METHODS FOR MODULATING DEVELOPMENT AND FUNCTION OF

PHOTORECEPTOR CELLS

FIELD OF THE DISCLOSURE

The present invention relates to compositions and methods for administering gene modifiers for treating ocular disease.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No. EY017653 awarded by the National Institute of Health and the National Eye Institute. The Government has certain rights in the invention.

BACKGROUND OF THE DISCLOSURE

Genetic heterogeneity is observed for many Mendelian, single gene disorders. While environmental influences provide minor contributions, variations in phenotypic outcome are generally attributable to allelic heterogeneity or genetic modifier genes, allelic variants distinct from the mutant gene, which can affect disease onset, progression, and outcome by either increasing or reducing disease severity. As such, prior to the invention described herein, there was a pressing need to identify genetic modifiers of disease.

SUMMARY OF THE DISCLOSURE

The present invention relates to the delivery of compositions that modify or restore the signaling pathways and/or function of photoreceptors for use in the treatment and prevention of ocular diseases or disorders. For example, the invention provides for methods of treating or reducing the severity of an ocular disease or disorder in a subject in need thereof by locally administering to an ocular or adnexal tissue of the subject a composition comprising a nucleic acid comprising a nucleic acid encoding a nuclear hormone receptor or fragment thereof. The present invention further provides compositions comprising a nucleic acid encoding a nuclear hormone receptor or fragment thereof for local administration to the ocular or adnexal tissue of a subject for treating or reducing the severity of an ocular disease or disorder. Preferably, the nuclear hormone receptor or fragment thereof increases the amount of photoreceptors, increases the activity of photoreceptors, or normalizes the activity of photoreceptors in an ocular cell. In some embodiments, the nucleic acid encodes Nrldl, Nr2e3, Rora, Nuprl, Nr2Cl, or a fragment thereof. The nuclear hormone receptors of the present invention regulate a level of a photopigment. Examples of photopigments include, but are not limited to rhodopsin, S-opsin, M-opsin, L-opsin, or L/M-opsin.

In some preferred embodiments, the nuclear hormone receptor includes a

photoreceptor- specific nuclear hormone receptor. For example, photoreceptor- specific nuclear hormone receptor comprises Nr2e3.

The subject is preferably a mammal in need of such treatment, e.g., a subject that is suffering from or at risk of developing an ocular disease or disorder. The ocular disease or disorder is characterized by retinal degeneration, e.g., photoreceptor degeneration.

Photoreceptor degeneration can be characterized by loss or death of photoreceptor cells, such as rod and cone cells in the retina. In one example, the ocular disease or disorder is characterized by Nr2e3- associated retinal degeneration. For example, the ocular disease or disorder is selected from enhanced S-cone syndrome (ESCS), Goldmann-Favre syndrome, age-related macular degeneration (AMD), retinitis pigmentosa (RP), including, among others, autosomal dominant retinitis pigmentosa.

Administration of the gene modifier ameliorates clinical, morphological, and functional defects associated with the primary gene mutation. Preferably, retinal integrity and visual function are restored in the subject. Additionally, the methods and compositions provided herein are useful for alleviating or reducing at least one symptom associated with the ocular disease or disorder. Symptoms include retinal spotting, retinal dysplasia (i.e., presence of waves, whorls, and rosettes), increased number of S-opsin-expressing cone cells, degeneration of photoreceptor cells (i.e., rods and cones), and decreased rod cell, long cone cell, and M-cone-cell function in the subject. These clinical, morphological, and functional defects can be measured using standard methods in the art and as described herein. Retinal dysplasia, for example, can be identified or determined using histology and immuno staining. Retinal spotting can be identified or quantified by fundus photography. Retinal integrity, visual acuity/function, and retinal degeneration can be determined by electroretinogram analysis. Electroretinography measures the electrical responses of various cell types in the retina, including the photoreceptors (rods and cones), inner retinal cells (bipolar and amacrine cells) and the ganglion cells. Electrodes are usually placed on the cornea and the skin near the eye to record the ERG. During a recording, the patient's eyes are exposed to standardized stimuli and the resulting signal is displayed showing the time course of the signal's amplitude (voltage). Signals are very small, and typically are measured in microvolts or nanovolts. The ERG is composed of electrical potentials contributed by different cell types within the retina, and the stimulus conditions (flash or pattern stimulus, whether a background light is present, and the colors of the stimulus and background) can elicit stronger response from certain components. Clinically used mainly by ophthalmologists, the electroretinogram (ERG) is used for the diagnosis of various retinal diseases.

Photopic and scotopic responses are measured under different stimuli conditions to differentiate between visual signal and response from rod and cones. Under photopic conditions (i.e., luminance greater than 3 cd/m ), the rods are saturated and only the cones are producing visual signal. Under scotopic conditions (i.e., luminance less than 0.03 cd/m ), the light levels are too low to activate the cones, but the rods respond. Other components of the visual response can be identified and compared to determine restoration of photoreceptor or visual function, such as the a- wave, b-wave, c-wave, d-wave and m-wave.

As used herein, "normalize" a level or activity refers to bringing the level or activity of a photoreceptor or photopigment to within the normal range, i.e., the level or activity range as determined from a subject not suffering from the ocular disease or disorder. For example, the methods and composition described herein induce normalization of visual response as measured by electroretinogram, such that the amplitude in response to scotopic condition is at least greater than 100 microvolts; and the amplitude in response to photopic condition is at least greater than 50 microvolts.

As used herein, an "increase" in a level or activity of a nuclear hormone receptor, a downstream signaling component (i.e., photo transducin), or a photoreceptor can be measured by methods known in the art, such as RT-PCR, Western blot, transactivation assays, or electroretinography. An increase in expression level or activity can be 1%, 2%, 5%, 10%, 25%, 50%, 75%, 1-fold, 2-fold, 5-fold, or 10-fold reduced when compared to expression level or activity before treatment, or to expression level or activity in subjects that are suffering from the ocular disease or disorder that have not received treatment. Similarly and as described herein, a "decrease" in a level or activity of a nuclear hormone receptor, a downstream signaling component (i.e., photo transducin), or a photoreceptor can be measured by methods known in the art, such as RT-PCR, Western blot, transactivation assays, or electroretinography, can be measured by methods known in the art, such as RT-PCR or transactivation assays. A reduction in expression level or activity can be 1%, 2%, 5%, 10%, 25%, 50%, 75%, 1-fold, 2-fold, 5-fold, or 10-fold reduced when compared to expression level or activity before treatment, or to expression level or activity in subjects that are suffering from the ocular disease or disorder that have not received treatment.

The mammal can be any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a horse, as well as livestock or animals grown for food consumption, e.g., cattle, sheep, pigs, chickens, and goats. In a preferred embodiment, the mammal is a human.

Preferably, composition of the present invention reduces the expression or activity of a cone photoreceptor specific transducin, wherein the cone photoreceptor specific transducin comprises Gnatl. Alternatively or in addition, the composition of the present invention reduces the expression or activity of an S-cone-specific opsin, wherein the S-cone specific opsin comprises Opnlsw.

A suitable nucleic acid sequence of human Nrldl is set forth in SEQ ID NO: 5 or a fragment thereof. Alternatively, a suitable nucleic acid sequence encodes human Nrldl comprising the amino acid sequence SEQ ID NO: 6, or a fragment thereof. A suitable nucleic acid sequence of human Nr2e3 is set forth in SEQ ID NO: 7 or a fragment thereof. Alternatively, a suitable nucleic acid sequence encodes human Nr2e3 comprising the amino acid sequence SEQ ID NO: 8, or a fragment thereof. A suitable nucleic acid sequence of human Rora is set forth in SEQ ID NO: 9 or a fragment thereof. Alternatively, a suitable nucleic acid sequence encodes human Rora comprising the amino acid sequence SEQ ID NO: 10, or a fragment thereof. A suitable nucleic acid sequence of human Nuprl is set forth in SEQ ID NO: 28 or a fragment thereof. Alternatively, a suitable nucleic acid sequence encodes human Nuprl comprising the amino acid sequence SEQ ID NO: 29, or a fragment thereof. A suitable nucleic acid sequence of human Nr2cl is set forth in SEQ ID NO: 11 or a fragment thereof. Alternatively, a suitable nucleic acid sequence encodes human Nr2cl comprising the amino acid sequence SEQ ID NO: 12, or a fragment thereof.

Administration is local to the ocular or adnexal tissues. Preferably, the composition is administered intravitreally, sub-retinally, or topically. Topical ophthalmic formulations include eye drops. Preferably, the methods do not include systemic administration. Local ocular administration has several advantages as the eye is an immune-privileged environment and compounds administered to the eye function locally and have little or no systemic dissemination.

The composition is administered at a concentration of 0.001 μg to 100 μg, e.g., 0.01 μg, 0.1 μg, 0.5 μg, 1.0 μg, 1.5 μg, 2.0 μg, 5.0 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, or 100 μg. The composition is administered in a volume of 0.01 μΐ to 10 μΐ, e.g., 0.1 μΐ, 0.25 μΐ, 0.5 μΐ, 1 μΐ, 1.5 μΐ, 2 μΐ, 2.5 μΐ, 3 μΐ, 3.5 μΐ, 4 μΐ, 4.5 μΐ, 5 μΐ, 6 μΐ, 7 μΐ, 8 μΐ, 9 μΐ, or 10 μΐ. The composition is administered once per day, once per week, once per month, every 3 months, every 6 months, or every 12 months. The composition is administered for the duration of 1 day, 1 week, 1 month, 3 months, 6 months, 1 year, 2 years, or 5 years.

The composition comprising a nucleic acid, i.e., a nucleic acid vector, is administered via electroporation. Alternatively, the composition is administered via biodegradable Nile red poly(lactide-co-glycolide) (PLGA) nanoparticle-based gene delivery, small molecule- based gene delivery, naked DNA delivery, viral-based gene delivery, e.g., adeno-associated virus delivery, or genome editing systems, e.g., CRISPR.

Optionally, the method further comprises the administration of a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" is art-recognized and refers to compositions, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" is art-recognized, and refers to, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be

"acceptable" in the sense of being compatible with the other ingredients of the supplement and not injurious to the patient. Optionally, a pharmaceutically acceptable carrier is non- pyrogenic. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyro gen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

The polynucleotides, polypeptides (e.g., large proteins), peptides (e.g., small or medium- sized proteins), antibodies, or other biological agents are purified and/or isolated.

