergone limited genetic admixture with other naturally occurring AAV viruses, thereby defining a recognizably distinct population at the genetic level.

Examples of AAV clades and isolates that may be used in the invention include the following:

- 10 046019

Clade A: AAV1 NC_002077, AF063497, AAV6 NC_001862, Hu. 48 AY530611, Hu 43

AY530606, Hu 44 AY530607, Hu 46 AY530609,

Clade B: Hu. 19 AY530584, Hu. 20 AY530586, Hu 23 AY530589, Hu22 AY530588,

Hu24 AY530590, Hu21 AY530587, Hu27 AY530592, Hu28 AY530593, Hu 29 AY530594, Hu63

AY530624, Hu64 AY530625, Hu13 AY530578, Hu56 AY530618, Hu57 AY530619, Hu49

AY530612, Hu58 AY530620, Hu34 AY530598, Hu35 AY530599, AAV2 NC_001401, Hu45

AY530608, Hu47 AY530610, Hu51 AY530613, Hu52 AY530614, Hu T41 AY695378, Hu S17

AY695376, Hu T88 AY695375, Hu T71 AY695374, Hu T70 AY695373, Hu T40 AY695372, Hu

T32 AY695371, Hu T17 AY695370, Hu LG15 AY695377,

Clade C: Hu9 AY530629, Hu10 AY530576, Hull AY530577, Hu53 AY530615, Hu55

AY530617, Hu54 AY530616, Hu7 AY530628, Hu18 AY530583, Hu15 AY530580, Hu16

AY530581, Hu25 AY530591, Hu60 AY530622, Ch5 AY243021, Hu3 AY530595, Hu1

AY530575, Hu4 AY530602 Hu2, AY530585, Hu61 AY530623,

Clade D: Rh62 AY530573, Rh48 AY530561, Rh54 AY530567, Rh55 AY530568, Cy2 AY243020, AAV7 AF513851, Rh35 AY243000, Rh37 AY242998, Rh36 AY242999, Sub AY243016, Cy4AY 243018, Cy3AY243019, Cy5AY243017, Rh13 AY243013,

Clade E: Rh38 AY530558, Hu66 AY530626, Hu42 AY530605, Hu67 AY530627, Hu40

AY530603, Hu41 AY530604, Hu37 AY530600, Rh40 AY530559, Rh2 AY243007, YOU

AY243023, Bb2 AY243022, Rh10 AY243015, Hu17 AY530582, Hu6 AY530621, Rh25

AY530557, Pi2 AY530554, Pi1 AY530553, Pi3 AY530555, Rh57 AY530569, Rh50 AY530563,

Rh49 AY530562, Hu39 AY530601, Rh58 AY530570, Rh61 AY530572, Rh52 AY530565, Rh53

AY530566, Rh51 AY530564, Rh64 AY530574, Rh43 AY530560, AAV8 AF513852, Rh8

AY242997, Rh1 AY530556,

Clade F: Hu14 (AAV9) AY530579, Hu31 AY530596, Hu32 AY530597, clonal isolate AAV5 Y18065, AF085716, AAV 3 NC_001729, AAV 3B NC_001863, AAV4 NC_001829, Rh34 AY243001, Rh3 3 AY243002, Rh32 AY243003.

One skilled in the art may select the appropriate AAV serotype, clade, clone, or isolate for use in the present invention based on their current general knowledge.

However, it should be understood that the invention also covers the use of the AAV genome of other serotypes that may not yet be identified or characterized. AAV serotype determines the tissue specificity of infection (or tropism) of the AAV virus. Accordingly, preferred AAV serotypes for use in AAV viruses administered to patients in accordance with the invention are AAV serotypes that have a natural tropism for or high efficiency in infecting target cone photoreceptor cells.

Wild-type AAVs containing viral genes insert their genomic material into chromosome 19 of the host cell. The AAV single-stranded DNA genome contains two inverted terminal repeats (ITRs) and two open reading frames containing structural (cap) packaging (rep) genes.

Typically, the genome of an AAV serotype or naturally occurring AAV isolate or clade contains at least one inverted terminal repeat (ITR) sequence. The vectors of the invention typically contain two ITRs, preferably one at each end of the genome. The ITR sequence acts in cis to provide a functional origin of replication and mediates integration and excision of the vector from the cell genome. Preferred ITR sequences are AAV2 sequences and variants thereof. The AAV genome typically contains packaging genes, such as the rep and/or cap genes, which encode packaging functions for the AAV viral particle. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof. The cap gene encodes one or more capsid proteins, such as VP1, VP2 and VP3 or variants thereof. These proteins make up the capsid of the AAV viral particle. Capsid variants are discussed below.

For therapeutic purposes, ITRs can be provided in cis in addition to the therapeutic gene. The AAV virus can thus be modified: the viral genes can be removed from the genome, resulting in recombinant AAV (rAAV). rAAV contains a therapeutic gene and at least one ITR. Removal of viral genes renders rAAV unable to actively insert its genome into host cell DNA. Instead, rAAV genomes are fused via ITR to form circular, episomal structures, or are inserted into pre-existing chromosomal breaks. To produce viruses, the structural and packaging genes, now removed from rAAV, are supplied in trans, as a helper plasmid.

Preferably, the AAV genome will be derivatized for administration to patients. Such derivatization is standard in the art, and the present invention covers the use of any known AAV genome derivative and derivatives that could be produced by

- 11 046019 application of techniques known in the field.

AAV genome derivatives include any truncated or modified form of the AAV genome that allows expression of a Rep-1 transgene from a vector of the invention in vivo. Typically, it is possible to significantly truncate the AAV genome to include minimal viral sequence that still retains the above function. This is preferred for safety reasons to reduce the risk of recombination of the vector with the wild-type vector, and also to avoid stimulation of the cellular immune response by the presence of viral gene proteins in the target cell.

Typically, the derivative will include at least one inverted terminal repeat (ITR) sequence, preferably more than one ITR, such as two or more ITRs. One or more ITRs may be derived from AAV genomes of different serotypes, or may be a chimeric or mutant ITR. A preferred mutant ITR is an ITR having a trs (terminal resolution site) deletion. This deletion allows continuous genome replication to generate a single-stranded genome that contains both coding and complementary sequences, namely the self-complementary AAV genome. This provides a bypass for DNA replication in the target cell, and thus allows for accelerated expression of the transgene.

One or more ITRs will preferably flank an expression cassette construct containing a promoter and transgene of the invention. The inclusion of one or more ITRs is preferred to assist in packaging the vector of the invention into viral particles. In preferred embodiments, the ITR elements will be the only sequences retained from the native AAV genome in the derivative. Thus, the derivative will preferably not include the rep and/or cap genes of the native genome and any other sequences of the native genome. This is preferred for the reasons described above and also to reduce the possibility of integration of the vector into the genome of the host cell. In addition, reducing the size of the AAV genome provides increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the transgene.

With reference to the AAV2 genome, the following portions could thus be deleted in a derivative of the invention: one inverted terminal repeat (ITR) sequence, replication (rep) and capsid (cap) genes. However, in some embodiments, including in vitro embodiments, the derivatives may further include one or more rep and/or cap genes, or other viral sequences of the AAV genome.

The derivative may be a chimeric, shuffled, or capsid-modified derivative of one or more naturally occurring AAV viruses. The invention covers the provision of capsid protein sequences from different AAV serotypes, clades, clones or isolates in the same vector. The invention covers the packaging of the genome of one serotype into the capsid of another serotype, namely pseudotyping.

Chimeric, shuffled or capsid-modified derivatives will typically be selected to provide one or more desired functionality for the viral vector. Thus, these derivatives may exhibit increased gene delivery efficiency, reduced immunogenicity (humoral or cellular), altered range of tropism, and/or improved targeting of a specific cell type, compared to an AAV viral vector containing the naturally occurring AAV genome, such as AAV2. Increased efficiency of gene delivery may be influenced by improved receptor or coreceptor binding on the cell surface, improved internalization, improved migration within the cell and into the nucleus, improved decapsidation of the viral particle, and improved conversion of the single-stranded genome to the double-stranded form. Increased efficacy may also relate to an altered range of tropism or targeting of a specific cell population such that the vector dose is not carried away by administration into tissues where it is not needed.

Chimeric capsid proteins include proteins formed by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This can be accomplished, for example, through a genetic marker rescue approach in which non-infectious capsid sequences from one serotype are co-transfected with capsid sequences from another serotype, and directed selection is used to select capsid sequences having the desired properties. The capsid sequences of different serotypes can change as a result of homologous recombination in the cell to produce new chimeric capsid proteins.

Chimeric capsid proteins also include proteins formed by engineering capsid protein sequences to transfer specific capsid protein domains, surface loops, or specific amino acid residues between two or more capsid proteins, for example, between two or more capsid proteins of different serotypes.

Shuffled or chimeric capsid proteins can also be formed by DNA shuffling or by error-prone PCR. AAV capsid hybrid genes can create

- 12 046019 is given by randomly fragmenting sequences of related AAV genes, for example, genes encoding capsid proteins of many different serotypes, and then subsequently reassembling the fragments in a self-priming polymerase reaction, which can also cause crossing over in regions of sequence homology. A library of AAV fusion genes thus created by shuffling capsid genes from several serotypes can be screened to identify viral clones having the desired functionality. Likewise, error-tolerant PCR can be used to randomly mutate AAV capsid genes to create a distinct library of variants that can then be selected for a desired property.

Capsid gene sequences can also be genetically modified to introduce specific deletions, substitutions, or insertions relative to the native wild-type sequence. In particular, capsid genes may be modified by insertion of an unrelated protein or peptide sequence within the open reading frame of the capsid coding sequence or at the N and/or C terminus of the capsid coding sequence.

The unrelated protein or peptide may advantageously be a protein that acts as a ligand for a particular cell type, thereby conferring improved binding to the target cell or improving the specificity of targeting of the vector to a particular cell population.

The unrelated protein may also be a protein that helps purify the viral particle as part of the production process, such as an epitope or affinity tag. The insertion site will generally be selected so as not to interfere with other functions of the viral particle, eg internalization, migration of the viral particle. One skilled in the art can identify suitable insertion sites based on his or her general knowledge.

The invention further covers providing AAV genome sequences in a different order and configuration than the order and configuration of the native AAV genome. The invention also covers the replacement of one or more AAV sequences or genes with sequences from another virus or chimeric genes consisting of sequences from more than one virus. Such chimeric genes may consist of sequences from two or more related viral proteins from different virus species.

The vector of the invention takes the form of a viral vector containing the promoters and expression constructs of the invention.

To avoid ambiguity, the invention also provides an AAV viral particle containing a vector of the invention. The AAV particles of the invention include transcapsidated forms, where the AAV genome or derivative having an ITR of one serotype is packaged into a capsid of another serotype. The AAV particles of the invention also include mosaic forms, where a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral envelope.

The AAV particle also includes chemically modified forms bearing ligands adsorbed on the capsid surface. For example, such ligands may include antibodies to target a specific cell surface receptor.

The AAV2 genome, like the genomes of all AAV serotypes, can be enclosed in a number of different capsid proteins. AAV2 can be packaged into its native AAV2 capsid (AAV2/2), or it can be pseudotyped with other capsids (e.g., the AAV2 genome in the AAV1 capsid, resulting in AAV2/1, or the AAV2 genome in the AAV8 capsid, resulting in to obtain AAV2/8).

In a preferred embodiment, the AAV capsid is derived from AAV8. In a particularly preferred embodiment, where the operably linked sequence is the CNGA3 gene, it is preferred that the capsid is AAV8 or another capsid other than AAV5.

AAV transduces cells through serotype-specific receptor-mediated endocytosis. The main factor influencing the kinetics of rAAV transgene expression is the rate of decapsidation of the viral particle within the endosome. This, in turn, depends on the type of capsid enclosing the genetic material. After decapsidation, the linear single-stranded rAAV genome is stabilized into a double-stranded molecule through de novo synthesis of the complementary strand. When using self-complementary DNA, this step can be bypassed by generating double-stranded transgenic DNA. Self-complementary AAV2/8 gene expression was found to have a faster onset and higher amplitude compared to single-stranded AAV2/8. Thus, by avoiding the time delay associated with second-strand synthesis, gene expression levels are increased compared to transgene expression from standard single-strand constructs. Subsequent studies examining the effects of self-complementary DNA in other AAV pseudotypes yielded similar results. One caveat to this technique is that since AAV has a packaging capacity of approximately 4.8 kb, the self-complementary recombinant genome must be of appropriate size (namely 2.3 kb or less).

In addition to modifying the packaging container, pseudotyping the AAV2 genome with other capsids

- 13 046019 mi AAV can change the cellular specificity and kinetics of transgene expression. For example, when AAV2 is pseudotyped using the AAV4 capsid, transgene expression is specifically targeted to RPE (retinal pigment epithelium) cells. In addition, AAV2/8 has been reported to transduce photoreceptors more efficiently than AAV2/2 or AAV2/5.

Getting vectors

The vector of the invention can be prepared by standard means known in the art for producing vectors for therapy. Thus, well-developed publicly available transfection, packaging and purification methods can be used to obtain a suitable vector preparation.

As discussed above, the vector of the invention may contain the complete genome of a naturally occurring AAV virus in addition to the promoter of the invention or a variant thereof. However, typically a derivatized genome will be used, eg a derivative that has at least one inverted terminal repeat (ITR) sequence, but which may lack AAV genes such as rep or cap.