Specifically, as used herein, an "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, or protein, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally- occurring state. A purified or isolated polypeptide is free of the amino acids or sequences that flank it in its naturally-occurring state. Purified also defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents.

Similarly, by "substantially pure" is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated. "Conservatively modified variations" of a particular polynucleotide sequence refers to those polynucleotides that encode identical or essentially identical amino acid sequences, or where the polynucleotide does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the

corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent substitutions" or "silent variations," which are one species of

"conservatively modified variations." Every polynucleotide sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted. Thus, silent substitutions are an implied feature of every nucleic acid sequence which encodes an amino acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques.

Similarly, "conservative amino acid substitutions," in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties are also readily identified as being highly similar to a particular amino acid sequence, or to a particular nucleic acid sequence which encodes an amino acid. Such conservatively substituted variations of any particular sequence are a feature of the present invention.

Individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are "conservatively modified variations" where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. See, e.g., Creighton (1984) Proteins, W.H. Freeman and Company, incorporated herein by reference.

By "isolated nucleic acid" is meant a nucleic acid that is free of the genes which flank it in the naturally- occurring genome of the organism from which the nucleic acid is derived. The term covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule, but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner, such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleic acid molecules according to the present invention further include molecules produced synthetically, as well as any nucleic acids that have been altered chemically and/or that have modified backbones. For example, the isolated nucleic acid is a purified cDNA or RNA polynucleotide.

Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid and the phrase "nucleic acid sequence" refers to the linear list of nucleotides of the nucleic acid molecule, the two phrases can be used interchangeably.

By the terms "effective amount" and "therapeutically effective amount" of a formulation or formulation component is meant a sufficient amount of the formulation or component, alone or in a combination, to provide the desired effect. For example, by "an effective amount" is meant an amount of a compound, alone or in a combination, required to reduce or prevent ocular disease in a mammal. Ultimately, the attending physician or veterinarian decides the appropriate amount and dosage regimen.

The terms "treating" and "treatment" as used herein refer to the administration of an agent or formulation to a clinically symptomatic individual afflicted with an adverse condition, disorder, or disease, e.g., ocular disease, so as to effect a reduction in severity and/or frequency of symptoms, eliminate the symptoms and/or their underlying cause, and/or facilitate improvement or remediation of damage.

The terms "preventing" and "prevention" refer to the administration of an agent or composition to a clinically asymptomatic individual who is susceptible or predisposed to a particular adverse condition, disorder, or disease, and thus relates to the prevention of the occurrence of symptoms and/or their underlying cause.

The transitional term "comprising," which is synonymous with "including,"

"containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase

"consisting of excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a series of photomicrographs and a pie chart showing that rd7 phenotypes are suppressed in N6 B6.Cg-Mor7 ^AKRI3 :Nr2e3 ^rd7/rd7 mice. Figures 1A and IB are photomicrographs showing hematoxylin and eosin staining of retinal sections from affected (a) and suppressed (b) F ₂ B6.Cg-Mor7 ^AKR/3 :Nr2e3 ^rd7/rd7 P30 animals. Retinal dysplasia was absent in the suppressed rd7 homozygote animals. Figures 1C and ID are photomicrographs showing labeling of retinal sections with OPN1SW- specific antibody. The blue cone population is restored to a normal level in (c) affected and (d) suppressed F ₂ B6.Cg- or7 ^AKR/J :Nr2e3 ^rd7/rd? animals. Figure IE is a pie chart showing the distribution of the 95 retinal genes that map to the Mor7 interval. GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer, OS: outer segments, RPE: retinal pigment epithelium.

Figure 2 is a series of chromatograms and sequence alignments showing strain specific expression of Nrldl . Figure 2A shows C57BL/6J and AKR/J chromatograms of polymorphisms identified in the ligand-binding domain of Nrldl, resulting in arginine to glutamine substitution in AKR/J at position 408. Figure 2B is a ClustalW2 sequence alignment of amino acid sequences from C57BL/6J, AKR/J, rat, chimpanzee and human. Stars indicate identity in all sequences, while dots indicate conserved amino acids. Figure 2C are C57BL/6J and AKR/J chromatograms of polymorphisms identified in the Nrldl 5'UTR region. Figure 2D is a ClustalW2 sequence alignment across species that reveals the consensus is in accordance with AKR/J sequence. Stars indicate nucleotide conservation in all species. Figure 2E is a bar chart showing Nrldl relative expression in P30.5 AKR/J and C57BL/6J retinas (n=3, p=0.0024).

Figure 3 is a series of photomicrographs and line graphs showing gene delivery of Nr2e3 or Nrldl suppresses pan-retinal spotting, retinal dysplasia and function in Nr2e3 ^rd7/rd7 mice. Figures 3A-3F show fundus photographs of control and rd7 injected retinas: (a) B6 (uninjected), (b) rd7 (uninjected), (c) GFP injected, (d) GFP.Nr2e3 ^B6 injected, (e)

GFP.Nrldl ^AKR/J injected, (f) GFP.Nrldl ^m injected. Figures 3G-3J show DAPI staining (blue) show rescue of defects in retinal morphology 30 days after electroporation into rd7 neonatal retinas with (g) GFP control, (h) Nr2e3 ^B6 injected, (i) GFP control, (j) Nrldl™ ⁵ injected. L: left, R: right, GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. Scale bar=50 μιη. Figures 3K and 3L show representative (k) scotopic and (1) photopic electroretinograms from animals 4 month after injection with GFP (blue) or GFP.Nrldl ^AKR/J (red).

Figure 4 is a bar chart showing that expression of phototransduction genes Opnlsw and Gnat2 is rescued in rd7 retinas upon Nrldl delivery. Quantitative real time PCR shows that Nrldl delivery results in down-regulation of Opnlsw and Gnat2 in rd7 retinas normalizing gene expression (n=3, p<0.05).

Figure 5 is a series of photomicrographs showing rescue of retinal degeneration 1 (rdl), a mouse model for severe autosomal dominant retinitis pigmentosa. B6 = control/wild type eye. rdl L eye = left eye of rdl mouse, rdl R eye (+Nrldl) = right eye of rdl mouse rescued with Nrldl .

Figure 6 is a series of photomicrographs showing the GFP expression in P30 rd7 retina electroporated with GFP. Nrldl AKR/J . (a) GFP expression in the outer nuclear layer (ONL) and inner nuclear layer (INL) of the retina, (b) GFP expression colocalizes with nuclear marker DAPI. ONL: outer nuclear layer; INL: inner nuclear layer; and RPE: retinal pigment epithelium. Figure 7 is a series of pictomicrographs showing histology (left panels) and fundus photography (right panels) of eyes from rd7 mice. A) rd7 mice exhibit white spots over the entire retina (right panel) and retinal waves (left panel) in comparison to B) control mice.

Figure 8 is two fundus photography images showing the retinas of rd7/rd7 mice injected with either blank vector (right panel) or Nr2e3 vector (left panel).

Figure 9 is two electroretinograms depicting the results of scotopic analysis of rd7 mice comparing mice that received blank vector or Nr2e3 vector at A) 2 months or B) 6 months.

Figure 10 is a schematic showing the domains of nuclear hormone receptors, such as

Nr2e3.

DETAILED DESCRIPTION OF THE INVENTION

The identification of genetic basis for disease has led to a greater understanding of many ocular diseases. Primary mutations in more than 300 genes have been associated with vision loss, and phenotypic variation is often observed for several ocular diseases caused by known primary mutations. Thus, genetic background greatly affects the phenotypic outcome of a disease and polymorphisms in other genes, modifier genes, affect severity, age of onset, and disease progression.

Described in detail below is the identification of genetic modifiers of ocular disease, i.e., genes that rescue disease caused by a mutation in another gene. The nuclear hormone receptor, Nr2e3, is a retinal-specific transcription factor with critical functions in

photoreceptor cell development and differentiation. Mutations in Nr2e3 have been associated with several eye disorders characterized by progressive retinal degeneration, such as enhanced s-cone syndrome (ESCS) and retinitis pigmentosa. Prior to the invention described herein, there were no effective treatment options for this group of diseases.

The retinal degeneration 7 (rd7) mouse model, which lacks Nr2e3 expression, has been a valuable tool for identifying genetic modifiers of Nr2e3-associated hereditary retinal degeneration. As described in detail below, the genetic background of the Nr2e3 mutation highly influences the disease phenotype. For example, B6.Cg-Nr2e3 ^rd7/rd7 mice undergo degeneration, while inbreed mouse strains CAST/EiJ, AKR/J and NOD . NON-H2 ^{nb 1} confer resistance to Nr2e3- associated degeneration (Haider et al., 2008 Mamm Genome, 19: 145- 154). One of the modifier alleles was identified as the nuclear receptor gene Nrldl (also known as Rev-erb alpha). This discovery led to the identification of nucleic acids containing genes that modulate the biological pathways for normal development and normal function of photoreceptor cells in the retina. For example, these nucleic acids contain genes that restore the function of photoreceptor cells and/or prevent the onset or progression of retinal degeneration associated with various ocular diseases.

Nuclear Hormone Receptors

Nuclear receptors represent an evolutionarily conserved group of transcription factors that regulate genes involved in diverse functions such as homeostasis, reproduction, development, metabolism and immune response. Nuclear receptors bind to lipophilic-ligands such as steroid hormones, thyroid hormone, vitamin D and retinoids, which modulate transcriptional activity. Nuclear receptors also function with co-activators or co-repressors to activate or repress the transcription of genes involved in the development and maintenance of specific cell types. In the retina, nuclear hormone receptors regulate the development and patterning of many cell types, such as photoreceptor cells (i.e., rods and cones).

The present invention provides nucleic acids comprising genes or encoding proteins that modulate the biological pathways associated with photoreceptor development and function. Preferably, the nucleic acids comprise nuclear hormone receptors. The nucleic acid and amino acid sequences of exemplary nuclear hormone receptors, such as Nrldl, Nr2e3, Rora, Nuprl, and Nr2cl are listed below.

Nrldl, also known as Rev-erb alpha and nuclear receptor family 1 group D member 1, is a nuclear hormone receptor that has a diverse role in regulating gene networks in several biological processes and in many tissue types. Nrldl is expressed in retinal progenitor cells during development, and in ganglion cells and photoreceptors of adult retinas.

Human Nrldl mRNA (Genbank Accession No. HQ692861.1 (GL325495532), incorporated herein by reference) is provided below (SEQ ID NO: 5):

1 atgacgaccc tggactccaa caacaacaca ggtggcgtca tcacctacat tggctccagt 61 ggctcctccc caagccgcac cagccctgaa tccctctata gtgacaactc caatggcagc 121 ttccagtccc tgacccaagg ctgtcccacc tacttcccac catcccccac tggctccctc 181 acccaagacc cggctcgctc ctttgggagc attccaccca gcctgagtga tgacggctcc 241 ccttcttcct catcttcctc gtcgtcatcc tcctcctcct tctataatgg gagcccccct 301 gggagtctac aagtggccat ggaggacagc agccgagtgt cccccagcaa gagcaccagc 361 aacatcacca agctgaatgg catggtgtta ctgtgtaaag tgtgtgggga cgttgcctcg 421 ggcttccact acggtgtgca cgcctgcgag ggctgcaagg gctttttccg tcggagcatc 481 cagcagaaca tccagtacaa aaggtgtctg aagaatgaga attgctccat cgtccgcatc 541 aatcgcaacc gctgccagca atgtcgcttc aagaagtgtc tctctgtggg catgtctcga 601 gacgctgtgc gttttgggcg catccccaaa cgagagaagc agcggatgct tgctgagatg 661 cagagtgcca tgaacctggc caacaaccag ttgagcagcc agtgcccgct ggagacttca 721 cccacccagc accccacccc aggccccatg ggcccctcgc caccccctgc tccggtcccc 781 tcacccctgg tgggcttctc ccagtttcca caacagctga cgcctcccag atccccaagc 841 cctgagccca cagtggagga tgtgatatcc caggtggccc gggcccatcg agagatcttc 901 acctacgccc atgacaagct gggcagctca cctggcaact tcaatgccaa ccatgcatca 961 ggtagccctc cagccaccac cccacatcgc tgggaaaatc agggctgccc acctgccccc 1021 aatgacaaca acaccttggc tgcccagcgt cataacgagg ccctaaatgg tctgcgccag 1081 gctccctcct cctaccctcc cacctggcct cctggccctg cacaccacag ctgccaccag 1141 tccaacagca acgggcaccg tctatgcccc acccacgtgt atgcagcccc agaaggcaag 1201 gcacctgcca acagtccccg gcagggcaac tcaaagaatg ttctgctggc atgtcctatg 1261 aacatgtacc cgcatggacg cagtgggcga acggtgcagg agatctggga ggatttctcc 1321 atgagcttca cgcccgctgt gcgggaggtg gtagagtttg ccaaacacat cccgggcttc 1381 cgtgaccttt ctcagcatga ccaagtcacc ctgcttaagg ctggcacctt tgaggtgctg 1441 atggtgcgct ttgcttcgtt gttcaacgtg aaggaccaga cagtgatgtt cctaagccgc 1501 accacctaca gcctgcagga gcttggtgcc atgggcatgg gagacctgct cagtgccatg 1561 ttcgacttca gcgagaagct caactccctg gcgcttaccg aggaggagct gggcctcttc 1621 accgcggtgg tgcttgtctc tgcagaccgc tcgggcatgg agaattccgc ttcggtggag 1681 cagctccagg agacgctgct gcgggctctt cgggctctgg tgctgaagaa ccggcccttg 1741 gagacttccc gcttcaccaa gctgctgctc aagctgccgg acctgcggac cctgaacaac 1801 atgcattccg agaagctgct gtccttccgg gtggacgccc agtga

The amino acid sequence for human Nrldl is provided below (SEQ ID NO: 6).

Similar to other nuclear hormone receptors, Nrldl has a DNA -binding domain, a hinge region, and a ligand-binding domain. Specifically, the DNA-binding domain is located at amino acid positions 127- 215; the hinge region is located at amino acid positions 206-284; and the ligand binding domain is located at amino acid positions 285-614, preferably at amino acids 418-611.

1 mttldsnnnt ggvityigss gsspsrtspe slysdnsngs fqsltqgcpt yfppsptgsl 61 tqdparsfgs ippslsddgs psssssssss sssfyngspp gslqvameds srvspsksts 121 nitklngmvl lckvcgdvas gfhygvhace gckgffrrsi qqniqykrcl knencsivri 181 nrnrcqqcrf kkclsvgmsr davrfgripk rekqrmlaem qsamnlannq lssqcplets 241 ptqhptpgpm gpspppapvp splvgfsqfp qqltpprsps peptvedvis qvarahreif 301 tyahdklgss pgnfnanhas gsppattphr wenqgcppap ndnntlaaqr hnealnglrq 361 apssypptwp pgpahhschq snsnghrlcp thvyaapegk apansprqgn sknvllacpm 421 nmyphgrsgr tvqeiwedfs msftpavrev vefakhipgf rdlsqhdqvt llkagtfevl 481 mvrfaslfnv kdqtvmflsr ttyslqelga mgmgdllsam fdfseklnsl alteeelglf 541 tavvlvsadr sgmensasve qlqetllral ralvlknrpl etsrftklll klpdlrtlnn 601 mhsekllsfr vdaq

Nr2e3 is also known as Nuclear Receptor Subfamily 2 Group E Member 3, photocep tor- specific nuclear receptor (PNR); rd7, retina- specific nuclear receptor, and ESPS. Nr2e3 is a nuclear receptor critical for development and maintenance of rod and cone photoreceptor cells. Loss of Nr2e3 causes retinal diseases primarily characterized by photoreceptor degeneration. Mutations of Nr2e3 have been associated with several retinal diseases. Nr2e3 functions as a dual activator and suppressor of gene expression, and together with transcription factors CRX and Nrldl modulates photoreceptor cell fate and differentiation. Nr2e3 is also expressed in mature photoreceptors where it regulates expression of genes essential for proper function, for example, phototransducin genes.

Human Nr2e3 mRNA sequence is provided below (SEQ ID NO: 7):

1 ctcagcagca gagttaagta gtattgcttt aattgcaaca agctgagcta atgtgggaag 61 aatgcagtag agtggcacag aagaatgcta acccgaaact ctaagcctgt tccctggaat 121 cttccatctg gatggaggag agaaagttga cctggagtga ggttcaatgt aaggacaaga 181 tctgcacccg gagaagctct ctctcggaga gcacaggcgg cctgaggagt caaaacaggt 241 ggcctgtgga gtcagcacag gcagcctgga ggaggtgagg aactgaagtt tggacagatt 301 gagtcacttt ctcagggaca catggctggc cggtgatgga gaaggcgtga gcccctcgct 361 ccagtgccgc gtgtgcggag acagcagcag cgggaagcac tatggcatct atgcctgcaa 421 cggctgcagc ggcttcttca agaggagcgt acggcggagg ctcatctaca ggtgccaggt 481 gggggcaggg atgtgccccg tggacaaggc ccaccgcaac cagtgccagg cctgccggct 541 gaagaagtgc ctgcaggcgg ggatgaacca ggacgccgtg cagaacgagc gccagccgcg 601 aagcacagcc caggtccacc tggacagcat ggagtccaac actgagtccc ggccggagtc 661 cctggtggct cccccggccc cggcagggcg cagcccacgg ggccccacac ccatgtctgc 721 agccagagcc ctgggccacc acttcatggc cagccttata acagctgaaa cctgtgctaa 781 gctggagcca gaggatgctg atgagaatat tgatgtcacc agcaatgacc ctgagttccc 841 ctcctctcca tactcctctt cctccccctg cggcctggac agcatccatg agacctcggc 901 tcgcctactc ttcatggccg tcaagtgggc caagaacctg cctgtgttct ccagcctgcc 961 cttccgggat caggtgatcc tgctggaaga ggcgtggagt gaactctttc tcctcggggc 1021 catccagtgg tctctgcctc tggacagctg tcctctgctg gcaccgcccg aggcctctgc 1081 tgccggtggt gcccagggcc ggctcacgct ggccagcatg gagacgcgtg tcctgcagga 1141 aactatctct cggttccggg cattggcggt ggaccccacg gagtttgcct gcatgaaggc 1201 cttggtcctc ttcaagccag agacgcgggg cctgaaggat cctgagcacg tagaggcctt 1261 gcaggaccag tcccaagtga tgctgagcca gcacagcaag gcccaccacc ccagccagcc 1321 cgtgaggttt gggaaattgc tcctgctcct cccgtctttg aggtttatca ctgcggaacg 1381 catcgagctc ctctttttcc gcaagaccat agggaatact ccaatggaga agctcctttg 1441 tgatatgttc aaaaactagt gggggtggag gtgaaatgtt tccaagcact ctggaaaaca 1501 atctactgaa acgaaacatt tgcctactct ttgccccagc aattcctcgt aggtgtgtgt 1561 acccagcaga aatgcccacc gaaagatatt gtaagaatat tcatagcagc tttattcata 1621 atagccccaa actgtatatt gatggtagga tgaattaaca agttgtggta tattcatata 1681 atgaaaaata atttaaaaag aatgaattac ggatacatgt ggcaacacag gtaaacttca 1741 cagacataaa agttgaatga aagaagccag gccgaagttc catttatgca gagttcagga 1801 acaggcaaga ctaattgaca ataatagaag ttggaatagt ggttacttct gggtggtggg 1861 ggattgatac agagggggct catgggagcc ctctggtgta ccagaaatgt tgattttgat 1921 ctgggcagtg gtttcacaaa tgtattcata cgtaataatt cattgagctg tgcactttat 1981 tttgttagac ctcaataaaa aagtaaaaaa aaaaaaaaa

The amino acid sequence for human Nr2e3 is provided below (SEQ ID NO: 8).

With regard to Nr2e3, the DNA-binding domain is located at amino acid positions 1-42; the hinge region is located at, for example, amino acid positions 43-103; and the ligand binding domain is located at amino acid positions 104-309.