In such embodiments, to facilitate assembly of the derivatized genome into an AAV viral particle, additional genetic constructs providing AAV and/or helper virus functions may be provided in the host cell in combination with the derivatized genome. These additional constructs will typically contain genes encoding AAV structural capsid proteins, namely cap, VP1, VP2, VP3, and genes encoding other functions required for the AAV life cycle, such as rep. Selection of the structural capsid proteins provided on the additional construct will determine the serotype of the packaged viral vector.

A particularly preferred packaged viral vector for use in the invention comprises a derivatized AAV2 genome in combination with an AAV8 capsid protein.

As mentioned above, replication-defective AAV viruses, and thus helper virus functions, preferably helper adenovirus functions, can be provided on one or more additional constructs to promote AAV replication. There are also systems known to one skilled in the art that use a single construct that contains rep, cap, and Ad helper functions, thus no additional helper constructs are required.

All of the above-mentioned additional constructs may be provided as plasmids or other episomal elements in the host cell, or alternatively, one or more constructs may be integrated into the genome of the host cell.

The transcription control unit of the invention has the ability to restore loss of cone photoreceptor function, which may occur, for example, as a result of mutations in the CNGA3 gene. The term restoration generally means any improvement or slowing of the progression of a retinal disorder or dystrophic phenotype, for example, restoring the presence of the CNGA3 protein in the cone photoreceptor, improving ERG activity or slowing the loss of ERG activity, improving retinal sensitivity or slowing/stopping the progressive loss of retinal sensitivity, slowing or stopping the loss photoreceptor cells, improving vision or slowing/stopping vision loss.

The properties of the transcription control unit of the invention can also be tested using techniques based on the techniques in the Examples section. In particular, a transcriptional control unit of the invention can be assembled into a vector of the invention and delivered into the retina of a CNGA3-deficient test animal, such as a mouse, and the effects are observed and compared with a control. Preferably, the control will be another eye of the same animal that is either untreated or treated with a control vector, such as a vector containing a reporter gene, compared to the sequence of the invention. Electroretinography-based analysis of retinal responses to light can then be used to confirm that photoreceptor cells in eyes that are treated are more sensitive to light than photoreceptors from eyes that are not treated or treated with a control vector. The sensitivity of the treated eye to light may, for example, be at least 1.1, 1.2, 1.5, 2, 5, 10, 20, 50, 100, 200, 500 or 1000 times greater than the sensitivity of the untreated eye. control-treated eye.

Therapies and medical applications

In one aspect, the invention provides nucleic acid molecules (such as TCUs, promoters and fragments thereof, codon optimized genes, expression constructs and vectors), as well as methods for treating and/or preventing retinal disorders or dystrophies in a patient in need thereof.

The retina consists of a layer of retinal pigment epithelial (RPE) cells and three layers of neurosensory cells; namely (outer to inner), outer nuclear layer (containing rod and cone photoreceptor cells), inner nuclear layer (containing bipolar cells) and ganglion cell layer. Retinal disorders or dystrophies can be defined as diseases of the retina characterized by the progressive loss of photoreceptor cells and concomitant loss of vision. Retinal disorders or dystrophies may be hereditary disorders

- 14 046019 retinal or dystrophy.

In one embodiment, nucleic acid molecules and methods for treating and/or preventing cone-rod dystrophy and/or cone dystrophy are provided. Cone-rod dystrophies can be defined as diseases characterized by the progressive loss of cone photoreceptor cells and concomitant vision loss and may be hereditary. In one embodiment, the retinal disorder is achromatopsia or macular degeneration. Macular degeneration may be age-related macular degeneration (AMD), such as wet form or neovascular AMD, or geographic atrophy, an inherited condition of macular degeneration or non-hereditary cone degeneration.

The terms patient and subject may be used interchangeably. The patient is preferably a mammal. The mammal may be a commercially raised animal such as a horse, cow, sheep or pig, a laboratory animal such as a mouse or rat, or a domestic animal such as a cat, dog, rabbit or guinea pig. The patient is more preferably human. The subject may preferably be a man or a woman. The subject is preferably identified as being at risk for or having a retinal disorder or dystrophy.

The terms treat, being treated, treating or being treated, as used herein, refer to both therapeutic treatment and prophylactic or preventive measures where the goal is to prevent or slow down (reduce) an undesirable physiological condition, disorder or diseases, or obtaining useful or desirable clinical results. For the purposes of this invention, useful or desirable results include relief of symptoms; reducing the severity of a condition, disorder or disease; stabilization (i.e., no worsening) of the status of the condition, disorder, or disease; delaying the onset or slowing the progression of a condition, disorder or disease; improvement of a condition, disorder or course of a disease; and remission (either partial or complete), whether detectable or undetectable, or exacerbation or improvement of a condition, disorder or disease, but not limited to. Treatment involves inducing a clinically significant response without excessive levels of side effects.

The patient may be asymptomatic and/or may have a predisposition to the disease. In this regard, the invention also provides a method or use that includes the step of identifying whether a subject is at risk of developing or has retinal disorders such as cone dystrophies, including but not limited to achromatopsia. A prophylactically effective amount of a nucleic acid, such as a vector as disclosed herein, can be administered to such a subject. A prophylactically effective amount is an amount that prevents the onset of one or more symptoms of the disease. Accordingly, in some embodiments, a nucleic acid, such as a vector as disclosed herein, can be administered to prevent or delay the onset of one or more symptoms of retinal disorders such as cone-rod dystrophies, including but not limited to achromatopsia. Alternatively, a nucleic acid, such as a vector as disclosed herein, can be administered once a subject has developed symptoms of a disease, namely to treat existing symptoms of a disease. Such a subject can be administered a therapeutically effective amount of a nucleic acid, such as a vector, as disclosed herein. As used herein, the term therapeutically effective amount means an amount of a nucleic acid as defined herein that, when administered to a mammal, is effective in producing the desired therapeutic effect. In one embodiment, the invention provides methods for treating and/or preventing a retinal disorder or dystrophy, wherein a nucleic acid, such as a vector as disclosed herein, is administered to a patient in need of a therapeutically effective amount. In some embodiments, the retinal disorder or dystrophy is a cone dystrophy, including but not limited to achromatopsia.

The invention also provides the use of a nucleic acid, such as a vector as disclosed herein, in the manufacture of a medicament for the treatment or prevention of retinal disorders or dystrophies, such as cone dystrophies, including but not limited to achromatopsia. The invention also provides a method of treating or preventing retinal disorders such as cone dystrophies, particularly achromatopsia, in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a nucleic acid, such as a vector, as disclosed herein.

Methods of administration

In general, direct retinal, subretinal or intravitreal delivery of a nucleic acid such as a vector as disclosed herein is preferred, typically by injection. Thus, delivery to the retinal space, subretinal space or intravitreal space is preferred.

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Thus, the invention also provides a method of treating or preventing cone dystrophies, in particular achromatopsia, in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a nucleic acid, such as a vector as disclosed herein, by injection directly into the retina, subretinal or intravitreal injection.

In a related aspect, the invention provides the use of a nucleic acid, such as a vector as disclosed herein, in a method of treating or preventing retinal disorders, such as cone dystrophies, particularly achromatopsia, by administering said vector to a patient by injection directly into the retina, subretinal or intravitreal injection.

The invention further provides the use of a nucleic acid, such as a vector as disclosed herein, in the manufacture of a medicament for the treatment or prevention of retinal disorders, such as cone dystrophies, in particular achromatopsia, by injection directly into the retina, subretinal or intravitreal injections.

The invention also provides a nucleic acid, such as a vector as disclosed herein, for use in the treatment of retinal disorders such as cone dystrophies, in particular achromatopsia, wherein said vector is administered directly into the retinal space, subretinal space or intravitreal space.

Administration of a nucleic acid, such as a vector as disclosed herein, is typically accomplished by injection directly into the retina or subretinal injection. This involves direct delivery to cone photoreceptor cells.

Delivery is usually direct to or subretinal to the degenerating retina of a patient suffering from retinal disorders such as cone-rod dystrophies, in particular achromatopsia.

A nucleic acid, such as a vector as disclosed herein, can transduce the above target cells without introducing any other cell populations. Intravitreal injection can also be used to deliver the vector of the invention.

The dosage of a nucleic acid such as a vector as disclosed herein can be determined according to various parameters, especially according to the age, weight and condition of the patient to be treated; by administration; and the required circuit. In addition, the attending physician will be able to determine the required route of administration and dosage for a particular patient.

A typical single dose is 10 ¹⁰ to 10 ¹² genome particles, depending on the amount of remaining retinal tissue that requires transduction. A genome particle is defined herein as an AAV capsid that contains a single-stranded DNA molecule that can be quantified by a sequence-specific method (such as real-time PCR). This dose may be offered as a single dose, but may be repeated for the second eye or in cases where a nucleic acid, such as a vector as disclosed herein, may not be targeted to the correct area of the retina for any reason (such as postoperative complication). Treatment is preferably one continuous treatment per eye, but repeat injections may be considered, for example in the future and/or with different AAV serotypes.

Host cells

The invention further provides a host cell containing a nucleic acid, such as a vector or viral vector, or an AAV viral particle disclosed herein. Any suitable host cell can be used to produce nucleic acids, such as the vectors disclosed herein. Typically, such cells will be transfected mammalian cells, but other cell types, such as insect cells, can also be used. With regard to mammalian cell-based production systems, HEK293 and HEK293T are the preferred AAV vectors. You can also use BNK (Baby Hamster Kidney) or CHO (Chinese Hamster Ovary) cells.

Pharmaceutical compositions and dosages

The invention further provides a pharmaceutical composition comprising a nucleic acid, such as a vector disclosed herein, as well as a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, buffer, stabilizer and/or other substances well known to those skilled in the art. . Such substances must be non-toxic and must not interfere with the effectiveness of the active substance. Also provided is a pharmaceutical composition that contains a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, buffer, stabilizer and/or other substances well known in the art, and a nucleic acid sequence, plasmid, vector or viral vector as described herein .

The exact nature of the carrier or other substance can be determined by one skilled in the art in accordance with

- 16 046019 by injection, namely in this document by injection directly into the retina, subretinal injection or intravitreal injection.

The pharmaceutical composition is usually in liquid form. Liquid pharmaceutical compositions typically include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Saline solution, magnesium chloride solution, dextrose or other saccharide, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, a surfactant such as pluronic acid (PF68) 0.001% may be used.

For injection at the site of the lesion, the active substance will be in the form of an aqueous solution that is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art may well prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's solution for injection, Ringer's lactate injection, Hartmann's solution. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as needed.

For delayed release, the vector can be included in a pharmaceutical composition that is formulated for slow release, such as microcapsules formed from biocompatible polymers or liposomal carrier systems according to methods known in the art.

Dosages and dosage regimens can be determined within the standard skill of the physician responsible for administering the composition. The dosage of the active agent(s) may vary depending on the reason for use, the individual, and the route of administration. The dosage may be adjusted based on the weight of the subject, the age and health of the subject, and the tolerance of the compound(s) or composition.

Combination therapies

The nucleic acids, TCUs, promoters, codon optimized genes, expression constructs, vectors and/or pharmaceutical compositions disclosed herein can be used in combination with any other therapy to treat and/or prevent retinal disorders such as cone dystrophies, including , but not limited to achromatopsia.

Sets

Nucleic acids, TCUs, promoters, codon optimized genes, expression constructs, vectors and/or pharmaceutical compositions disclosed in the art can be packaged in a kit.

Examples

Materials and methods Plasmid construction

To create an improved version of the green cone opsin promoter, a 1250 bp locus control region (LCR) was used. (Smallwood et al., 2002), combined with different fragments of the green opsin core promoter (bp -480 to +40) into the original plasmid pAAV/CMV.eGFP, creating the plasmid construct pAAV/hG1.7.eGFP and pAAV /hG1.4.eGFP. Vector constructs carrying promoters with the M8 mutation were obtained by site-directed mutagenesis.

For therapeutic genetic constructs, pAAV/coCNGA3 was created by cloning the codon- and Kozak-optimized CNGA3 sequence from plasmid pUC57 (obtained via GenScript) into plasmid pAAV carrying different green (M) cone opsin promoter constructs.

Codon optimization

Codon optimization was achieved using the OptimumGeneTM codon optimization tool, proprietary to GenScript.

Protocol for obtaining AAV2/8 and AAV2/5 viruses

Recombinant AAV2 virus serotype 8 and AAV2 virus serotype 5 were produced using triple transfection of 293T cells as previously described (Gao et al., 2002). 145 cm ² plates with 293T cells (20 plates per viral batch) were transfected with a mixture containing the following test plasmid: viral plasmid capsid: helper plasmid DNA in a ratio of 1:1:3, polyethylenimine (PEI - Polysciences Inc., Eppelheim, Germany) and DMEM, after 10 min incubation. Transfected cells were plated for 24 hours. 48 hours after transfection, cells were collected, concentrated by _{centrifugation} , and resuspended in TD buffer (140 mM NaCl, 5 mM KCl, 0.7 mM _K2HPO4 , 3.5 mM _MgCl2 , 25 mM Tris base [pH 7.5]). Lysis was then carried out by 3-4 cycles of freeze-thaw followed by treatment with Benzonase (Sigma Aldrich, Dorset, UK), and the cell debris was then removed through successive steps of centrifugation and syringe filter filtration.