1 mcpvdkahrn qcqacrlkkc lqagmnqdav qnerqprsta qvhldsmesn tesrpeslva 61 ppapagrspr gptpmsaara lghhfmasli taetcaklep edadenidvt sndpefpssp 121 ysssspcgld sihetsarll fmavkwaknl pvfsslpfrd qvilleeaws elfllgaiqw 181 slpldscpll appeasaagg aqgrltlasm etrvlqetis rfralavdpt efacmkalvl 241 fkpetrglkd pehvealqdq sqvmlsqhsk ahhpsqpvrf gklllllpsl rfitaeriel 301 lffrktignt pmekllcdmf kn Rora is also known as RAR-related Orphan Receptor A, ROR-alpha, retinoic acid receptor-related orphan receptor alpha, and Nuclear Receptor Subfamily 1 Group F Member 1 (Nrlf 1). Rora is also a member of the NRl subfamily of nuclear hormone receptors. Rora regulates a number of genes involved in lipid metabolism and photoreceptor development, including PCP2, OPN1SW, OPN1SM, and ARR3. There are four transcripts variants encoding different isoforms. The present invention encompasses all four transcript variants.

Human Rora mRNA sequence is provided below (SEQ ID NO: 9):

1 ccatctgtct gatcaccttg gactccatag tacactgggg caaagcacag ccccagtttc 61 tggaggcaga tgggtaacca ggaaaaggca tgaatgaggg ggccccagga gacagtgact 121 tagagactga ggcaagagtg ccgtggtcaa tcatgggtca ttgtcttcga actggacagg 181 ccagaatgtc tgccacaccc acacctgcag gtgaaggagc cagaagggat gaactttttg 241 ggattctcca aatactccat cagtgtatcc tgtcttcagg tgatgctttt gttcttactg 301 gcgtctgttg ttcctggagg cagaatggca agccaccata ttcacaaaag gaagataagg 361 aagtacaaac tggatacatg aatgctcaaa ttgaaattat tccatgcaag atctgtggag 421 acaaatcatc aggaatccat tatggtgtca ttacatgtga aggctgcaag ggctttttca 481 ggagaagtca gcaaagcaat gccacctact cctgtcctcg tcagaagaac tgtttgattg 541 atcgaaccag tagaaaccgc tgccaacact gtcgattaca gaaatgcctt gccgtaggga 601 tgtctcgaga tgctgtaaaa tttggccgaa tgtcaaaaaa gcagagagac agcttgtatg 661 cagaagtaca gaaacaccgg atgcagcagc agcagcgcga ccaccagcag cagcctggag 721 aggctgagcc gctgacgccc acctacaaca tctcggccaa cgggctgacg gaacttcacg 781 acgacctcag taactacatt gacgggcaca cccctgaggg gagtaaggca gactccgccg 841 tcagcagctt ctacctggac atacagcctt ccccagacca gtcaggtctt gatatcaatg 901 gaatcaaacc agaaccaata tgtgactaca caccagcatc aggcttcttt ccctactgtt 961 cgttcaccaa cggcgagact tccccaactg tgtccatggc agaattagaa caccttgcac 1021 agaatatatc taaatcgcat ctggaaacct gccaatactt gagagaagag ctccagcaga 1081 taacgtggca gaccttttta caggaagaaa ttgagaacta tcaaaacaag cagcgggagg 1141 tgatgtggca attgtgtgcc atcaaaatta cagaagctat acagtatgtg gtggagtttg 1201 ccaaacgcat tgatggattt atggaactgt gtcaaaatga tcaaattgtg cttctaaaag 1261 caggttctct agaggtggtg tttatcagaa tgtgccgtgc ctttgactct cagaacaaca 1321 ccgtgtactt tgatgggaag tatgccagcc ccgacgtctt caaatcctta ggttgtgaag 1381 actttattag ctttgtgttt gaatttggaa agagtttatg ttctatgcac ctgactgaag 1441 atgaaattgc attattttct gcatttgtac tgatgtcagc agatcgctca tggctgcaag 1501 aaaaggtaaa aattgaaaaa ctgcaacaga aaattcagct agctcttcaa cacgtcctac 1561 agaagaatca ccgagaagat ggaatactaa caaagttaat atgcaaggtg tctacattaa 1621 gagccttatg tggacgacat acagaaaagc taatggcatt taaagcaata tacccagaca 1681 ttgtgcgact tcattttcct ccattataca aggagttgtt cacttcagaa tttgagccag 1741 caatgcaaat tgatgggtaa atgttatcac ctaagcactt ctagaatgtc tgaagtacaa 1801 acatgaaaaa caaacaaaaa aattaaccga gacactttat atggccctgc acagacctgg 1861 agcgccacac actgcacatc ttttggtgat cggggtcagg caaaggaggg gaaacaatga 1921 aaacaaataa agttgaactt gtttttctca

The amino acid sequence for human Rora is provided below (SEQ ID NO: 10). The DNA binding domain is located at amino acid positions 99-193; the hinge region is located at amino acid positions 172-304; and the ligand-binding domain is located at amino acid positions 305-544.

1 mnegapgdsd letearvpws imghclrtgq armsatptpa gegarrdelf gilqilhqci 61 lssgdafvlt gvccswrqng kppysqkedk evqtgymnaq ieiipckicg dkssgihygv 121 itcegckgff rrsqqsnaty scprqkncli drtsrnrcqh crlqkclavg msrdavkfgr 181 mskkqrdsly aevqkhrmqq qqrdhqqqpg eaepltptyn isangltelh ddlsnyidgh

241 tpegskadsa vssfyldiqp spdqsgldin gikpepicdy tpasgffpyc sftngetspt

301 vsmaelehla qniskshlet cqylreelqq itwqtflqee ienyqnkqre vmwqlcaiki

361 teaiqyvvef akridgfmel cqndqivllk agslevvfir mcrafdsqnn tvyfdgkyas

421 pdvfkslgce dfisfvfefg kslcsmhlte deialfsafv Imsadrswlq ekvkieklqq

481 kiqlalqhvl qknhredgil tklickvstl ralcgrhtek lmafkaiypd ivrlhfpply

541 kelftsefep amqidg

Nr2cl is also known as Nuclear Receptor Subfamily 2 Group C Member 1, TR2, and nuclear receptor TR2.

Human Nr2cl mRNA sequence is provided below (SEQ ID NO: 11):

1 gcttctcccc gttgctaatg cgcaggcgct ggcgggatag cgcgccgccg agccgagaaa 61 gaggtcacga actctgaccc cccagaaata cccaaacaca gaaagctctc tccgccgtga 121 atctcgatcc cacatcccgt cggctttctt caacctctct tcccggagcg ccccccaatc 181 cacgagtggc agccgcggga ctgtcgcgtc ggcgcccgac gccggagtca gcagggcgca 241 aaagcgccgg tagatcatgg caaccataga agaaattgca catcaaatta ttgaacaaca 301 gatgggagag attgttacag agcagcaaac tgggcagaaa atccagattg tgacagcact 361 tgatcataat acccaaggca agcagttcat tctgacaaat cacgacggct ctactccaag 421 caaagtcatt ctggccaggc aagattccac tccgggaaaa gttttcctta caactccaga 481 tgcagcaggt gtcaaccagt tattttttac cactcctgat ctgtctgcac aacacctgca 541 gctcctaaca gataattctc cagaccaagg accaaataag gtttttgatc tttgcgtagt 601 atgtggagac aaagcatcag gacgtcatta tggagcagta acttgtgaag gctgcaaagg 661 attttttaaa agaagcatcc gaaaaaattt agtatattca tgtcgaggat caaaggattg 721 tattattaat aagcaccacc gaaaccgctg tcaatactgc aggttacaga gatgtattgc 781 gtttggaatg aagcaagact ctgtccaatg tgaaagaaaa cccattgaag tatcacgaga 841 aaaatcttcc aactgtgccg cttcaacaga aaaaatctat atccgaaagg accttcgtag 901 cccattaact gcaactccaa cttttgtaac agatagtgaa agtacaaggt caacaggact 961 gttagattca ggaatgttca tgaatattca tccatctgga gtaaaaactg agtcagctgt 1021 gctgatgaca tcagataagg ctgaatcatg tcagggagat ttaagtacat tggccaatgt 1081 ggttacatca ttagcgaatc ttggaaaaac taaagatctt tctcaaaata gtaatgaaat 1141 gtctatgatt gaaagcttaa gcaatgatga tacctctttg tgtgaatttc aagaaatgca 1201 gaccaacggt gatgtttcaa gggcatttga cactcttgca aaagcattga atcctggaga 1261 gagcacagcc tgccagagct cagtagcggg catggaagga agtgtacacc taatcactgg 1321 agattcaagc ataaattaca ccgaaaaaga ggggccactt ctcagcgatt cacatgtagc 1381 tttcaggctc accatgcctt ctcctatgcc tgagtacctg aatgtgcact acattgggga 1441 gtctgcctcc agactgctgt tcttatcaat gcactgggca ctttcgattc cttctttcca 1501 ggctctaggg caagaaaaca gcatatcact ggtgaaagct tactggaatg aactttttac 1561 tcttggtctt gcccagtgct ggcaagtgat gaatgtagca actatattag caacatttgt 1621 caattgtctt cacaatagtc ttcaacaaga tgccaaggta attgcagccc tcattcattt 1681 cacaagacga gcaatcactg atttataaat gcttaactat agaatggctt atgactaccc 1741 aaaacagtgc cccatcaaca aatggggaaa attgcctttt gagctcagga ataatttata 1801 aattggggac taccttttag ttctttagca tattctattt cttattgttt tatataattt 1861 ttaaatcatt tgcttcctcc ttatgtttaa cagcagaggg gtaatcacct taaaatgtca 1921 tcaaaaatag atctactaga aggcagcatc acattcccat cttacttatg gactcctacc 1981 cctggttcat gtcttatatg cctgtaatgg ttataaagcc taccttcagg aaagctatgg 2041 ttgactaatt actaatggat gggttttaaa catgtccctc tacaataaat taaaatcttt 2101 attgtaaaac tttaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa

The amino acid sequence for human Nr2cl is provided below (SEQ ID NO: 12). T he DNA binding domain is located at amino acid positions 108-194; the hinge region is located at amino acid positions, for example, 195-367; and the ligand-binding domain is located at amino acid positions 368-589. 1 MATIEEIAHQ I IEQQMGEIV TEQQTGQKIQ IVTALDHNTQ GKQFILTNHD GSTPSKVILA

61 RQDSTPGKVF LTTPDAAGVN QLFFTTPDLS AQHLQLLTDN SPDQGPNKVF DLCVVCGDKA

121 SGRHYGAVTC EGCKGFFKRS IRKNLVYSCR GSKDCI INKH HRNRCQYCRL QRCIAFGMKQ

181 DSVQCERKPI EVSREKSSNC AASTEKIYIR KDLRSPLTAT PTFVTDSEST RSTGLLDSGM

241 FMNIHPSGVK TESAVLMTSD KAESCQGDLS TLANVVTSLA NLGKTKDLSQ NSNEMSMIES

301 LSNDDTSLCE FQEMQTNGDV SRAFDTLAKA LNPGESTACQ SSVAGMEGSV HLITGDSSIN

361 YTEKEGPLLS DSHVAFRLTM PSPMPEYLNV HYIGESASRL LFLSMHWALS IPSFQALGQE

421 NSISLVKAYW NELFTLGLAQ CWQVMNVATI LATFVNCLHN SLQQDKMSTE RRKLLMEHIF

481 KLQEFCNSMV KLCIDGYEYA YLKAIVLFSP DHPSLENMEL IEKFQEKAYV EFQDYITKTY

541 PDDTYRLSRL LLRLPALRLM NATITEELFF KGLIGNIRID SVIPHILKME PADYNSQIIG

601 HSI

Nuprl is also known as nuclear protein 1. Human Nuprl mRNA sequence is provided below (SEQ ID NO: 28):

1 caaagcgtta ggagaagaag agaggcaggg aagacaagcc aggcacgatg gccaccttcc

61 caccagcaac cagcgccccc cagcagcccc caggcccgga ggacgaggac tccagcctgg

121 atgaatctga cctctatagc ctggcccatt cctacctcgg aggtggaggc cggaaaggtc

181 gcaccaagag agaagctgct gccaacacca accgccccag ccctggcggg cacgagagga

241 aactggtgac caagctgcag aattcagaga ggaagaagcg aggggcacgg cgctgagaca

301 gagctggaga tgaggccaga ccatggacac tacacccagc aatagagacg ggactgcgga

361 ggaaggagga cccaggacag gatccaggcc ggcttgccac accccccacc cctaggactt

421 attcccgctg actgagtctc tgaggggcta ccaggaaagc gcctccaacc ctagcaaaag

481 tgcaagatgg ggagtgagag gctgggaatg gaggggcaga gccaggaaga tcccccagaa

541 aagaaagcta cagaagaaac tggggctcct ccagggtggc agcaacaata aatagacacg

601 cacggcagcc acaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa

The amino acid sequence for human Nuprl is provided below (SEQ ID NO: 29):

1 matfppatsa pqqppgpede dssldesdly slahsylggg grkgrtkrea aantnrpspg 61 gherklvtkl qnserkkrga rr

The present invention is also directed to nucleic acids that encode a biologically active fragment or a variant of Nrldl, Nr2e3, Rora, Nuprl, or Nr2cl. A biologically active fragment or variant is a "functional equivalent" - a term that is well understood in the art and is further defined in detail herein. The requisite biological activity of the fragment or variant, using any method disclosed herein or known in the art to establish activity of a nuclear hormone receptor, has the following activity relative to the wild-type native polypeptide: about 50%, about 55%, about 60 %, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, and any range derivable therein, such as, for example, from about 70% to about 80%, and more preferably from about 81% to about 90%; or even more preferably, from about 91% to about 99%.

A fragment, in the case of these sequences and all others provided herein, is defined as a part of the whole that is less than the whole. Moreover, a fragment ranges in size from a single nucleotide or amino acid within a polynucleotide or polypeptide sequence to one fewer nucleotide or amino acid than the entire polynucleotide or polypeptide sequence. Finally, a fragment is defined as any portion of a complete polynucleotide or polypeptide sequence that is intermediate between the extremes defined above.

For example, fragments of any of the nuclear hormone receptors disclosed herein is about 10 amino acids, about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 350 amino acids, about 400 amino acids, about 450 amino acids, about 500 amino acids, about 550 amino acids, about 600 amino acids, about 650 amino acids, or about 700 amino acids long. For example, fragments of any of the nuclear hormone receptors disclosed herein is about 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, 450 nucleotides, 500 nucleotides, 550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides, 1100 nucleotides, 1200 nucleotides, 1300 nucleotides, 1400 nucleotides or 1500 nucleotides long.

In some preferred embodiments, the fragments of the present invention comprise or consist primarily of the specific domains that are required for or contribute to functional activity of Nrldl, Nr2e3, Rora, Nuprl, or Nr2cl. For example, nuclear hormone receptors comprise evolutionary conserved domains shared with all members of the family, including: the highly variable A/B domain, N terminal DNA binding domain, a flexible hinge region and the ligand-binding and dimerization domain in the C terminus.

Variants encompassed by the present invention include nucleic acid or amino acid sequences comprising the following degrees of sequence identity to Nrldl, Nr2e3, Rora, Nuprl, or Nr2cl: about 50%, about 55%, about 60 %, about 65%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and any range derivable therein, such as, for example, from about 70% to about 80%, and more preferably from about 81% to about 90%; or even more preferably, from about 91% to about 99% identity.

It should be appreciated that any variations in the coding sequences of the present nucleic acids that, as a result of the degeneracy of the genetic code, express a polypeptide of the same sequence, are included within the scope of this invention.

The term "% identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example, % identity is relative to the entire length of the coding regions of the sequences being compared.

Nucleic acid and amino acid sequence identity can be determined using standard methods, typically based on certain mathematical algorithms. In a preferred embodiment, the percent identity between two nucleic acid or amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at

http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of Meyers and Miller

(CABIOS, 4: 11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The nucleotide and amino acid sequences of the present invention can further be used as a "query sequence" to perform a search against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (Altschul et al. (1990) J. Mol. Biol. 275:403-10). BLAST nucleotide searches can be performed with the NBLAST program to obtain nucleotide sequences homologous to nucleic acids of the present invention, e.g., Nrldl, Nr2e3, Rora, Nuprl, or Nr2cl. BLAST protein searches can be performed with the XBLAST program to obtain amino acid sequences homologous to the appropriate reference protein, e.g. , Nrldl, Nr2e3, Rora, Nuprl, or Nr2cl. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs {e.g., XBLAST and NBLAST) can be used.

Any of a number of known recombinant methods are used to produce a DNA molecule encoding the fragment or variant. For production of a variant, it is routine to introduce mutations into the coding sequence to generate desired amino acid sequence variants of the invention. Site-directed mutagenesis is a well-known technique for which protocols and reagents are commercially available {e.g., Zoller, MJ et al., 1982, Nucl Acids Res 70:6487-6500; Adelman, JP et al., 1983, DNA 2:183-93). These mutations include simple deletions or insertions, systematic deletions, insertions or substitutions of clusters of bases or substitutions of single bases.

Methods of Treatment

The present invention is based upon the discovery of a gene therapy approach in which a modifier gene is administered to the ocular or adnexal tissue for the treatment or prevention of disease. By "modifier gene" is meant a gene chat affects the phenotypic expression of another gene(s). For example, the modifier genes are administered via electroporation. Alternatively, the modifier gene is administered via biodegradable Nile red poly(lactide-co-glycolide) (PLGA) nanoparticle -based gene delivery, naked DNA delivery, small molecule-based gene delivery, or viral-based gene delivery, e.g., adeno-associated virus delivery. These techniques are utilized alone or in combination to ameliorate or reduce the severity of ocular disease or disorder.

In one embodiment, the invention also features a vector or a nucleic acid construct, e.g., a vector containing the nucleic acids described herein. The vector can further include one or more regulatory elements, e.g., a heterologous promoter.

A variety of known nucleic acid vectors may be used in these methods, e.g., recombinant viruses, such as recombinant adeno-associated virus (rAAV), recombinant adenoviruses, recombinant retroviruses, recombinant poxviruses, and other known viruses in the art, as well as plasmids, cosmids and phages, etc. Many publications well-known in the art discuss the use of a variety of such vectors for delivery of genes. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, latest edition; Kay, MA. et al, 2001, Nat. Med., 7:33-40; and Walther W et al, 2000, Drugs 60:249-71). The regulatory elements may be endogenously found upstream or downstream of the genes, or they may be exogenous regulatory elements that are not found to regulate the genes in nature, and introduced by recombinant DNA techniques known in the art. The regulatory elements can be operably linked to a gene or fragment thereof of the present invention, or a gene encoding a protein or fragment thereof of the present invention. Methods for assembly of the recombinant vectors are well-known. See, for example, WO 00/15822 and other references cited therein, all of which are incorporated by reference. Upon delivery of the vector to the subject, i.e., to the eye of the subject, the nucleic acid is optionally integrated into the genome of the cells of the eye, i.e., photoreceptor cells, cells of the outer nuclear layer, cells of the inner nuclear layer, or retinal pigment epithelium.

The vectors of present invention includs appropriate sequences operably linked to the coding sequence or ORF to promote expression of the nuclear hormone receptors of the present invention in a targeted host cell. "Operably linked" sequences include both expression control sequences such as promoters that are contiguous with the coding sequences and expression control sequences that act in trans or distally to control the expression of the polypeptide product.

Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency {e.g., Kozak consensus sequence); sequences that enhance nucleic acid or protein stability; and when desired, sequences that enhance protein processing and/or secretion. Many varied expression control sequences, including native and non-native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized herein, depending upon the type of expression desired.

Expression control sequences for eukaryotic cells typically include a promoter, an enhancer, such as one derived from an immunoglobulin gene, SV40, CMV, etc., and a polyadenylation sequence which may include splice donor and acceptor sites. The polyadenylation sequence generally is inserted 3' to the coding sequence and 5' to the 3' ITR sequence. PolyA from bovine growth hormone is a suitable sequence.

The promoter may be selected from a number of constitutive or inducible promoters that can drive expression of the selected transgene in an ocular setting, preferably in retinal neurons. A preferred promoter is "cell- specific", meaning that it is selected to direct expression of the selected transgene in a particular ocular cell type, such as photoreceptor cells. In some embodiments, an inducible promoter may be preferred.