Purification was carried out by ion exchange chromatography (using a method based on Davidoff et al., 2004). The eluate was concentrated in a 10 kDa Vivaspin 4 concentrator tube (Sartorius

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Stedim Biotech, Fisher Scientific, Loughborouh, UK), washed in PBS-MK, then concentrated to a volume of 100-150 µl, then aliquoted for storage at -80°C (long-term) or +4°C (short-term).

Generation and transduction of human stem cell-derived retinal organoids

Human H9 embryonic stem cell (ESC) lines were maintained under feeder cell-free conditions in 6-well plates containing E8 medium and coated with geltrex. hESCs were dissociated using dispase and collagenase solution. ESC clusters were collected and resuspended in E8 media. To differentiate the retinal neuroepithelium, human ESCs were maintained until cell confluence was reached, when media without FGF (Fibroblast Growth Factor) was added to the cultures for two days. Proneural induction media (improved DMEM/F12, MEM nonessential amino acids, N2 supplement, 100 mM Glutamine and Pen/Strep (penicillin/streptomycin)) were added until no eye vesicles were observed. Bubbles were removed manually and stored in 96-well plates in retinal differentiation media (DMEM, F12, Pen/Strep and B27 without retinoic acid), and after 6 weeks of differentiation, the medium was supplemented with FBS (Fetal Bovine Serum). , taurine and glutamax, and RA was added after 10 weeks.

AAV vectors (serotype SsH10) carrying green cone opsin promoter constructs driving eGFP were added to the medium at 1.2x1ο ¹¹ mg/well (1-3 organelles/well). Organoids were cultured for an additional 28 days before collection and analysis.

Immunohistochemistry

The eyes were prepared for fixation by puncturing the cornea and then immersed in 1% formaldehyde (PFA, pH 7.4). Retinal organoids were fixed by immersion in 1% paraformaldehyde (PFA, pH 7.4). The eyes were left fixed at room temperature for up to an hour before being removed from the solution and completely immersed in a matrix to be embedded at optimal cutting temperature (OCT), with the anteroposterior axis of the eye suspended in the horizontal-vertical axis in test tubes. for conclusion. They were then frozen and stored at -20°C until needed for sectioning.

10-18 µm sections were obtained using a Bright® OTF5000 cryostat (Bright Instrument Co Ltd, Cambridgeshire, UK), thereby allowing visualization of both the superior and inferior retina. Sections were collected immediately after sectioning onto polysine-coated microscope slides and allowed to air dry at room temperature. Slides were either stored at -20°C or directly analyzed.

For immunohistochemistry, sections were blocked in 5% goat serum and 1% bovine serum albumin in PBS (phosphate-buffered saline). Primary antibodies against cone opsin (Millipore) were incubated overnight at 4°C. Sections were incubated with secondary antibody for 2 h at RT (room temperature) and washed. Alexa-fluor-labeled secondary antibodies (Invitrogen-Molecular Probes) were used at a dilution of 1:500. Sections were stained with DAPI (4',6-diamidino-2-phenylindole) either as added to the mounting medium (0.1% DAPI in the medium) or by immersion in 0.2% DAPI in TBS (from the English tris-buffered saline - Tris-buffered saline solution) and PBS washes before placing in fluorescent preparation medium (DAKO). Embedded sections were stored at 4°C.

Images of embedded sections were obtained using a Leica DM5500Q confocal microscope (Leica Biosystems, Germany) or a Zeiss AxioObserver Z1 (Carl Zeiss Inc, Gottingen, Germany).

Subretinal injection

Subretinal injections were performed in Cpfl5 (CNGA3-deficient) (Hawes et al., 2006) and C57BL/6 mice approximately 2 weeks after birth. The operating microscope was used throughout eye surgery. A 1.5 cm long, 34-gauge hypodermic needle (Hamilton, Switzerland) was inserted tangentially through the sclera, creating a self-healing scleral tunnel wound (Tan et al., 2009). 1.0-1.5 μl of the viral suspension was injected into the upper and lower hemispheres of the subretinal space, and in each case an ophthalmoscopically visible bullous retinal detachment occurred. C57BL/6 was used for promoter studies and a virus titer of 1x10 ¹² was injected. Cpfl5 mice were used in CNGA3 restoration studies. Mice were injected with CNGA3 viral constructs into the target eye, with control viral constructs injected into the contralateral eye. All mice were injected bilaterally.

Efficiency of treatment in vivo

Recovery of retinal function was assessed by photopic electroretinography. ERG recordings were obtained from both eyes simultaneously. After dark adaptation overnight (approximately 12 hours), one drop of 2.5% phenylephrine and 1% tropicamide (Minims, Bausch & Lomb) was applied to the

- 18 046019 each eye to dilate the pupil, and the eyes were kept moist by applying a lubricant. ERG was performed using commercially available equipment (Espion ERG Diagnosys System). Recordings of photopic signals with one extreme were obtained with increasing light intensities of 0.1, 1, 3.16, 10, 31.6 and 75.28 cd/ ^m2 against a background of 30 cd/ ^m2 . Unless otherwise noted, the figures show mean photopic b-wave amplitudes (mean ± SD) at 4 weeks post-treatment when the vectors had reached maximum expression.

Example 1: Optimization of the Cone-Specific Transcriptional Control Unit (TCU)

The human locus control region (LCR), which lies upstream of the red opsin gene, enhances the expression of both the red opsin gene (L-opsin) and the green opsin genes (M-opsin), which are located in tandem (see Fig. 1, top left). Strong cone-specific promoters have previously used the LCR and the red opsin promoter because they are physically adjacent elements. In an in vivo reporter gene expression study in transgenic mice, Smallwood et al. examined different levels of expression from either the red or green opsin promoter based on their physical proximity to the LCR. Smallwood et al., constructed several derivatives of a number of human red and green pigments to exhibit different levels of expression from either the red opsin promoter or the green opsin promoter, based on their physical proximity to the LCR (see Fig. 1, bottom left). Each derivative consisted of an alkaline phosphatase (AP) reporter gene under the control of the red opsin promoter and β-galactosidase (LacZ) under the control of the green opsin promoter. For each embryonic stem cell line, retinas were analyzed from either multiple chimeric founders or multiple progeny that stably inherited the transgene. In the latter case, the analysis was performed both before and after crossing with germline cre mice to remove the loxP-flanked PGK-neo cassette.

In a wild-type construct using a natural chromosomal rearrangement (pPMS107), 65–95% of cones express either AP (under the control of the red opsin promoter) or lacZ (under the control of the green opsin promoter), but not both. Insertion of a 9 kb spacer between two transcription units (namely, increasing the distance between the LCR and the green opsin promoter, construct pPMS108) results in a large shift from lacZ (green promoter)- to AP (red promoter)- expressing cells. Changing the positions of these two transcription units (pPMS101) and the location of the green opsin core promoter immediately after the LCR (pPMS101) shifted the expression profile of these transgenic mice almost exclusively in the direction of transcription under the control of the green opsin core promoter (more than 99%).

This disclosure provides an alternative cone-specific transcriptional control unit (TCU) using a human LCR and an optimized human green opsin promoter. A conserved core element of the human green opsin promoter (0.2 kb) was identified, and a range of TCUs using engineered green opsin promoters were generated using the LCR region and green opsin promoters of different sizes: 2.0 kb. n. (hG2.0), 1.7 kb. (hG1.7) and 1.4 kb. (hG1.4), see fig. 2. A 1.7 kb red opsin promoter fragment was also generated as a control. (PR1.7, similar to the promoter used by AGTC, Ye et al., 2016)).

In addition, a variety of AAVshh10 vectors containing GFP reporters driven by the promoters described above were generated and expression assessed in human ES-derived retinal cell cultures.

All constructs analyzed provided similar transduction profiles with respect to GFP expression levels and cone cell specificity. Constructs with smaller promoter fragments provide more space for packaging larger genes into the AAV vector.

Example 2: Optimized TCU Provides Strong Reporter Gene Expression in All Cone Subtypes

To analyze the ability of the optimized TCUs disclosed herein to stimulate reporter protein expression in cone cells, human ES-derived retinas were transduced using AAVshh10-hG1.4.GFP. Strong expression of the reporter gene was observed in all cone subtypes (Fig. 3B',C) (Fig. 3A).

Compared to the previously characterized human cone arrestin (CAR) promoter, expression levels mediated by hG1.4 were higher in human cones and there was no ectopic expression in rod cells or retinal pigment epithelial (RPE) cells. In mice, cone promoters based on the human red opisna promoters were not cone specific and also mediated expression in rods (Ye et al., 2016).

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Example 3: Additional TCU Optimization and Codon Optimization of the Human CNGA3 Gene

Replacing the sequence GGGCCG at positions +5 to +10 relative to the transcription start site of the green opsin gene with TCTAGA doubles the resulting expression level. This sequence change (M8), see SEQ ID NO: 16, was thus included in the hG1.4 and hG1.7 constructs.

In addition, the CNGA3 coding sequence was subjected to codon optimization to attempt to improve codon preference and CG content, and to improve any cryptic processing sites and possible stem-loop structures in the mRNA.

The constructs hG1.4(M8) (SEQ ID NO: 6) and hG1.7(M8) (SEQ ID NO: 15) were used in AAV2/8 vectors carrying codon optimized human CNGA3 cDNA (coCNGA3) and the efficiency of the vectors was determined in a CNGA3 knockout mouse model using electroretinography (ERG)-based assessments of cone function. One month after vector administration, there was no clear advantage over the vector without the M8 sequence, although the hG1.7(M8) vector appeared to show a slight improvement over the corresponding vector without the M8 sequence (Fig. 4, left bars). When ERG assessments in these animals were repeated 2 months after administration, amplitudes in animals injected with M8 vectors increased further (Fig. 4, right bars), while amplitudes in animals injected with standard vectors remained constant. (hG1.4) or increased slightly (hG1.7). Maximum expression levels have been demonstrated to be important for achieving good functional recovery in achromatopsia due to CNGA3, and these results indicate that inclusion of the M8 sequence in constructs is beneficial for treatment.

The codon-optimized CNGA3 gene restored photopic responses in the Cnga3 knockout mouse more efficiently than the wild-type human CNGA3 gene (Fig. 5). Constructs carrying the red opsin promoter (1.7L) regulating either the wild-type human CNGA3 gene or the codon-optimized human CNGA3 gene were packaged into serotype AAV2/8 and injected subretinal into Cnga3-deficient mice. Photopic ERG responses were assessed 1 month and 2 months after injection. ERG responses in animals receiving the codon-optimized vector were consistently higher than those in animals receiving the wild-type gene (Fig. 5).

Example 4 AAV2/8 vectors expressing CNGA3 can restore photopic responses in a CNGA3 knockout mouse, while the corresponding AA2/5 construct provides minimal restoration

To assess the ability of AAV2/5 and AAV2/8 vectors expressing CNGA3 to restore photopic responses, viral vectors AAV2/8-hG1.4(M8).coCNGA3 and AAV2/5-hG1.4(M8).coCNGA3 were injected subretinal into mice. cnga3 knockout, 1 month old. Photopic ERG responses were assessed 4 weeks after administration. We observed strong photopic responses in AAV2/8-treated eyes, with ERG b-wave amplitudes up to 70 μV (Fig. 6A and B). Eyes that received subretinal injections in both hemispheres (superior/inferior) showed the greatest improvement in ERG b-wave amplitude. For comparison, a wild-type mouse has amplitudes of about 100-120 µV under the same experimental settings. There were no responses from untreated eyes, and AAV2/5-treated eyes provided minimal responses (Fig. 6A and B). These data demonstrate that the AAV2/8-hG1.4(M8).coCNGA3 vector is able to provide high levels of CNGA3 expression and restore cone function in CNGA3 knockout mice to approximately 60% wild-type function, as measured by ERG b-wave amplitudes .

To further demonstrate that restoration of retinal sensitivity by AAV2/8 vectors expressing CNGA3 was long-lasting, Cnga3-deficient mice were subretinal injected at 2 weeks of age with either AAV2/8-hG1.4(M8).coCNGA3 (n = 14), or AAV2/8-hG1.7(M8).coCNGA3 (n = 13) (titer 1x 10 ¹² mg/ml for both), or left untreated (n = 3). Both transfections with both vectors lead to sustained restoration of retinal sensitivity up to six months after treatment (Fig. 7).

Example 5 AAV2/8 Vectors Expressing CNGA3 Promote Long-Term Cone Survival

The ability of AAV2/8 vectors expressing CNGA3 to stimulate cone survival was assessed by injecting Cnga3-deficient mice at 2 weeks of age with AAV2/8hG1.7(M8).coCNGA3. A 3- to 4-month-old C57BL/6J mouse that did not receive the injection served as a control for healthy cones. Retinas were isolated, stained with cone arrestin, and cleared.

Cnga3-deficient retinas transduced with AAV2/8-hG1.7(M8).coCNGA3 (see Fig. 8C) exhibited cone survival in vivo 3–4 months after treatment. The rate of survival was similar to that of healthy control cones (see Fig. 8A) and was significantly increased compared with uninjected Cnga3-deficient retinas (see Fig. 8B).

The ability of AAV2/8 vectors expressing CNGA3 to stimulate cone survival was long-lasting and could still be observed 13 months after vector treatment (see Fig. 9).