The rAAV used in the present invention may be constructed and produced using the materials and methods described herein and those well-known in the art. The methods that are preferred for producing any construct of this invention are conventional and include genetic engineering, recombinant engineering, and synthetic techniques readily understood by the ordinarily skilled artisan.

Briefly, to package an rAAV construct into an rAAV virion, a sequences necessary to express AAV rep and AAV cap or functional fragments thereof as well as helper genes essential for AAV production must be present in the host cells. See, for example U.S. Patent Pub. 2007/0015238, which describes production of pseudotyped rAAV virion vectors encoding AAV Rep and Cap proteins of different serotypes and AdV transcription products that provide helper functions. For example, AAV rep and cap sequences may be introduced into the host cell in any known manner including, without limitation, transfection, electroporation, liposome delivery, membrane fusion, biolistic deliver of DNA-coated pellets, viral infection and protoplast fusion. Devices specifically adapted for delivering DNA to specific regions within and around the eye for the purpose of gene therapy have been described recently (for example, U.S. Patent Pub. 2005/0277868, incorporated by reference) are used within the scope of this invention. Such devices utilize electroporation and electromigration, providing, e.g., two electrodes on a flexible support that can be placed behind the retina. A third electrode is part of a hollow support, which can also be used to inject the molecule to the desired area. The electrodes can be positioned around the eye, including behind the retina or within the vitreous.

These sequences may exist stably in the cell as an episome or be stably integrated into the cell's genome. They may also be expressed more transiently in the host cell. The level of RNA expression may be monitored by Northern blots quantitative RT-PCR. The level of protein expression may be monitored by Western blot, immunohistochemistry, immunoassay including enzyme immunoassay (EIA) such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA) or by other methods.

In another preferred embodiment, the nucleic acids of the present invention can be delivered via nanoparticles. The nanoparticles are, for example, lipid-based colloidal particles with a diameter of less than 100 nm. Nanoparticles intended for drug and gene delivery can be characterized for various parameters including particle size, size distribution, morphology, zeta potential, drug loading, syringeability and injectability, in vitro drug release, and stability. The formulation of the nanoparticles varies, with respect to lipid composition, nucleic acid to lipid ratio, and formulation method, depending on the intended use. Nanoparticle assembly methods are known in the art, and as described in Kompella et al., "Nanoparticles for Drug and Gene Delivery in Treating Diseases of the Eye"; Methods in Pharmacology and Toxicology, 2014, pages 291-316. In a preferred embodiment, the nanoparticle is a hybrid polyamidoamine (PAMAM) dendrimer hydrogel/poly(lactic-co- glycolic acid) (PLGA) suitable for topical administration.

Genome editing systems can also be used to deliver nucleic acids of the present invention to the eye. Examples of such genome editing systems include, but are not limited to: CRISPR/Cas sytems, zinc finger nucleases (ZFNS), and transcription activator-like effector nucleases (TALENS). In such systems, the nucleic acids of the present invention can be readily incorporated into the host cell genome and expressed. In some embodiments, mutated forms of disease-causing genes (i.e., Nr2e3) can be "edited", or selectively excised, and replaced with any of the nucleic acids described herein. Expression is modulated by endogenous or exogenous regulatory elements, and expressed of these nucleic acids improves or ameliorates the symptoms of the disease.

The methods and compositions described herein refer to the restoration or

normalization of visual responses. As used herein, the "restoration" or "normalization" refers to increasing or decreasing the expression level or activity of photoreceptors to that of a subject that does not suffer from an ocular disease or disorder, i.e., a subject that does not display photoreceptor degeneration. The restoration or normalization of photoreceptor activity or visual response can be measured or determined by electroretinography. Standards for normal ranges of photoreceptor or visual response as measured by electroretinography have been extensively studied and are established in the art (Perlman, Ido, Chapter XI: The Electroretinogram: ERG by Ido Perlman; Webvision: The Organization of the Retina and Visual System; and Kremers J et al., Prog Retin eye Res. 2003, 22(5):579-605; both references are incorporated herein by reference in their entireties).

As described in detail below, gene delivery of Nrldl into the eyes of rd7 mice efficiently ameliorated clinical, morphological, and functional defects associated with rd7 retinal degeneration. Following the administration of the cDNA or nanoparticles, a partial rescue of the rd7 phenotypes was observed both by fundus examinations as well as electroretinogram (ERG) analysis of photoreceptor function. As described herein, in vivo electroporation was utilized to deliver cDNA of modifier genes (such as Nrldl) into the eyes of rd7 mice. Additionally, biodegradable Nile red poly(lactide-co-glycolide) (PLGA) nanoparticles was utilized as a non- viral alternative delivery method.

Genetic heterogeneity is observed for many Mendelian, single gene disorders (Wolf, U., 1997 Hum Genet, 100: 305-321). While environmental influences may provide minor contributions, variations in phenotypic outcome are generally attributable to allelic heterogeneity or genetic modifier genes. Genetic modifiers are allelic variants, distinct from the mutant gene that can alter disease outcome by either increasing or reducing disease severity, and affect disease onset and progression (Houlston, R.S. & Tomlinson, LP., 1998 Eur J Hum Genet, 6: 80-88). Identification of genetic modifiers has a significant impact in prediction of disease progression and development of new therapeutic strategies. Mouse models provide a unique platform to uncover genetic modifiers that influence phenotypic variation in human disease (Hamilton, B.A. & Yu, B.D., PLoS Genet, 8: el002644). For example, mouse models of cystic fibrosis in combination with genome-wide studies in patients have uncovered several pathways that can modify the disease, leading to a better understanding of cystic fibrosis etiology and novel strategies for therapeutic development (Cutting, G.R., 2005 Annu Rev Genomics Hum Genet, 6: 237-260; Cutting, G.R. Ann N Y Acad Sci, 1214: 57-69; Collaco, J.M. & Cutting, G.R., 2008 Curr Opin Pulm Med, 14: 559- 566). Significant disease variability is also prevalent for many inherited retinal degenerative diseases; however, the underlying causes for such variations have not been defined for most cases (Zernant et al., 2005 Invest Ophthalmol Vis Sci, 46: 3052-3059; Passerini et al., 2007 Eur J Ophthalmol, 17: 1000-1003; Walia et al., 2008 Arch Ophthalmol, 126: 379-384; Fahim et al., 2011 PLoS One, 6: e23021; Haideret al., 2002 Hum Mol Genet, 11: 1195-1206).

Described herein is the identification of a modifier gene of retinal degeneration.

Retinal degenerative diseases leading to progressive and significant vision loss are the primary cause of blindness throughout the world. Advances in whole genome sequencing technology have led to the identification of mutations responsible for syndromic and nonsyndromic retinal diseases in at least 191 distinct genes. The nuclear hormone receptor NR2E3, also known as photoreceptor- specific nuclear receptor (PNR), has emerged as a significant regulator of photoreceptor cell development and function and is associated with numerous retinal degenerations. Specifically, NR2E3 functions in photoreceptor

differentiation by suppressing expression of cone-specific genes and activating rod-specific genes, thereby committing precursors to a rod cell fate (Cheng et al., 2004 Hum Mol Genet, 13: 1563-1575; Haider et al., 2006 Vis Neurosci, 23: 917-929; Peng et al., 2005 Hum Mol Genet, 14: 747-764; Corbo, J.C. & Cepko, C.L., 2005 PLoS Genet, 1: el l; Cheng et al., 2006 Hum Mol Genet, 15: 2588-2602; Webber et al., 2008 Exp Eye Res, 87: 35-48). NR2E3 is also expressed in mature photoreceptors where it regulates expression of genes essential for proper function, such as phototransduction genes (Webber et al., 2008 Exp Eye Res, 87: 35- 48; Haider et al., 2009 Exp Eye Res, 89: 365-372). Mutations in human NR2E3 have been associated with several diseases characterized by progressive retinal degeneration, such as enhanced S-cone syndrome (ESCS), Goldmann-Favre syndrome and retinitis pigmentosa (Haider et al., 2000 Nat Genet, 24: 127-131; Schorderet, D.F. & Escher, P., 2009 Hum Mutat, 30: 1475-1485; Gerber et al., 2000 Hum Genet, 107: 276-284; Sharon et al., 2003 Arch Ophthalmol, 121: 1316-1323; Coppieters et al., 2007 Am J Hum Genet, 81: 147-157; Gire et al., 2007 Mol Vis, 13: 1970-1975). Significant phenotypic variation has been observed in patients with NR2EJ-associated retinal degeneration, with variable expressivity in patients harboring the same mutation and within the same pedigree, suggesting that genetic modifiers modulate disease outcome in these patients (Schorderet, D.F. & Escher, P., 2009 Hum Mutat, 30: 1475-1485; Escher et al., 2009 Hum Mutat, 30: 342-351; Bandah et al., 2009 Arch Ophthalmol, 127: 297-302).

The retinal degeneration 7 (rd7) mouse is a model for Nr2e3 associated retinal disease (Akhmedov et al., 2000 Proc Natl Acad Sci U S A, 97: 5551-5556; Haider et al., 2001 Hum Mol Genet, 10: 1619-1626). The Nr2e3 ^rd7/rd? mouse model was utilized to study the genetic heterogeneity observed in Nr2e3 associated retinal degeneration and to identify genetic modifiers that contribute to such variation. Homozygous rd7 mice develop retinal dysplasia, with whorls and rosettes apparent at postnatal day 10 (P10) and retinal spots detectable by fundus examination at eye opening (P14) (Akhmedov et al., 2000 Proc Natl Acad Sci U S A, 97: 5551-5556; Haider et al., 2001 Hum Mol Genet, 10: 1619-1626; Yanagi et al., 2002 Invest Ophthalmol Vis Sci, 43: 3489-3494). Similar to patients with Nr2e3 mutations, rd7 mice exhibit significant increase of S-cones and progressive degeneration of rod and cone photoreceptor cells (Haider et al., 2001 Hum Mol Genet, 10: 1619-1626). The Nr2e3 ^rd7/rd7 phenotype is highly variable depending on genetic background (Haider et al., 2008 Mamm Genome, 19: 145-154). Complete penetrance was observed in the B6.Cg-Nr2e3 ^rd7/rd? strain, while suppression occurs in crosses with the genetically divergent and inbred strains AKR/J, CAST/EiJ and NOD.NOH-H2 ^nbl ; revealing that modifier genes are conferring resistance or s suusscceeptibility to the Nr2e3 phenotypes (Haider et al., 2008 Mamm Genome, 19: 145 154).