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Example 6: AAV2/8 Vectors Expressing CNGA3 Preserve Synaptic Connectivity in Vivo

To evaluate the ability of AAV2/8 vectors expressing CNGA3 to improve synaptic integrity between cone cells and supporting neurons (bipolar cells), Cnga3-deficient mice were injected with AAV2/8-hG1.7(M8).coCNGA3 at 2 weeks of age. 3-4 months after treatment, retinas were isolated and stained with Gpr179 and PNA and then cleared. Synaptic connectivity was determined by measuring the signal intensity of the synapse marker Gpr179 on single-plane confocal images of retinal flat mounts. Leica Las X software was used for image processing. Gpr179 staining was monitored by freehand drawing a line on several Gpr179 stains associated with cone stalks (PNA staining was used to confirm cone stalks) and more than 10 Gpr179 stains associated with rod spherules (A). The output signal intensity (B; white line: Gpr179, red line: PNA). The signal intensity peaks from each source were averaged, and the ratio of GPr179 staining associated with cone stalks to GPr179 staining associated with rod spherules (CP/RS) was calculated. CP/RS from four different positions were used for statistical analysis (Bonferroni multiple comparison test (ns: p greater than 0.05, **: p less than or equal to 0.01, *: p less than or equal to 0.05)). Retinas from age-matched (3–4 months) uninjected Cnga3-deficient C57BL/6J mice served as negative and positive controls, respectively.

As shown in FIG. 10, transduction of Cnga3-deficient retinas with AAV2/8 vectors expressing CNGA3 results in synaptic connectivity similar to that of healthy control mice and significantly improved compared with uninjected Cnga3-deficient retinas.

Example 7 Delivery of coCNGA3 using AAV2/8 provides greater benefit than delivery using the new strong AAV serotypes AAV-Anc80, AAV-44.9 and AAV5

A number of different AAV serotypes and capsids are available for gene expression. For example, the newly designed capsid Anc80-L65 has been shown to have efficient photoreceptor tropism and is comparable or even superior to AAV8. In addition, the novel serotype AAV44.9 has also been shown to exhibit efficient transduction and high levels of expression in photoreceptor cells when assayed in combination with a fluorescent marker. Therefore, the ability of four different AAV vectors—Anc80-L65, AAV44.9, AAV5, and AAV8—to express coCNGA3 and mediate robust ERG responses was compared.

Appropriate vectors carrying the hG1.4(M8)-coCNGA3 expression cassette (1.0 x 10 ¹² mg/ml) were delivered into the subretinal space of Cnga3-deficient mice at 2 weeks of age, and single-extreme photopic ERG was recorded at different time points. after injection (see Fig. 11). A light stimulus of 10 cd/ ^m2 was used for analysis. As shown in FIG. 11, AAV8-mediated gene transfer resulted in higher ERG responses compared to Anc80-L65 (see Fig. 11A), AAV44.9 (see Fig. 11B) or AAV5 (see Fig. 11C and Fig. 6) .

Example 8: Comparison of Optimized TCUs with Previously Known Cone-Specific Promoters

To compare the expression level of the TCUs disclosed herein with one of the strongest cone-specific promoters available (1.7L), 17-19 week old human embryoid bodies (hEBs) were transduced with the AAV vector AAVShH10 expressing eGFP under the control of hG1.4 (M8), hG1.7(M8) or P1.7L, respectively, and harvested 2 weeks later (n = 6-8 for each promoter). After dissociation, cells were analyzed for relative mean fluorescence intensity (MFI) in GFP-positive cells. Relative MFI in hEBs transduced with different vectors was assessed by flow cytometry and calculated as a ratio to MFI in EBs transduced with AAV ShH10-1.7L-eGFP. As shown in FIG. 12A, the hG1.7 construct provides an approximately 50% increase in GFP expression compared to the previously known 1.7L promoter.

In addition, the AAV2/8 vector driving hCNGA3 from the hCAR (human cone arrestin) promoter was analyzed in CNGA3 knockout mice. Recovery of photopic responses did not exceed 30% of wild-type levels (Fig. 12B).

It has been reported that the AAV2/5(Y719F) vector and the mouse blue cone opsin promoter can be used to rescue CNGA3 knockout mice. In that study, photopic ERG amplitudes reached 30% of wild type when injected at P12 and were assessed 10 weeks after treatment (Michalakis et al., 2010). Recently, the AAV5 vector and the 2.1 kb red opsin promoter. used to restore CNGA3-deficient sheep. In this study, there was a doubling of cone ERG flickering compared to the untreated variant (Banin et al., 2015). However, both of these promoters are known to function only in a subset of human cones (the blue cone opsin promoter is active only in blue cones and the 2.1 kb red opsin promoter is active only in red cones).

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A study using the AAV2/5 vector with the CBA promoter to express murine CNGA3 in CNGA3 knockout mice conducted by the Hauswirth laboratory showed a recovery of up to 70% of cone ERG b-wave amplitudes when mice were treated very early (P14 assessed at 3 weeks after treatment - virus titer 1E13) (Pang et al., 2012). The inventors achieved similar effectiveness in restoring photopic vision when treating the same animal model at a later age (1 month - virus titer 7E12). While achromatopsia in humans is a stationary disorder, the CNGA3 mouse model suffers from cone cell death in the first month, indicating that earlier treatment is beneficial. The non-specific CBA promoter is unlikely to be used in the CNGA3 trial, but supports the inventors' view that high levels of CNGA3 expression are important for optimal recovery.

Links

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Sequence information

SEQ ID NO: 1-1.2 kb fragment of the locus control region (LCR) of the M/L opsin TAGGAATAGAAGGGTGGGTGCAGGAGGCTGAGGGGTGGGGAAAGGGCATGGGTG

TTTCATGAGGACAGAGCTTCCGTTTCATGCAATGAAAAGAGTTTGGAGACGATGGTGGTG ACTGGACTATACACTTACACACGGTAGCGATGGTACACTTTGTATTATGTATATTTTTACCAC GATCTTTTTAAAGTGTCAAAGGCAAATGGCCAAATGGTTCCTTGTCCTATAGCTGTAGCAGC CATCGGCTGTTAGTGACAAAGCCCCTGAGTCAAGATGACAGCAGCCCCCATAACTCC C CCCTCACCCCCTCGTTCTTCATATCCTTCTCTAGTGCTCCCTCCACTTTCATCCACCCTTC TGCAAGAGTGTGGGACCACAAATGAGTTTTCACCTGGCCTGGGGACACACGTGCCCCCAC AGGTGCTGAGTGACTTTCTAGGACAGTAATCTGCTTTAGGCTAAAATGGGACTTGATCTTCT GTTAGCCCTAATCATCAATTAGCAGAGCCGGTGAAGGTGCAGAACCTACCGCCTTTCCAG G CCTCCTCCCACCTCTGCCACCTCCACTCTTCCTTCCTGGGATGTGGGGGCTGGCACACGT GTGGCCCAGGGCATTGGTGGGATTGCACTGAGCTGGGTCATTAGCGTAATCCTGGACAAG GGCAGACAGGGCGAGCGGAGGGCCAGCTCCGGGGCTCAGGCAAGGCTGGGGGCTTCCC CCAGACACCCCACTCCTCCTCTGCTGGACCCCCACTTCATAGGGCACTTCGTGTTCTCAAA GGGCTT CCAAATAGCATGGTGGCCTTGGATGCCCAGGGAAGCCTCAGAGTTGCTTATCTC CCTCTAGACAGAAGGGGAATCTCGGTCAAGAGGGAGAGGTCGCCCTGTTCAAGGCCACC CAGCCAGCTCATGGCGGTAATGGGACAAGGCTGGCCAGCCATCCCACCCTCAGAAGGGA CCCGGTGGGGCAGGTGATCTCAGAGGAGGCTCACTTCTGGGTCTCACATTCTTG

SEQ ID NO: 2 - fragment of the M opsin promoter, 2.0 kb in size, fragment of SEQ ID NO: 3, 500 bp in size, UTR in italics without the M8 sequence TAAAAAGCAAGTCTTGCCAGGGCAGTGGTGTGCACCTGTGGTCCCAGCTACTCAG

GATGCTGAGGCAGGAGGATTACTTGTGCCCAGCAAGTAGAGGCTGCAGTGACCTGTGACT GTGCTACTGCCCTCCAACCTGGGTGACAGAGTGAGACCTTGTCTCAAAAAAAAAAGAGCG GGGGGGGGGGGCCGGGCCGGGCGTGGTGGCTCACAGCTGTAATCCCAGCACTTTGGGA AGCCAAGGCGGGTGGATCACTTGAGGTCAGGAGTTTGAGACCATCATGGTCAACACTGCG AAAC ACTGTCCCTACTAAAAATACAAAAATTAGCCGGGCATGGTGGCACACACCTGTAATC CCAGCTACTGGGGAGGCTGAGGCAGGAGAATTGCTTGAGCCGGGGAGACGGAGGTTGCA GTGAGCCGAGACTGCGCCACTGCACTCCAGCCTGACTGACAAGAGTGAGATTGTCTCAAA AAAAAAAAAAAAGTAATCACTAGAAAAGAAGCTACATATGTACATAACATCCAAATAACCAA GAGGAAAAAAATGGGACTTG ATTAATCAAAACAAAAACAAAAAAGAAAGAAAGAAAGGG

- 23 046019

C GAGGCTGCCGTGATCGCACCACTGCCCTCCAGCCAGGGTGGCAGACTGAGACCCCATC TCAAAAAATAAATAAATAAAAGCAAACAAGAAAAAAAAAGGCTTGAAACATATCTGATAGATA AAGGGCTAATCAACACAATATATAAAGAACTGCAAATCAGTAAACTAAGAGCAAATAACCCA ATATAAAGACATTAAAGGGTAGCCACGGACATCTCAGACGACGAAAAACAAAAGACAGTAA ACGTATAATAAAACATGTA ATTGCAAGGTGATCCGGGAATAGTAAGCGAAAAGCAACAATTA AATACTATTTTCTCATCCACCAGAACGCCAAAAATTAAAAAGCCTAACAATGTCCAGGGCTG GCGAAATGTGGCAGAAGGTGATGTCACATACCCTGCAAGTGGGAATCTAAACAGATTCA GGGTTTTGGTTTTTTTTTAATCGCAATTAGGTGGCCTGTTAAATTTTTTTTCTTGAGACAGAG TTTTGCTCTTGTTGCCCA GGCTGGAGTGCAATGGCTCGATCTTGGCTCACCGCAACCTCGA CCTCCCAGGTACAAGCGATTCTCCTGTCTCAGCCTCCCAAGTAGCTGGGAGTACAGGTATT TGCCACTAAGCCCAGCTAATTGTTTTTTATTTAGTAGAAACGGGGTTTCACCATGTTAGTCA GGCTGGTCGGGAACTCCTGACCTCAGGAGATCTACCCGCCTTGGCCTCCCAAAGTGCTGG GATTACAGGCGTG TGCCACTGTGCCCAGCCACTTTTTTTTAGACAGAGTCTTGGTCTGTTG CCCAGGCTAGAGTTCAGTGGCGCCATCTCAGCTCACTGCAACCTCCGCCTCCCAGATTCA AGCGATTCTCCTGCCTCGACCTCCCAGTAGCTGGGATTACAGGTTTCCAGCAAATCCCTCT GAGCCGCCCCCGGGGGCTCGCCTCAGGAGCAAGGAAGCAAGGGGTGGGAGGAGGAGGT CTAAGTCCCAGGCCC AATTAAGAGATCAGATGGTGTAGGATTTGGGAGCTTTTAAGGTGAA GAGGCCCGGGCTGATCCCACTGGCCGGTATAAAGCACCGTGACCCTCAGGTGACGCACC AGGGCCGGCTGCCGTCGGGGACAGGGCTTTCCATAGCC

SEQ ID NO: 3 - M opsin fragment, 500 bp in size, UTR in italics, M8 mutation is underlined ACAGGTATTTGCCACTAAGCCCAGCTAATTGTTTTTTATTTAGTAGAAACGGGGTTT

CACCATGTTAGTCAGGCTGGTCGGGAACTCCTGACCTCAGGAGATCTACCCGCCTTGGCC TCCCAAAGTGCTGGGATTACAGGCGTGTGCCACTGTGCCCAGCCACTTTTTTTTAGACAGA GTCTTGGTCTGTTGCCCAGGCTAGAGTTCAGTGGCGCCATCTCAGCTCACTGCAACCTCC GCCTCCCAGATTCAAGCGATTCTCCTGCCTCGACCTCCCAGTAGCTGGGATTACAGGTT TC CAGCAAATCCCTCTGAGCCGCCCCCGGGGGCTCGCCTCAGGAGCAAGGAAGCAAGGGGT GGGAGGAGGAGGTCTAAGTCCCAGGCCCAATTAAGAGATCAGATGGTGTAGGATTTGGGA GCTTTTAAGGTGAAGAGGCCCGGGCTGATCCCACTGGCCGGTATAAAGCACCGTGACCCT CAGGTGACGCACCATCTAGAGCTGCCGTCGGGGACAGGGCTTTCCATAGCC

- 24 046019

SEQ ID NO: 4 - variant of the hG1.7(M8) construct, 1.2 kb. LCR M/L opsin fragment, M opsin fragment, 500 bp in size, UTR in italics, M8 mutation TAGGAATAGAAGGGTGGGTGCAGGAGGCTGAGGGGTGGGGAAAGGGCATGGGTG is underlined

TTTCATGAGGACAGAGCTTCCGTTTCATGCAATGAAAAGAGTTTGGAGACGATGGTGGTG ACTGGACTATACACTTACACACGGTAGCGATGGTACACTTTGTATTATGTATATTTTTACCAC GATCTTTTTAAAGTGTCAAAGGCAAATGGCCAAATGGTTCCTTGTCCTATAGCTGTAGCAGC CATCGGCTGTTAGTGACAAAGCCCCTGAGTCAAGATGACAGCAGCCCCCATAACTCC C CCCTCACCCCCTCGTTCTTCATATCCTTCTCTAGTGCTCCCTCCACTTTCATCCACCCTTC TGCAAGAGTGTGGGACCACAAATGAGTTTTCACCTGGCCTGGGGACACACGTGCCCCCAC AGGTGCTGAGTGACTTTCTAGGACAGTAATCTGCTTTAGGCTAAAATGGGACTTGATCTTCT GTTAGCCCTAATCATCAATTAGCAGAGCCGGTGAAGGTGCAGAACCTACCGCCTTTCCAG G CCTCCTCCCACCTCTGCCACCTCCACTCTTCCTTCCTGGGATGTGGGGGCTGGCACACGT GTGGCCCAGGGCATTGGTGGGATTGCACTGAGCTGGGTCATTAGCGTAATCCTGGACAAG GGCAGACAGGGCGAGCGGAGGGCCAGCTCCGGGGCTCAGGCAAGGCTGGGGGCTTCCC CCAGACACCCCACTCCTCCTCTGCTGGACCCCCACTTCATAGGGCACTTCGTGTTCTCAAA GGGCTT CCAAATAGCATGGTGGCCTTGGATGCCCAGGGAAGCCTCAGAGTTGCTTATCTC CCTCTAGACAGAAGGGGAATCTCGGTCAAGAGGGAGGTCGCCCTGTTCAAGGCCACC CAGCCAGCTCATGGCGGTAATGGGACAAGGCTGGCCAGCCATCCCACCCTCAGAAGGGA CCCGGTGGGGCAGGTGATCTCAGAGGAGGCTCACTTCTGGGTCTCACATTCTTGACAGGT ATTTGCCACTAAG CCCAGCTAATTGTTTTTTATTTAGTAGAAACGGGGTTTCACCATGTTAG TCAGGCTGGTCGGGAACTCCTGACCTCAGGAGATCTACCCGCCTTGGCCTCCCAAAGTGC TGGGATTACAGGCGTGTGCCACTGTGCCCAGCCACTTTTTTTTAGACAGAGTCTTGGTCTG TTGCCCAGGCTAGAGTTCAGTGGCGCCATCTCAGCTCACTGCAACCTCCGCCTCCCAGAT TCAAGCGATTCT CCTGCCTCGACCTCCCAGTAGCTGGGATTACAGGTTTCCAGCAAATCCC TCTGAGCCGCCCCCGGGGGCTCGCCTCAGGAGCAAGGAAGCAAGGGGTGGGAGGAGGA GGTCTAAGTCCCAGGCCCAATTAAGAGATCAGATGGTGTAGGATTTGGGAGCTTTTAAGGT GAAGAGGCCCGGGCTGATCCCACTGGCCGGTATAAAGCACCGTGACCCTCAGGTGACGC ACCATCTAGAGCTGCCGTC GGGGACAGGGCTTTCCATAGCC

SEQ ID NO: 5 - M opsin fragment 200 bp, UTR in italics, M8 mutation GATCGATTACAGGTTTCCAGCAAATCCCTCTGAGCCGCCCCCGGGGGCTCGCCTC is underlined

AGGAGCAAGGAAGCAAGGGGTGGGAGGAGGAGGTCTAAGTCCCAGGCCCAATTAAGAGA TCAGATGGTGTAGGATTTGGGAGCTTTTAAGGTGAAGAGGCCCGGGCTGATCCCACTGGC CGGTAATAAAGCACCGTGACCCTCAGGTGACGCACCATCTAGAGCTGCCGTCGGGGACAG GGCTTTCCATAGCC

- 25 046019

SEQ ID NO: 6 - hG1.4(M8) construct: LCR M/L opsin fragment, size 1.2 kb, M opsin fragment 200 bp, UTR in italics, underlined mutation M8 TAGGAATAGAAGGGTGGGTGCAGGAGGCTGAGGGGTGGGGAAAGGGCATGGGTG

TTTCATGAGGACAGAGCTTCCGTTTCATGCAATGAAAAGAGTTTGGAGACGATGGTGGTG ACTGGACTATACACTTACACACGGTAGCGATGGTACACTTTGTATTATGTATATTTTTACCAC GATCTTTTTAAAGTGTCAAAGGCAAATGGCCAAATGGTTCCTTGTCCTATAGCTGTAGCAGC CATCGGCTGTTAGTGACAAAGCCCCTGAGTCAAGATGACAGCAGCCCCCATAACTCC C CCCTCACCCCCTCGTTCTTCATATCCTTCTCTAGTGCTCCCTCCACTTTCATCCACCCTTC TGCAAGAGTGTGGGACCACAAATGAGTTTTCACCTGGCCTGGGGACACACGTGCCCCCAC AGGTGCTGAGTGACTTTCTAGGACAGTAATCTGCTTTAGGCTAAAATGGGACTTGATCTTCT GTTAGCCCTAATCATCAATTAGCAGAGCCGGTGAAGGTGCAGAACCTACCGCCTTTCCAG G CCTCCTCCCACCTCTGCCACCTCCACTCTTCCTTCCTGGGATGTGGGGGCTGGCACACGT GTGGCCCAGGGCATTGGTGGGATTGCACTGAGCTGGGTCATTAGCGTAATCCTGGACAAG GGCAGACAGGGCGAGCGGAGGGCCAGCTCCGGGGCTCAGGCAAGGCTGGGGGCTTCCC CCAGACACCCCACTCCTCCTCTGCTGGACCCCCACTTCATAGGGCACTTCGTGTTCTCAAA GGGCTT CCAAATAGCATGGTGGCCTTGGATGCCCAGGGAAGCCTCAGAGTTGCTTATCTC CCTCTAGACAGAAGGGGAATCTCGGTCAAGAGGGAGAGGTCGCCCTGTTCAAGGCCACC CAGCCAGCTCATGGCGGTAATGGGACAAGGCTGGCCAGCCATCCCACCCTCAGAAGGGA CCCGGTGGGGCAGGTGATCTCAGAGGAGGCTCACTTCTGGGTCTCACATTCTTGGATCGA TTACAGGTTTCCA C

SEQ ID NO: 7-CNGA3 cDNA without codon optimization

ATGGCCAAGATCAACACCCAATACTCCCACCCCTCCAGGACCCACCTCAAGGTAAA GACCTCAGACCGAGATCTCAATCGCGCTGAAAATGGCCTCAGCAGAGCCCACTCGTCAAG TGAGGAGACATCGTCAGTGCTGCAGCCGGGGATCGCCATGGAGACCAGAGGACTGGCTG ACTCCGGGCAGGGCTCCTTCACCGGCCAGGGGATCGCCAGGCTGTCGCGCCTCATCTTC TTGCTGCGCA GGTGGGCTGCCAGGCATGTGCACCACCAGGACCAGGGACCGGACTCTTT TCCTGATCGTTTCCGTGGAGCCGAGCTTAAGGAGGTGTCCAGCCAAGAAAGCAATGCCCA GGCAAATGTGGGCAGCCAGGAGCCAGCAGACAGAGGGAGAAGCGCCTGGCCCCTGGCC

- 26 046019

AAATGCAACACTAACACCAGCAACAACACGGAGGAGGAGAAGAAGACGAAAAAGAAGGAT GCGATCGTGGTGGACCCGTCCAGCAACCTGTACTACCGCTGGCTGACCGCCATCGCCCT GCCTGTCTTCTATAACTGGTATCTGCTTATTTGCAGGGCCTGTTTCGATGAGCTGCAGTCC GAGTACCTGATGCTGTGGCTGGTCCTGGACTACTCGGCAGATGTCCTGTATGTCTTGGAT GTG CTTGTACGAGCTCGGACAGGTTTTCTCGAGCAAGGCTTAATGGTCAGTGATACCAACA GGCTGTGGCAGCATTACAAGACGACCACGCAGTTCAAGCTGGATGTGTTGTCCCTGGTCC CCACCGACCTGGCTTACTTAAAGGTGGGCACAAACTACCCAGAGTGAGGTTCAACCGCC TACTGAAGTTTTCCCGGCTCTTTGAATTCTTTGACCGCCAGAGACAAGCAACTACCC CAATATG TTCAGGATTGGGAACTTGGTCTTGTACATTCTCATCATCATCCACTGGAATGCCT GCATCTACTTTGCCATTTCCAAGTTCATTGGTTTTGGGACAGACTCCTGGGTCTACCCAAAC ATCTCAATCCCAGAGCATGGGCGCCTCTCCAGGAAGTACATTTACAGTCTCTACTGGTCCA CCTTGACCCTTACCACCATTGGTGAGACCCCACCCCCCGTGAAAGATGAGGAGTATCTCTT TGTGGTCG TAGACTTCTTGGTGGGTGTTCTGATTTTTGCCACCATTGTGGGCAATGTGGGC TCCATGATCTCGAATATGAATGCCTCACGGGCAGAGTTCCAGGCCAAGATTGATTCCATCA AGCAGTACATGCAGTTCCGCAAGGTCACCAAGGACTTGGAGACGCGGGTTATCCGGTGGT TTGACTACCTGTGGGCCAACAAGAAGACGGTGGATGAGAAGGAGGTGCTCAAGAGCCTCC CAGACAAGCT GAAGGCTGAGATCGCCATCAACGTGCACCTGGACACGCTGAAGAAGGTTC GCATCTTCCAGGACTGTGAGGCAGGGCTGCTGGTGGAGCTGGTGCTGAAGCTGCGACCC ACTGTGTTCAGCCCTGGGGATTATATCTGCAAGAAGGGAGATATTGGGAAGGAGATGTACA TCATCAACGAGGGCAAGCTGGCCGTGGTGGCTGATGATGGGGTCACCCAGTTCGTGGTC CTCAGCGATGGC AGCTACTTCGGGGAGATCAGCATTCTGAACATCAAGGGGAGCAAGTCG GGGAACCGCAGGACGGCCAACATCCGCAGCATTGGCTACTCAGACCTGTTCTGCCTCTCA AAGGACGATCTCATGGAGGCCCTCACCGAGTACCCCGAAGCCAAGAAGGCCCTGGAGGA GAAAGGACGGCAGATCCTGATGAAAGACAACCTGATCGATGAGGAGCTGGCCAGGGCGG GCGCGGACCCCAAGGACCTTGA GGAGAAAGTGGAGCAGCTGGGGTCCTCCCTGGACACC CTGCAGACCAGGTTTGCACGCCTCCTGGCTGAGTACAACGCCACCCAGATGAAGATGAAG CAGCGTCTCAGCCAACTGGAAAGCCAGGTGAAGGGTGGTGGGGACAAGCCCCTGGCTGA TGGGGAAGTTCCCGGGGATGCTACAAAAACAGAGGACAAACAACAGTGA

SEQ ID NO: 8 - codon optimized CNGA3 cDNA

ATGGCAAAAATCAATACCCAGTACAGCCACCCCTCACGAACTCACCTGAAAGTCAA AACAAGCGATAGAGACCTGAACAGAGCCGAGAACGGCCTGTCCAGGGCCCACAGCTCCT CTGAGGAAACTAGTTCAGTGCTGCAGCCTGGAATCGCTATGGAGACCAGAGGGCTGGCTG ACTCTGGCCAAGGAAGTTTCACAGGGCAGGGCATCGCCAGGCTGTCTAGACTGATTTTTCT GCTGAG GAGATGGGCCGCTAGGCATGTGCACCATCAGGACCAGGGACCCGATAGTTTCC CTGACAGGTTCAGGGGGGCCGAACTGAAGGAGGTCAGCTCCCAGGAATCTAACGCACAG GCCAATGTGGGCAGTCAGGAGCCCGCTGATAGAGGACGGTCCGCATGGCCTCTGGCCAA GTGCAACACTAATACCTCTAACAATACAGAGGAAGAGAAGAAAACTAAGAAAAAGGATGCC

- 27 046019

ATCGTGGTCGACCCTTCTAGTAACCTGTACTATAGGTGGCTGACAGCTATCGCACTGCCAG TGTTCTACAATTGGTATCTGCTGATTTGCAGAGCTTGTTTTGACGAACTGCAGAGTGAGTAT CTGATGCTGTGGCTGGTGCTGGATTACTCAGCAGACGTGCTGTATGTGCTGGATGTCCTG GTGCGCGCACGAACTGGGTTCCTGGAGCAGGGCCTGATGGTGAGCGACACCA G