As described herein, the nuclear hormone receptor Rev-erb alpha, hereafter referred to as Nrldl, was identified as a genetic modifier of Nr2e3 ^rd7/rd? . A locus on chromosome 11 linked to Nr2e3 ^rd7/rd? suppression was genetically fine mapped in the AKR/J background. Through sequence analysis, two strain specific variations in the Nrldl gene within this locus were identified. Furthermore, mRNA expression of Nrldl is increased in AKR/J retinas compared to C57BL/6J (B6), suggesting that differential levels of NR1D1 modulate rd7 penetrance. As described below, delivery of the Nrldl gene to the retinas of B6.Cg- ^~ Nr2e3 ^rd7/rdy mice rescues clinical spotting, retinal dysplasia, and molecular changes associated with Nr2e3 loss, confirming that increased Nrldl expression is sufficient for suppressing rd7. Most importantly, it was determined that by increasing Nrldl expression through gene delivery, key genes within the Nr2e3-directed network are now re-regulated, thereby restoring sufficient normalization of the network to ameliorate disease. Thus, NR1D1 is a potent therapeutic target for /W2e3-associated retinal degeneration that can compensate for Nr2e3 loss by regulating key molecular pathways associated with disease.

It was determined that Nrldl is a genetic modifier gene able to ameliorate Nr2e3 associated retinal degeneration. The Nr2e3 ^rd7/rd? mouse model is completely penetrant in the C57BL/6J genetic background, while suppression of retinal degeneration is observed in the AKR/J background (Akhmedov et al., 2000 Proc Natl Acad Sci U S A, 97: 5551-5556;

Haider et al., 2008 Mamm Genome, 19: 145-154). The genetic modifier locus linked with rd7 suppression was fine-mapped to a 3.3cM region containing Nrldl and subsequently identified two Nrldl sequence variations between C57BL/6J and AKR/J: one in the promoter region and one in the ligand-binding domain of Nrldl. The present study demonstrates that a single delivery of the Nrldl gene to the retina of B6.Cg-Nr2e3 ^rd/7rdy animals is sufficient to suppress /W2e3-associated retinal degeneration. Rescue of the rd7 phenotype was observed by fundus and histological examinations of eyes electroporated with Nrldl expression vector. As described below, gene delivery experiments with Nrldl alleles from both AKR/J and C57BL/6J were able to rescue rd7, suggesting that increased Nrldl levels, rather than the nature of the allele, are responsible for the observed suppression.

NR1D1 has been described as a co-factor of NR2E3 and functions in the same transcriptional network as NR2E3 (Cheng et al., 2004 Hum Mol Genet, 13: 1563-1575; Haider et al, 2009 Exp Eye Res, 89: 365-372; Mollema et al, 2011 PLoS One, 6: el7494). A group of genes that are co-targeted by both NR2E3 and NR1D1 was previously identified. Further, previous results show that acute knockdown of NR1D1 by shRNA targeting in the mouse retina results in phenotypes similar to rd7, such as pan-retinal spotting and loss of photoreceptor function (Mollema et al., 2011 PLoS One, 6: el7494). The present study confirms that NR1D1 and NR2E3 act synergistically to regulate genes involved in retinal development and function. These processes are strictly and temporally regulated by key transcription factors directing expression of gene networks both during development and in the mature retina. Misregulation or mutations in genes involved in these processes, such as Nr2e3, disrupt this balance and result in retinal defects. Importantly, delivery of Nrldl resulted in molecular changes that restore a balance in the transcriptional networks by normalizing gene expression, thus leading to rescue of retinal integrity and function in rd7 animals. Specifically, it was observed that Nrldl delivery results in rescue of expression of the cone photo transduction genes Opnlsw and Gnat2, which are misregulated in rd7. This data indicates that NR1D1 suppresses defects associated with NR2E3 loss through a compensatory mechanism.

The data presented herein illustrate the powerful potential of modifier genes for treatments for inherited retinal disease. As described herein, rescue of retinal integrity and function was achieved through a gene therapy approach by delivering a modifier gene rather than replacing the disease-causing gene. Gene therapy clinical trials have resulted in tremendous success for treating patients with Leber's congenital amaurosis (LCA), an inherited retinal degeneration disease (Maguire et al., 2008 N Engl J Med, 358: 2240-2248; Cideciyan et al., 2009 Hum Gene Ther, 20: 999-1004; Cideciyan et al., 2009 N Engl J Med, 361: 725-727; Simonelli et al., 2010 Mol Ther, 18: 643-650; Jacobson et al., Arch

Ophthalmol, 130: 9-24). These studies have led to great advancements towards the use of gene therapy in the clinic; however, prior to the invention described herein, gene replacement studies have not yielded a plethora of therapeutics. The alternate approach described herein identifies genetic modifiers that suppress disease caused by a number of different genes that converge on specific nodes or pathways within a signaling network. As genes function in networks and not singularly, the impact of any gene delivery is on the network as a whole rather than just a single gene. These studies illustrate that viable therapeutic options which have broad impact emanate from genetic modifier genes that are capable of modulating a disease state by impacting entire gene networks that regulate specific biological processes rather than a single gene.

Example 1: Identification of Genetic Modifiers as Therapeutic Targets for Retinal Disease: Rev-erb alpha Ameliorates Nr2e3-Associated Retinal Degeneration

As described in detail in the Examples below, retinal degenerative diseases are the leading cause of blindness throughout the world. Described herein is the identification of genetic modifiers of retinal degeneration in the rd7 mouse model, which lacks a functional Nr2e3 gene, and the investigation of their potential as therapeutic targets. Described in detail below is the identification of Nrldl (also known as Rev-erb alpha) as a genetic modifier of Nr2e3 associated retinal degeneration. The rd7 modifier locus was mapped to a 3.3 cM region in chromosome 11 and it was determined that Nrldl was one of three nuclear receptor genes within the modifier locus. Sequence variants between affected and suppressed animals were identified only in Nrldl . In vivo delivery of Nrldl to rd7 retinas rescued retinal degeneration associated with Nr2e3 loss. The rescue was observed not only at the clinical and histological level, but importantly at the functional and molecular level as well such that Nrldl was able to restore normal vision to rd7 animals by normalizing key genes within the Nr2eJ-directed transcriptional network. Together, these findings uncover Nrldl as a therapeutic target for the treatment of retinal degenerative diseases caused by loss of function of Nr2e3. The materials and methods utilized in this example are described in detail below. Animal maintenance

Animals were housed in vivariums at the Schepeiis Eye Research Institute and the Nebraska Medical Center, use and procedures were approved by the Animal Care and Use Committee and conducted in compliance with the Animal Welfare Act Regulations.

C57BL/6J and AKR/J mice were obtained from Jackson Laboratories, Bar Harbor, ME. B6.Cg-Nr2e3 ^rd7/rd7 has been previously described (Haider et al., 2008 Mamm Genome, 19: 145-154). B6.Cg-Mor7 ^AKR :Nr2e3 ^rd7/rd7 mice were generated by outcrossing B6.Cg- Nr2e3 ^rd7/rd? x AKR/J F ₂ mice to C57BL/6J, followed by backcrossing of the F ₂ progeny to C57BL/6J for six consecutive generations. Genotyping for the Nr2e3 ^{r r} mutation was performed as previously described (Haider et al., 2001 Hum Mol Genet, 10: 1619-1626). Construction of expression vectors

cDNA from C57BL/6J or AKR/J mice was used to amplify the Nrldl ^B6 and Nrldl ^AKR/J alleles, as well as the Nr2e3 ^B6 allele with the following primers: Nrldl (F:

TTTTTAAGCTTCATCACAACCTCCAGTTTGTGTC (SEQ ID NO: 1);

R: TTTTTAAGCTTGGCGTCCACCCGGAAGGACAGCA (SEQ ID NO: 2) ) and Nr2e3 (F: TTTTTAAGCTTGCAAGCAGGCTACCC TTAGGACC (SEQ ID NO: 3); R:

TTTTTAAGCTTGAACATGTCACACAGGAGCTTCT (SEQ ID NO: 4)). Amplified sequences were cloned into the pAcGFPl-Nl plasmids.

In vivo electroporation

Nrldl allele specific constructs (designated as GFP.Nrldl ^B6 and GFP.Nrldl ^AKK ' ³ ) were delivered subretinally into the right eye of P0.5 Nr2e3 ^rd7/rd7 mice using the

electroporation method developed by Matsuda (Matsuda, T. & Cepko, C.L. 2007 Proc Natl Acad Sci U S A, 104: 1027-1032). The Nr2e3 allele from C57B6L/J (GFP.Nr2e3 ^B6 ) was electroporated into rd7 animals as a positive control, while electroporation of empty GFP expression vector or no injection to the left eye served as a negative control, ^g of naked cDNA was injected subretinally, followed by in vivo electroporation immediately after the injection. Tweezer electrodes were used to hold the head of the pup and five square 80V pulses of 50ms duration, with 950 ms intervals, were applied using a square wave electroporator. Mice were aged to P30.5 and phenotyped by indirect ophthalmoscopy, electroretinogram (ERG), and immunohistochemistry (Haider et al., 2006 Vis Neurosci, 23: 917-929).

Clinical examination

Animals were examined by indirect ophthalmology at P30 as previously described (Haider et al., 2008 Mamm Genome, 19: 145-154). Pupils of animals were dilated with 1% Atropine and a Keeler Vantage indirect ophthalmoscope with a 60-diopter lens was used for fundus examinations.

Quantitative real time PCR

Gene expression analysis was performed using quantitative RT-PCR as previously described (Haider et al., 2009 Exp Eye Res, 89: 365-372). In brief, retinas were dissected rapidly after eye enucleation and placed in Trizol (Invitrogen) for RNA extraction. Two micrograms of total RNA was reverse transcribed using Retroscript (Ambion). Real-time PCR was performed in technical triplicates with a minimum of three biological replicates using SYBR Green PCR master mix (Applied Biosystems). Reactions were quantified using a Roche 480 LightCycler real time PCR instrument. Relative expression levels were normalized to the amount of β-actin expressed and fold change relative to wild-type C57BL/6J control was calculated using the delta Ct method.