TTCCGCATCGGCAACCTGGTGCTGTATATTCTGATCATTATCCACTGGAATGCTTGTATCTA CTTTGCAATCAGCAAGTTCATTGGATTTGGGACCGACAGCTGGGTGTATCCAAACATTTCC ATCCCCGAACATGGACGACTGAGCAGGAAGTACATCTATTCACTGTACTGGAGCACACTGA CTCTGACCACAATTGGGGAGACCCCCCCTCCAGTGAAGGATGAAGAGTACCTGTTCGTGG A ACTGAAGGCAGAGATTGCCATCAATGTCCATCTGGATACTCTGAAAAAGGTGCGGATCTT CCAGGACTGCGAAGCAGGACTGCTGGTCGAGCTGGTGCTGAAGCTGCGCCCTACCGTGT TTAGCCCAGGCGATTATATCTGTAAAAAGGGGGACATTGGCAAAGAAATGTACATTATCAA CGAGGGGAAGCTGGCTGTCGTGGCAGACGATGGCGTGACCCAGTTCGTCGTGCTGAGCG ATGGCAGC TATTTTGGGGAAATTTCCATCCTGAATATCAAAGGCTCCAAGTCTGGAAACCG GCGCACAGCTAATATTCGGTCCATCGGATATTCTGACCTGTTCTGCCTGTCTAAGGACGAT CTGATGGAGGCACTGACTGAATACCCCGAGGCCAAAAGGCTCTGGAAGAGAAAGGCCG GCAGATCCTGATGAAGGATAACCTGATTGACGAAGAGCTGGCACGAGCTGGAGCAGACCC TAAAGATCTGGAAGAGAA GGTGGAGCAGCTGGGATCAAGCCTGGATACCCTGCAGACACG CTTCGCTCGACTGCTGGCAGAATACAATGCCACCCAGATGAAAATGAAGCAGCGCCTGAG TCAGCTGGAGTCACAGGTGAAAGGCGGAGGGGACAAGCCCCTGGCAGATGGCGAAGTCC CTGGCGACGCTACAAAAACAGAAGATAAACAGCAGTAA

SEQ ID NO: 9 - PDE6C cDNA, NM_006204.3

ATGGGTGAGATCAACCAAGTTGCCGTGGAGAAATACCTGGAGGAGAACCCTCAGTT

TGCCAAGGAGTACTTTGACAGGAAGTTGCGGGTGGAGGTGCTGGGAGAAATCTTCAAGAA CAGCCAGGTGCCAGTCCAGTCCAGCATGTCCTTCTCTGAGCTGACCCAGGTGGAGGAGTC AGCCCTGTGCTTGGAGCTGCTGTGGACCGTGCAGGAGGAGGGGCACCCCAGAGCAG GGGGTTCACAGGGCCCTGCAGAGGCTGGCCCACCTGCTCCAGGCTGACCGCTGCAGCAT GTTCCTGTGCCGGTCCCGGAACGGCATACCTGAGGTGGCCTCTAGGTTGCTGGATGTCAC CCCCACCTCCAAGTTTGAGGACAACCTGGTGGGCCCTGACAAAGAAGTTGTGTTTCCATTG GACATTGGGATAGTGGGTTGGGCTGCTCACACGAAGAAAACTCATAATGTCCCAGATGTGA

- 28 046019

AAAAGAACAGCCATTTTTCTGACTTCATGGACAAGCAAACTGGGTATGTCACTAAGAACCT GCTGGCAACCCCGATCGTGGTGGGCAAGGAGGTTCTTGCTGTGATCATGGCAGTTAACAA AGTAAATGCATCTGAATTTTCCAAACAGGATGAAGAGGTCTTTTCCAAATACCTCAACTTTG TGTCTATCATCCTAAGGCTTCATCACACCAGCTACATGTACAATATTGAATCCCGAAG AAGC CAGATCCTTATGTGGTCAGCCAATAAAGTATTTGAAGAACTCACAGATGTTGAGCGACAGT TTCACAAAGCGCTCTACACGGTTAGATCATATCTGAACTGTGAACGATACTCCATTGGACT GCTGGACATGACCAAGGAGAAGGAATTCTACGATGAATGGCCAATCAAGCTTGGAGAAGT AGAGCCTTATAAAGGTCCAAAGACACCTGATGGCAGGGAAGTCAACTTTTATAAAATC ATT GATTACATTTTACATGGAAAAGAAGAGATCAAAGTGATTCCGACGCCTCCTGCAGACCACT GGACACTCATTAGTGGGTTGCCAACATATGTTGCTGAAAATGGATTTATCTGTAACATGATG AATGCCCCTGCGGATGAATACTTCACATTTCAGAAAGGACCTGTAGACGAAACTGGTTGGG TCATTAAGAATGTTTTGTCCCTGCCTATTGTCAACAAGAAAGAAGATATTGTGGGAGTGGCT ACATTTTACAACAGGAAGGATGGAAAACCTTTCGATGAGCATGATGAATACATTACCGAGA CTCTCACACAATTTCTTGGATGGTCTCTTTTAAATACTGACACCTACGATAAGATGAATAAG CTAGAAAACAGAAAGGACATTGCTCAGGAAATGCTCATGAACCAAACCAAAGCCACTCCTG AAGAAATTAAGTCCATTTTGAAATTTCAAGAGAAGTTAAATGTTGATGTAATTGACGACTGT GAAGAAAAAACA ACTTGTTGCAATTTTGAAAGAGGACTTGCCAGACCCACGCTCAGCAGAAC TGTACGAATTCCGCTTCAGTGACTTCCCCCTTACAGCACGGATTGATTAAATGTGGAAT ACGACTGTTTTTTGAAATAAATGTGGTGGAGAAATTCAAAGTACCTGTAGAGGTTCTTACCA GATGGATGTACACTGTGAGGAAAGGGTACCGAGCTGTCACTTACCACAATTGGCGGCATG GGTTCAAC GTGGGGCAGACCATGTTTACTTTGCTGATGACAGGAAGATTAAAGAAGTACTA CACAGATCTCGAAGCCTTGCCATGCTTGCTGCTGCTTTCTGCCATGATATTGACCACAGA GGCACCAATAATTTGTACCAGATGAAATCCACGTCTCCATTAGCAAGACTTCATGGTTCTTC TATTTTGGAGAGGCACCACCTGGAGTACAGTAAGACTCTGTTGCAGGATGAGAGTTTAAAC ATCTTCCA GAACCTAAATAAGCGGCAGTTTGAAACAGTTATTCATTTGTTCGAGGTCGCAAT AATAGCAACTGACCTGGCTTTATATTTCAAGAAGAGGACCATGTTTCAAAAAATTGTTGATG CCTGTGAACAAATGCAAACGGAAGAAGAAGCCATCAAATATGTAACTGTTGATCCAACCAA GAAAGAGATTATCATGGCAATGATGATGACGGCATGTGACTTGTCTGCTATTACCAAGCCC TGGGAT GCAAAGTCAGGTAGCACTTATGGTTGCAAATGAATTTTGGGAACAAGGAGATC TGGAGAGAACAGTGTTGCAGCAACAACCCATTCCTATGATGGACAGAAACAAAAGAGATGA ATTACCTAAACTTCAAGTTGGATTTATTGATTTTGTTTGTACTTTTGTATATAAGGAGTTCTCA CGGTTTCACAAAGAAATCACACCTATGCTGAGTGGTCTTCAGAATAACAGAGTAGAATGGA AATC ACTAGCTGATGAGTATGATGCAAAGATGAAGGTCATTGAAGAGGAGGCAAAAAAGCA AGAAGGAGGAGCCGAAAAAGCTGCTGAAGATTCAGGAGGTGGTGATGACAAAAAGTCCAA AACATGTTTAATGTTGTAA

SEQ ID NO: 10 - PDE6H cDNA, NM_006205.2

- 29 046019

ATGAGTGACAACACTACTCTGCCTGCTCCAGCTTCAAACCAGGGTCCTACCACCCC ACGCAAAGGCCCTCCCAAGTTCAAGCAGAGGCAGACTCGCCAATTCAAGAGTAAACCCTCC AAAGAAAGGTGTGAAAGGATTTGGAGATGACATTCCAGGAATGGAGGGGCTAGGAACAGA TATCACAGTGATTTGTCCATGGGAGGCATTCAGCCACCTGGAATTGCATGAGCTCGCTCAG TTTGGGATT ATCTGA

SEQ ID NO: 11 - GNAT2 cDNA, NM_005272.3

ATGGGAAGTGGAGCCAGTGCTGAGGACAAAGAACTGGCCAAGAGGTCCAAGGAGC TAGAAAAGAAGCTGCAGGAGGATGCTGATAAGGAAGCCAAGACTGTCAAGCTGCTACTGC TGGGTGCTGGGGAGTCAGGAAAGAGCACCATCGTCAAACAGATGAAGATCATTCACCAGG ATGGCTATTCACCAGAAGAATGCCTGGAGTTCAAGGCTATCATCTATGGAAATGTGCTGCA GTCCATCCTGG CTATCATCCGGGCCATGACCACACTGGGCATCGATTATGCTGAACCAAG CTGTGCGGATGACGGGCGACAGCTCAACAACCTGGCTGACTCCATTGAGGAGGGAACCAT GCCTCCTGAGCTCGTGGAGGTCATTAGGAGGTTGTGGAAGGATGGTGGGGTGCAAGCCT GCTTCGAGAGAGCTGCAGAATACCAGCTTAATGACTCCGCATCTTACTACCTGAACCAATT AGAACGAATTACAGA CCCTGAGTACCTCCCTAGTGAGCAAGATGTGCTCCGATCCAGAGTC AAAACCACGGGCATCATTGAAACCAAGTTTTCCGTCAAAGACTTGAATTTCAGGATGTTTGA TGTGGGAGGGCAGAGATCCGAGAGAAAGAAGTGGATCCACTGCTTCGAGGGAGTCACCT GCATCATTTTCTGTGCAGCCCTCAGTGCCTATGATATGGTGCTGGTGGAAGATGACGAAGT GAATCGTATGCATGA GTCTTTGCATCTGTTCAACAGCATATGTAACCACAAAATTCTTTGCGG CTACTTCCATTGTCCTCTTTCTCAACAAGAAGGACCCTTTGAGGAAAAAATCAAGAAAGTC CATCTCAGCATTTGTTTTCCAGAGTATGATGGTAACAACTCCTATGATGATGCGGGGAATTA CATAAAGAGCCAGTTCCTTGACCCTCAATATGCGAAAAGATGTCAAAGAAATCTACAGTCACA TGACCTGTGCTAC AGATACACAGAATGTCAAATTTGTGTTTGATGCAGTTACAGATATTATC ATCAAAGAAAACCTCAAGGACTGCGGCCTCTTCTAA

SEQ ID NO: 12 - KCNV2 cDNA, NM_133497.3

ATGCTCAAACAGAGTGAGAGGAGACGGTCCTGGAGCTACAGGCCCTGGAACACGA CGGAGAATGAGGGCAGCCAACACCGCAGGAGCATTTGCTCCCTGGGTGCCCGTTCCGGC TCCCAGGCCAGCATCCACGGCTGGACAGGGGGCAACTATAACTACATCGAGGAAGAC GAAGACGGCGAGGAGGAGGACCAGTGGAAGGACGACCTGGCAGAAGAGGACCAGCAGG CAGGGGAGGTCACCA CCGCCAAGCCCGAGGGCCCCAGCGACCCTCCGGCCCTGCTGTC CACGCTGAATGTGAACGTGGGTGGCCACAGCTACCAGCTGGACTACTGCGAGCTGGCCG GCTTCCCCAAGACGCGCCTAGGTCGCCTGGCCACCTCCACCAGCCGCAGCCGCCAGCTA AGCCTGTGCGACGACTACGAGGAGCAGACAGACGAATACTTCTTCGACCGCGACCCGGC CGTCTTCCAGCTGGTCTACAAT TTCTACCTGTCCGGGGTGCTGCTGGTGCTCGACGGGCT GTGTCCGCGCCGCTTCCTGGAGGAGCTGGGCTACTGGGGCGTGCGGCTCAAGTACACGC CACGCTGCTGCCGCATCTGCTTCGAGGAGCGGCGCGACGAGCTGAGCGAACGGCTCAAG

- 30 046019

ATCCAGCACGAGCTGCGCGCAGGCGCAGGTCGAGGAGGCGGAGGAACTCTTCCGCG ACATGCGCTTCTACGGCCCGCAGCGGCGCCGCCTCTGGAACCTCATGGAGAAGCCATTCT CCTCGGTGGCCGCCAAGGCCATCGGGGTGGCCTCCAGCACCTTCGTGCTCGTCTCCGTG GTGGCGCTGGCGCTCAACACCGTGGAGGAGATGCAGCAGCACTCGGGGCAGGGCGAGG GCGGCCCAGAC CTGCGGCCCATCCTGGAGCACGTGGAGATGCTGTGCATGGGCTTCTTC ACGCTCGAGTACCTGCTGCGCCTAGCCTCCACGCCCGACCTGAGGCGCTTCGCGCGCAG CGCCCTCAACCTGGTGGACCTGGTGGCCATCCTGCCGCTCTACCTTCAGCTGCTGCTCGA GTGCTTCACGGGCGAGGGCCACCAACGCGGCCAGACGGTGGGCAGCGTGGGTAAGGTGGGTCAGGTGTTGCGCG TCATGCGCCTCATGCGCATCTTCCGCATCCTCAAGCTGGCGCGC CACTCCACCGGACTGCGTGCCTTCGGCTTCACGCTGCGCCAGTGCTACCAGCAGGTGGG CTGCCTGCTGCTCTTCATCGCCATGGGCATCTTCACTTTCTCTGCGGCTGTCTACTCTGTG GAGCACGATGTGCCCAGCACCAACTTCACTACCATCCCCCACTCCTGGTGGTGGGCCGCG GTGAGCATCTCCACCGTGGGCT ACGGAGACATGTACCCAGAGACCCACCTGGGCAGGTTT TTTGCCTTCCTCTGCATTGCTTTTGGGATCATTCTCAACGGGATGCCCATTTCCATCCTCTA CAACAAGTTTTCTGATTACTACAGCAAGCTGAAGGCTTATGAGTATACCACCATACGCAGG GAGAGGGGAGAGGTGAACTTCATGCAGAGAGCCAGAAAGAAGATAGCTGAGTGTTTGCTT GGAAGCAACCCACAGCTCACCCCAA GACAAGAGAATTAG

SEQ ID NO: 13 - CACNA2D4 cDNA, NM_172364.4

ATGGTCTGTGGCTGCTCTGCCCTCCTTCCCCTCCCCAACCCCAGGCCCACCATGC CTGCAACTCCCAACTTCCTCGCAAACCCCAGCTCCAGCAGCCGCTGGATTCCCCTCCAGC CAATGCCCGTGGCCTGGGCCTTTGTGCAGAAGACCTCGGCCCTCCTGTGGCTGCTGCTTC TAGGCACCTCCCTGTCCCCTGCGTGGGGACAGGCCAAGATTCCTCTGGAAACAGTGAAGC TATGG GCTGACACCTTTCGGCGGGGACCTGTATAACACTGTGACCAAATACTCAGGCTCTCT CTTGCTGCAGAAGAAGTACAAGGATGTGGAGTCCAGTCTGAAGATCGAGGTGGATGG CTTGGAGCTGGTGAGGAAGTTCTCAGAGGACATGGAGAACATGCTGCGGAGGAAAGTCGA GGCGGTCCAGAATCTGGTGGAAGCTGCCGAGGAGGCCGACCTGAACCACGAATTCAATG AATCCCT GGTGTTCGACTATTACAACTCGGTCCTGATCAACGAGAGGGACGAGAAGGGCA ACTTCGTGGAGCTGGGCGCCGAGTTCCTCCTGGAGTCCAATGCTCACTTCAGCAACCTGC CGGTGAACACCTCCATCAGCAGCGTGCAGCTGCCCACCAACGTGTACAACAAAGACCCAG ATATTTTAAATGGAGTCTACATGTCTGAAGCCTTGAATGCTGTCTTCGTGGAGAACTTCCAG AGAGACCCAACG TTGACCTGGCAATATTTTGGCAGTGCAACTGGATTCTTCAGGATCTATC CAGGTATAAAATGGACACCTGATGAGAATGGAGTCATTACTTTTGACTGCCGAAACCGCGG CTGGTACATTCAAGCTGCTACTTCTCCCAAGGACATAGTGATTTTGGTGGACGTGAGCGGC AGTATGAAGGGGCTGAGGATGACTATTGCCAAGCACACCATCACCACCATCTTGGACACC CTGGGGAAATGACTTCAT TAATATCATAGCGTACAATGACTACGTCCATTACATCGAGC CTTGTTTTAAAGGGATCCTCGTCCAGGCGGACCGAGACAATCGAGAGCATTTCAAACTGCT GGTGGAGGAGTTGATGGTCAAAGGTGTGGGGGTCGTGGACCAAGCCCTGAGAGAAGCCT

- 31 046019

TCCAGATCCTGAAGCAGTTCCAAGAGGCCAAGCAAGGAAGCCTCTGCAACCAGGCCATCA TGCTCATCAGCGACGGCGCCGTGGAGGACTACGAGCCGGTGTTTGAGAAGTATAACTGGC CAGACTGTAAGGTCCGAGTTTTCACTTACCCTCATTGGGAGAGAAGTGTCTTTTGCTGACCG CATGAAGTGGATTGCATGCAACAACAAAGGCTACTACACGCAGATCTCAACGCTGGCGGA CA CCCAGGAGAACGTGATGGAATACCTGCACGTGCTCAGCCGCCCCATGGTCATCAACCA CGACCACGACATCATCTGGACAGAGGCCTACATGGACAGCAAGCTCCTCAGCTCGCAGGC TCAGAGCCTGACACTGCTCACCACTGTGGCCATGCCAGTCTTCAGCAAGAAGAACGAAAC GCGATCCCATGGCATTCTCCTGGGTGTGGTGGGCTCAGATGTGGCCCTGAGAGAGCTGAT GAAGCT GGCGCCCCGGTACAAGCTTGGAGTGCACGGATACGCCTTTCTGAACACCAACAA TGGCTACATCCTCTCCCATCCCGACCTCCGGCCCCTGTACAGAGAGGGGAAGAAACTAAA ACCCAAACCTAACTACAACAGTGTGGATCTCTCCGAAGTGGAGTGGGAAGACCAGGCTGA ATCTCTGAGAACAGCCATGATCAATAGGGAAACAGGTACTCTCTCGATGGATGTGGGAAGGTT CCGATAT AAAGGGAAGCGAGTTCTTTTCCTGACCAATGACTACTTCTTCACGGACATCA GCGACACCCCTTTCAGTTTGGGGGTGGTGCTGTCCCGGGGCCACGGAGAATACATCCTTC TGGGGAACACGTCTGTGGAAGAAGGCCTGCATGACTTGCTTCACCCAGACCTGGCCCTGG CCGGTGACTGGATCTACTGCATCACAGATATTGACCCAGACCACGGAAGCTCAGCCAGC TAGAGGCCATGATCCGCTTC CTCACCAGGAAGGACCCAGACCTGGAGTGTGACGAGGAG CTGGTCCGGGAGGTGCTGTTTGACGCGGTGGTGACAGCCCCCATGGAAGCCTACTGGAC AGCGCTGGCCCTCAACATGTCCGAGGAGTCTGAACACGTGGTGGACATGGCCTTCCTGGG CACCCGGGCTGGCCTCCTGAGAAGCAGCTTGTTCGTGGGCTCCGAGAAGGTCTCCGACA GGAAGTTCCTGACACCTGAGGAC GAGGCCAGCGTGTTCACCCTGGACCGCTTCCCGCTGT GGTACCGCCAGGCCTCAGAGCATCCTGCTGGCAGCTTCGTCTTCAACCTCCGCTGGGCAG AAGGACCAGAAAGTGCGGGTGAACCCATGGTGGTGACGGCAAGCACAGCTGTGGCGGTG ACCGTGGACAAGAGGACAGCCATTGCTGCAGCCGCGGGCGTCCAAATGAAGCTGGAATTC CTCCAGCGCAAATTCTGGGCGGCAACGCGG CAGTGCAGCACTGTGGATGGGCCGTGCAC ACAGAGCTGCGAGGACAGTGATCTGGACTGCTTCGTCATCGACAACAACGGGTTCATTCT GATCTCCAAGAGGTCCCGAGACGGGAAGATTTCTGGGGGAGGTGGATGGTGCTGTCC TGACCCAGCTGCTCAGCATGGGGGTGTTCAGCCAAGTGACTATGTATGACTATCAGGCCA TGTGCAAACCCTCGAGTCACCACCACAGTG CAGCCCAGCCCCTGGTCAGCCCAATTTCTG CCTTCTTGACGGCGACCAGGTGGCTGCTGCAGGAGCTGGTGCTGTTCCTGCTGGAGTGG AGTGTCTGGGGCTCCTGGTACGACAGAGGGGCCGAGGCCAAAAGTGTCTTCCATCACTCC CACAAACACAAGAAGCAGGACCCGCTGCAGCCCTGCGACACGGAGTACCCCGTGTTCGT GTACCAGCCGGCCATCCGGGAGGCCAACGGGAT CGTGGAGTGCGGGCCCTGCCAGAAG GTATTTGTGGTGCAGCAGATTCCCAACAGTAACCTCCTCCTCCTGGTGACAGACCCCACCT GTGACTGCAGCATCTTCCCACCAGTGCTGCAGGAGGCGACAGAAGTCAAATATAATGCCT CTGTCAAATGTGACCGGATGCGCTCCCAGAAGCTCCGCCGGCGACCAGACTCCTGCCAC GCCTTCCATCCAGAGGAGAATGCCCAGGACTGCGGCGGC GCCTCGGACACCTCAGCCTC

- 32 046019

GCCGCCCCTACTCCTGCTGCCTGTGTGTGCCTGGGGGCTACTGCCCCAACTCCTGCGGT GA

SEQ ID NO: 14 - CNGB3 cDNA, NM_019098.4

ATGTTTAAAATCGCTGACAAAAGTCAACAAGGTGAAGCCTATAGGAGAGAACAATGA GAATGAACAAAGTTCTCGTCGGAATGAAGAAGGCTCTCACCCAAGTAATCAGTCTCAGCAA ACCACAGCACAGGAAGAAAACAAAGGTGAAGAGAAATCTCTCAAAACCAAGTCAACTCCAG TCACGTCTGAAGAGCCACACCAACATACAAGACAAACTCTCCAAGAAAAATTCCTCTGG AGATCT GACCACAAACCCTGACCCTCAAAATGCAGCAGAACCAACTGGAACAGTGCCAGA GCAGAAGGAAATGGACCCCGGGAAAGAAGGTCCAAACAGCCCACAAAACAAACCGCCTGC AGCTCCTGTTATAAATGAGTATGCCGATGCCCAGCTACACAACCTGGTGAAAAGAATGCGT CAAAGAACAGCCCTCTACAAGAAAAAGTTGGTAGAGGGAGATCTCTCCTCACCCGAAGCC AGCCCACAAACTGC AAAGCCCACGGCTGTACCACCAGTAAAAGAAAGCGATGATAAGCCA ACAGAACATTACTACAGGCTGTTGTGGTTCAAAGTCAAAAAGATGCCTTTAACAGAGTACTT AAAGCGAATTAAACTTCCAAACAGCATAGATTCATACACAGATCGACTCTATCTCCTGTGGC TCTTGCTTGTCACTCTTGCCTATAACTGGAACTGCTGGTTTATACCACTGCGCCTCGTCTTC CCATATCAAACCGCAGACAA CATACACTACTGGCTTATTGCGGACATCATATGTGATATCAT CTACCTTTATGATATGCTATTTATCCAGCCCAGACTCCAGTTTGTAAGAGGAGGAGACATAA TAGTGGATTCAAATGAGCTAAGGAAACACTACAGGACTTCTCCAAAATTTCAGTTGGATGTC GCATCAATAATACCATTTGATATTTGCTACCTCTTCTTTGGGTTTAATCCAATGTTTAGAGCA AATAGGATGTTAAAGTACACTTCAT TTTTTGAATTTAATCATCACCTAGAGTCTATAATGGAC AAAGCATATATCTACAGAGTTATTCGAACAACTGGATACTTGCTGTTTATTCTGCACATTAAT GCCTGTGTTTATTACTGGGCTTCAAACTATGAAGGAATTGGCACTACTAGATGGGTGTATG ATGGGGAAGGAAACGAGTATCTGAGATGTTATTATTGGGCAGTTCGAACTTTAATTACCATT GGTGGCCTACCAGAACCACAAACT TTATTTGAAATTGTTTTTCAACTCTTGAATTTTTTTTCT GGAGTTTTTGTGTTCTCCAGTTTAATTGGTCAGATGAGAGATGTGATTGGAGCAGCTACAG CCAATCAGAACTACTTCCGCGCCTGCATGGATGACACCATTGCCTACATGAACAATTACTC CATTCCTAAACTTGTGCAAAAGCGAGTTCGGACTTGGTATGAATATACATGGGACTCTCAAA GAATGCTAGATGAGTCTGATTTGC TTAAGACCTACCAACTACGGTCCAGTTAGCCCTCGC CATTGATGTGAACTTCAGCATCATCAGCAAAGTCGACTTGTTCAAGGGTTGTGATACACAG ATGATTTATGACATGTTGCTAAGATTGAAATCCGTTCTCTATTTGCCTGGTGACTTTGTCTG CAAAAAGGGAGAAATTGGCAAGGAAATGTATATCATCAAGCATGGAGAAGTCCAAGTTCTT GGAGGCCCTGATGGTACTAAAGTTCT TTACTCTGAAAGCTGGGTCGGTGTTTGGAGAA ATCAGCCTTCTAGCAGCAGGAGGAGGAAACCGTCGAACTGCCAATGTGGTGGCCCACGG GTTTGCCAATCTTTTAACTCTAGACAAAAAGACCCTCCAAGAAATTCTAGTGCATTATCCAG ATTCTGAAAGGATCCTCATGAAGAAAGCCAGAGTGCTTTTTAAAGCAGAAGGCTAAGACCGC AGAAGCAACCCCTCCAAGAAAAGATCTTGCCCTCCTCT TCCCACCGAAAGAAGAGACACCC AAACTGTTTTAAAACTCTCCTAGGAGGCACAGGAAAAGCAAGTCTTGCAAGACTACTCAAATT GAAGCGAGAGCAAGCAGCTCAGAAGAAAGAAAATTCTGAAGGAGGAGAGGAAGAAGGAAA AGAAAATGAAGATAAACAAAAAGAAAATGAAGATAAACAAAAAGAAAATGAAGATAAAGGAA AAGAAAATGAAGATAAAGATAAAGGAAGAGAGCCAGAAGAGAAGCCACTGGA CAGACCTG AATGTACAGCAAGTCCTATTGCAGTGGAGGAAGAACCCCACTCAGTTAGAAGGACAGTTTT ACCCAGAGGGACTTCTCGTCAATCACTCATTATCAGCATGGCTCCTTCTGCTGAGGGCGGA GAAGAGGTTCTTACTATTGAAGTCAAAGAAAAGGCTAAGCAATAA

- 33 046019

SEQ ID NO: 15 - hG1.7(M8) construct, LCR M/L opsin fragment, size 1.2 kb, M opsin fragment 500 bp, UTR in italics, underlined mutation M8 TAGGAATAGAAGGGTGGGTGCAGGAGGCTGAGGGGTGGGGAAAGGGCATGGGTG

TTTCATGAGGACAGAGCTTCCGTTTCATGCAATGAAAAGAGTTTGGAGACGATGGTGGTG ACTGGACTATACACTTACACACGGTAGCGATGGTACACTTTGTATTATGTATATTTTTACCAC GATCTTTTTAAAGTGTCAAAGGCAAATGGCCAAATGGTTCCTTGTCCTATAGCTGTAGCAGC CATCGGCTGTTAGTGACAAAGCCCCTGAGTCAAGATGACAGCAGCCCCCATAACTCC C CCCTCACCCCCTCGTTCTTCATATCCTTCTCTAGTGCTCCCTCCACTTTCATCCACCCTTC TGCAAGAGTGTGGGACCACAAATGAGTTTTCACCTGGCCTGGGGACACACGTGCCCCCAC AGGTGCTGAGTGACTTTCTAGGACAGTAATCTGCTTTAGGCTAAAATGGGACTTGATCTTCT GTTAGCCCTAATCATCAATTAGCAGAGCCGGTGAAGGTGCAGAACCTACCGCCTTTCCAG G CCTCCTCCCACCTCTGCCACCTCCACTCTTCCTTCCTGGGATGTGGGGGCTGGCACACGT GTGGCCCAGGGCATTGGTGGGATTGCACTGAGCTGGGTCATTAGCGTAATCCTGGACAAG GGCAGACAGGGCGAGCGGAGGGCCAGCTCCGGGGCTCAGGCAAGGCTGGGGGCTTCCC CCAGACACCCCACTCCTCCTCTGCTGGACCCCCACTTCATAGGGCACTTCGTGTTCTCAAA GGGCTT CCAAATAGCATGGTGGCCTTGGATGCCCAGGGAAGCCTCAGAGTTGCTTATCTC CCTCTAGACAGAAGGGGAATCTCGGTCAAGAGGGAGAGGTCGCCCTGTTCAAGGCCACC CAGCCAGCTCATGGCGGTAATGGGACAAGGCTGGCCAGCCATCCCACCCTCAGAAGGGA CCCGGTGGGGCAGGTGATCTCAGAGGAGGCTCACTTCTGGGTCTCACATTCTTGGATCAC AGGTATTTGCCACT AAGCCCAGCTAATTGTTTTTTATTTAGTAGAAACGGGGTTTCACCATG TTAGTCAGGCTGGTCGGGAACTCCTGACCTCAGGAGATCTACCCGCCTTGGCCTCCCAAA GTGCTGGGATTACAGGCGTGTGCCACTGTGCCCAGCCACTTTTTTTTAGACAGAGTCTTGG TCTGTTGCCCAGGCTAGAGTTCAGTGGCGCCATCTCAGCTCACTGCAACCTCCGCCTCCC AGATTCAAGCGAT TCTCCTGCCTCGACCTCCCAGTAGCTGGGATTACAGGTTTCCAGCAAA TCCCTCTGAGCCGCCCCCGGGGGCTCGCCTCAGGAGCAAGGAAGCAAGGGGTGGGAGG AGGAGGTCTAAGTCCCAGGCCCAATTAAGAGATCAGATGGTGTAGGATTTGGGAGCTTTTA AGGTGAAGAGGCCCGGGCTGATCCCACTGGCCGGTATAAAGCACCGTGACCCTCAGGTG ACGCACCATCTAGAGCTGC CGTCGGGGACAGGGCTTTCCATAGCC

-

Claims (29)

SEQ ID NO: 16 - M8 TCTAGA mutation SEQ ID NO: 17 - SEQ ID NO: 22 - fragment M of the opsin promoter, 2.0 kb in size, underlined fragment SEQ ID NO: 3, 500 bp in size, UTR in italics, underlined mutation M8 TAAAAAGCAAGTCTTGCCAGGGCAGTGGTGTGCACCTGTGGTCCCAGCTACTCAG GATGCTGAGGCAGGAGGATTACTTGTGCCCAGCAAGTAGAGGCTGCAGTGACCTGTGACT GTGCTACTGCCCTCCAACCTGGGTGACAGAGTGAGACCTTGTCTCAAAAAAAAAAGAGCG GGGGGGGGGGGCCGGGCCGGGCGTGGTGGCTCACAGCTGTAATCCCAGCACTTTGGGA AGCCAAGG CGGGTGGATCACTTGAGGTCAGGAGTTTGAGACCATCATGGTCAACACTGCG AAACACTGTCCCTACTAAAAATACAAAAATTAGCCGGGCATGGTGGCACACACCTGTAATC CCAGCTACTGGGGAGGCTGAGGCAGGAGAATTGCTTGAGCCGGGGAGACGGAGGTTGCA GTGAGCCGAGACTGCGCCACTGCACTCCAGCCTGACTGACAAGAGTGAGATTGTCTCAAA AAAAAAAAAAAAGTAATCACT AGAAAGAAGCTACATATGTACATAACATCCAAATAACCAA GAGGAGAAAAAAATGGGACTTGATTAATCAAAACAAAAACAAAAAAGAAAGAAAGAAAGGG GGAGAAAATAAAACAAGGGCTGGGTGTGCTGGCTCATGCCTGTAATCCCAGCACTTTGGA AGCCAAGGTGGGTGGATCTCTTGAGCTCAGGAGGTCAAGACCAGCCTGGGCAACATGGC GAAACCCCGTCTCTATTAAAAAAATTAATACAA CAATTATCCTGGAGTGGTGGTGCACA CCTGTAGTCCCAGCTACCCAGGACGCTGAGACGGGAGGATCGCTTGATCCCGGGGATGT CGAGGCTGCCGTGATCGCACCACTGCCCTCCAGCCAGGGTGGCAGACTGAGACCCCATC TCAAAAAATAAATAAATAAAAGCAAACAAGAAAAAAAAAGGCTTGAAACATATCTGATAGATA AAGGGCTAATCAACACAATATATAAAGAACTGCAAATCAGTAAACT AAGAGCAAATAACCCA ATATAAAGACATTAAAGGGTAGCCACGGACATCTCAGACGACGAAAAACAAAAGACAGTAA ACGTATAATAAAACATGTAATTGCAAGGTGATCCGGGAATAGTAAGCGAAAAGCAACAATTA AATACTATTTTCTCATCCACCAGAACGCCAAAAATTAAAAAGCCTAACAATGTCCAGGGCTG GCGAGAATGTGGCAGAAGGTGATGTCACATACCCTGCAAGTGGGAATCTAAA CAGATTCA GGGTTTTGGTTTTTTTTTAATCGCAATTAGGTGGCCTGTTAAATTTTTTTTCTTGAGACAGAG TTTTGCTCTTGTTGCCCAGGCTGGAGTGCAATGGCTCGATCTTGGCTCACCGCAACCTCGA CCTCCCAGGTACAAGCGATTCTCCTGTCTCAGCCTCCCAAGTAGCTGGGAGTACAGGTATT TGCCACTAAGCCCAGCTAATTGTTTTTTATTTAGTAGAAACGGGGTT TCACCATGTTAGTCA GGCTGGTCGGGAACTCCTGACCTCAGGAGATCTACCCGCCTTGGCCTCCCAAAGTGCTGG GATTACAGGCGTGTGCCACTGTGCCCAGCCACTTTTTTTTAGACAGAGTCTTGGTCTGTTG CCCAGGCTAGAGTTCAGTGGCGCCATCTCAGCTCACTGCAACCTCCGCCTCCCAGATTCA AGCGATTCTCCTGCCTCGACCTCCCAGTAGCTGGGATTACAGGTTTC CAGCAAATCCCTCT GAGCCGCCCCCGGGGGCTCGCCTCAGGAGCAAGGAAGCAAGGGGTGGGAGGAGGAGGT CTAAGTCCCAGGCCCAATTAAGAGATCAGATGGTGTAGGATTTGGGAGCTTTTAAGGTGAA GAGGCCCGGGCTGATCCCACTGGCCGGTATAAAGCACCGTGACCCTCAGGTGACGCACC ATCTAGAGCTGCCGTCGGGGACAGGGCTTTCCATAGCC CLAIM 1. Transcription control unit (TCU) up to 2500 nucleotides long, containing in the 5' to 3' direction: (a) a locus control region (LCR) containing: (i) SEQ ID NO: 1 or (ii) a sequence having at least 90% sequence identity with the specified sequence (a)(i); and (b) a promoter element comprising: (i) at least the last 200 nucleotides, but not more than 1100 nucleotides of SEQ ID NO: 17; or (ii) a sequence having at least 90% sequence identity with the specified sequence (b)(i) and wherein the nucleotides corresponding to nucleotides 1934-1939 of SEQ ID NO: 2 are replaced by SEQ ID NO: 16; wherein said TCU exhibits cone photoreceptor-specific promoter activity. 2. TCU according to claim 1, where (b) contains: (i) at least the last 500 nucleotides of SEQ ID NO: 17 or (ii) a sequence having at least 90% sequence identity with the specified sequence (i) and where the nucleotides corresponding to nucleotides 1934-1939 of SEQ ID - 35 046019 NO: 2, replaced by SEQ ID NO: 16; wherein said TCU exhibits cone photoreceptor-specific promoter activity. 3. TCU according to claim 1 or 2, wherein the promoter element contains at least the last 200 nucleotides of SEQ ID NO: 17. 4. TCU according to any one of claims 1 to 3, wherein the promoter element contains at least the last 500 nucleotides of SEQ ID NO: 17. 5. TCU according to any one of claims 1 to 4, wherein (b) contains at least 200 nucleotides of SEQ ID NO: 3. 6. TCU according to any one of claims 1 to 5, wherein (b) contains a sequence of at least 10 contiguous nucleotides selected from nucleotides 1-35 of SEQ ID NO: 3, or a sequence containing at least 10 contiguous nucleotides selected from a sequence having at least 90% sequence identity to nucleotides 1-35 of SEQ ID NO: 3. 7. TCU according to any one of claims 1-6, where (b) contains: (i) SEQ ID NO: 3 or (ii) a sequence having at least 90% sequence identity with the specified sequence (i); wherein said TCU exhibits cone photoreceptor-specific promoter activity. 8. The TCU of claim 7, wherein the TCU comprises SEQ ID NO: 4 or SEQ ID NO: 15. 9. The TCU of claim 7, wherein the TCU comprises SEQ ID NO: 15 or a sequence having at least 75%, at least 80%, at least 85%, at least 90% identical, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15, wherein said TCU exhibits cone photoreceptor-specific promoter activity. 10. TCU according to any one of claims 1 to 5, wherein the promoter element contains SEQ ID NO: 5. 11. The TCU of claim 10, wherein the TCU comprises SEQ ID NO: 6. 12. TCU according to claim 1, where TCU contains: (a) a locus control region containing SEQ ID NO 1; and (b) a promoter element comprising SEQ ID NO: 3. 13. An expression construct comprising a TCU according to any one of claims 1 to 12 and an operably linked sequence. 14. The expression construct of claim 13, wherein the operably linked sequence is CNGA3, CNGB3, PDE6C, PDE6H, GNAT2, KCNV2, or CACNA2D4. 15. The expression construct of claim 14, wherein the operably linked sequence comprises SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14, or a sequence that has at least 80% sequence identity with SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14 and has the ability to restore cone photoreceptor function. 16. The expression construct of claim 14 or 15, wherein the operably linked sequence comprises SEQ ID NO: 8. 17. The expression construct of claim 14 or 15, wherein the operably linked sequence comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO : 8 and has the ability to restore cone photoreceptor function. 18. A vector containing a TCU according to any one of claims 1-12 or an expression construct according to any one of claims 13-17. 19. The vector according to claim 18, which is a viral vector. 20. The vector of claim 19, which is an adeno-associated virus (AAV) vector. 21. The vector according to claim 20, where the vector contains the AAV genome or a derivative thereof. 22. The vector of claim 21, wherein the vector contains an AAV genome or a derivative thereof and wherein the AAV capsid is derived from AAV8. 23. The vector of claim 22, wherein the AAV capsid is derived from AAV8 and the expression construct is defined as in any one of claims 13 to 17. 24. The vector of claim 23, wherein the operably linked sequence is CNGA3. 25. A vector according to any one of claims 21 to 24, wherein said derivative is a chimeric or capsid-modified derivative. 26. The vector according to any one of claims 21-24, wherein said AAV genome is derived from a naturally occurring AAV serotype or isolate or clade. 27. The vector of claim 26, wherein said AAV genome is derived from AAV serotype 2 (AAV2), AAV serotype 4 (AAV4), or AAV serotype 8 (AAV8) and/or wherein the capsid is derived from AAV8. 28. The vector of claim 27, wherein the genome is derived from AAV2 and the capsid is derived from AAV8. 29. A host cell that contains the vector according to any one of claims 18-28. -