Electroretino graph v

Electroretinogram analysis was performed on 7 mice of each strain (4 month-old), as described previously (Haider et al., 2008 Mamm Genome, 19: 145-154). Mice were anesthetized with an intraperitoneal injection of a saline carrier (10 mg/g body weight) containing ketamine (1 mg/mL) and xylazine (0.4 mg/mL). Mice were dark adapted for at least six hours and then anesthetized prior to recording. Dark-adapted responses were recorded to short wavelength ( max = 470 nm; Wratten 47A filter) flashes of light over a 4.0 log unit range of intensities (0.3 log unit steps) up to the maximum allowable by the photic stimulator. Light-adapted responses were obtained with white flashes (0.3 step) on the rod- saturating background after 10 min of exposure to the background light to allow complete light adaptation. Signal processing was performed using EM for Windows v7.1.2. Signals were sampled every 0.8 ms over a response window of 200 ms. Responses were computer averaged for each stimulus condition with up to 50 records for the weakest signals.

Preparation of cDNA loaded nanoparticles

The nanoparticles were formulated using the previously described double emulsion solvent evaporation method (Aukunuru et al., 2003 J Pharm Pharmacol, 55(9): 1199-206; Singh et al., 2009 Gene Ther, 16(5):645-59). The biodegradable polymer used was Poly(L- lactide-co-glycolide)(PLGA) Resomer 503H (50:50; i.v. 0.44 dl/g; Boehringer Ingelheim, Petersburg, VA). After preparing nanoparticles, drug loading, encapsulation efficiency and particle size were determined.

Administration of cDNA loaded nanoparticles

cDNA loaded Nile red PLGA nanoparticles are introduced into postnatal day 0 (P0) eyes by intravitreal or subretinal injection, and in some instances the injection was followed by electroporation. Particles were reconstituted in IX PBS and animals received 1.5 μg of modifier gene cDNA into the right eyes and the same dose of control (empty) nanoparticles into the left eyes at PO. The total volume injected was 0.5 μΐ. The efficacy of delivery was assessed using Nile red as a nanoparticle tracking dye, and green fluorescent protein expression as a marker of transfected cells. Mice were aged to P30.5 and phenotyped by indirect ophthalmoscopy, electroretinogram (ERG), and immunohistochemistry (Haider et al., 2006 Vis Neurosci, 23: 917-929).

Statistical analysis

Statistical analysis for Figures 2 and 4 was performed using the two-tailed Student' s t test, with significance defined as P <0.05. At minimum, 3 biological replicates were included in the each study.

A detailed description of the results of this example is provided below.

Genetic fine mapping of rd7 modifier locus on AKR/J chromosome 11

Previous results revealed that genetic background strongly influences penetrance of Nr2e3 ^rd7/rd7 phenotypes (Akhmedov et al., 2000 Proc Natl Acad Sci U S A, 97: 5551-5556; Haider et al., 2008 Mamm Genome, 19: 145-154). Specifically, complete suppression of rd7 retinal degeneration was observed in outcrosses of B6.Cg-Nr2e3 ^rd7/rdy mice to AKR/J, CAST/EiJ or NOD.NOH-H2 ^nbl mice ((Haider et al., 2008 Mamm Genome, 19: 145-154). A genome wide scan identified several modifier loci that were unique for each strain (Haider et al., 2008 Mamm Genome, 19: 145-154). To determine if a single modifier gene is able to ameliorate rd7 associated retinal degeneration, an incipient congenic strain that harbors the AKR/J modifier locus on chromosome 11, named Mor7 for modifier of rd7, was generated by backcrossing F ₂ progeny from the B6.Cg-Nr2e3 ^rd7/rd7 x AKR/J cross to the C57BL/6J inbred strain for six consecutive generations. Approximately 65% of the B6.Cg- Mor7 ^AKR :Nr2e3 ^rd7/rd? N6 F ₂ animals homozy gous for the rd7 mutation showed a suppressed phenotype, compared to 49% of F ₂ animals in the initial intercross of B6.Cg-Nr2e3 ^r<i7/r ' ⁱ⁷ x AKR/J, suggesting a single modifier gene may be sufficient to suppress rd7. A genome wide analysis of the F ₂ pups confirmed that approximately 95% of the B6.Cg-Mor7 ^KR Nr2e3 ^rd7/rdy genome harbored C57BL/6J alleles in the N6 generation (Silver, L.M. Mouse Genetics:

Concept and Applications, (1995)). Two-thirds of the B6.Cg-Mor7 ^AKR :Nr2e3 ^rd7/rd7 suppressed mice were heterozygotes across the Mor7 locus, indicating that the AKR/J Mor7 allele acts as a dominant protective allele. Consistent with previous results, the suppressed B6.Cg-Mor7 ^AKR :Nr2e3 ^rd7/rd7 mice harboring the modifier allele showed restored retinal morphology (Figure 1A and IB) and expression of S-cone opsin (OPN1SW), compared to affected littermates harboring the susceptible allele (Figure 1C and ID). Through fine mapping analysis, the Mor7 suppressor locus was refined to a 3.3 cM region in

chromosome 11. This region is flanked by markers Dl lMitl45 and Dl lMit360 and contains approximately 200 genes.

Identification of Nrldl as a genetic modifier of rd7

A candidate approach was utilized to identify the Mor7 gene responsible for conferring rd7 suppression. Through rigorous in silico analysis using several resources, it was determined that 95 of the approximately 200 genes that lie within the Mor7 locus are expressed in the retina, 10 of which are transcription factors (Figure IE). Three of these genes: thyroid hormone receptor alpha (Thra), retinoid acid receptor alpha (Rara) and rev-erb alpha (Nrldl) are, like Nr2e3, members of the nuclear hormone receptor family. As described herein, the Mor7 modifier gene functions in the same or parallel pathway as Nr2e3. Further, given that several members of this family have been described as key regulators of retinal development and function, Thra, Rara and Nrldl were considered strong candidates for Mor7 and their coding as well as upstream regions were sequenced to identify allelic variants between C57BL/6J and AKR/J. While allelic variants were not found in either Thra or Rara, two single nucleotide polymorphisms (SNPs) were identified in Nrldl (Figure 2). The SNPs identified in Nrldl were in both the translated and un-translated regions of the gene. A non-synonymous SNP at position 1222bp was identified in Nrldl, resulting in replacement of the consensus Arginine at position 408 by Glutamine in the AKR/J NR1D1 protein (Figure 2A and 2B). Sequence alignment of the Nrldl gene across species revealed that the consensus for the SNP in the coding region is in accordance with the C57BL/6J sequence (Figure 2B). This SNP is located within the highly conserved ligand-binding domain of this nuclear hormone receptor. Specifically, the SNP lies within the co-repressor N-CoR binding domain, also known as X domain (Zamir et al., 1996 Mol Cell Biol, 16: 5458-5465).

A second SNP was identified at position +494bp from the putative transcriptional start site (Figures 2C and 2D), within the Nrldl promoter region (Adelmant et al., 1996 Proc Natl Acad Sci U S A, 93: 3553-3558; Kakizawa et al., 2007 J Mol Endocrinol, 38: 555-568). Specifically, the AKR/J genome harbors a thymidine whereas a cytosine residue is found in C57BL/6J. Sequence alignment of the Nrldl gene across species revealed that the consensus nucleotide for this location corresponds to thymidine, which is in accordance with the AKR/J sequence (Figure 2D). As this SNP resides within the promoter region of Nrldl, it was examined whether Nrldl mRNA expression varies in B6 versus AKR/J retinas. Quantitative real time PCR confirmed that Nrldl mRNA expression is up-regulated by 3 fold in the AKR/J retina, compared to C57BL/6J (P=0.0024, Figure 2E). This difference in expression may account for the suppressed effect observed in AKR/J genetic background.

Nrldl delivery restores retinal integrity in rd7

NR1D1 regulates many processes such as differentiation, metabolism, and the circadian rhythm (Burris, T.P. 2008 Mol Endocrinol, 22: 1509-1520). More recently, results have demonstrated a role for NR1D1 in the retina. NR1D1 forms a complex with NR2E3, CRX and NRL, key transcriptional regulators of retinal development and function (Walia et al., 2008 Arch Ophthalmol, 126: 379-384). In addition, recent results have identified a number of targets co-regulated by NR2E3 and NR1D1 in the developing and adult retina (Cheng et al., 2004 Hum Mol Genet, 13: 1563-1575; Mollema et al., 2011 PLoS One, 6: el7494). Thus, Nrldl is a strong candidate to modify the effects of Nr2e3 associated retinal degeneration.

In vivo electroporation was performed to deliver Nrldl alleles from either C57BL/6J or AKR/J into the retina of neonatal rd7 mice to determine whether NR1D1 can modulate rd7 associated retinal degeneration. One month after injection, animals were examined clinically by indirect ophthalmoscopy for detection of the characteristic rd7 pan-retinal spotting. While spotting of the fundus was clearly observable in the eyes electroporated with the control GFP vector at P30.5, electroporation of GFP.Nr2e3 B6 resulted in suppression of the phenotype (Figures 3C and 3D). Delivery of either GFP.Nrldl ^B6 or GFP.Nrldl^ ³ also resulted in rescue of the pan-retinal spotting phenotype (Figures 3E and 3F). Further, the absence of retinal spotting correlated with absence of retinal dysplasia in histological sections

(Figures 3G-3J). A subset of the electroporated animals were aged to 4 months and electroretinograms (ERGs) were performed to examine visual function. Significant improvements in both scotopic (dark- adapted) and photopic (light- adapted) ERG response were observed in B6.Cg-Nr2e3 ^rd/7rd? eyes injected with compared to GFP injected eyes (Figures 3K and 3L). These studies demonstrate that the dosage of Nrldl is sufficient for rescue of Nr2eJ-associated retinal disease irrespective of allelic variants in the ligand-binding domain; thus the promoter SNP is likely the protective allele, mediating rescue of disease. Mechanism of Nrldl -mediated suppression through molecular rescue of rd7 mis-regulated genes

NR1D1, a regulator of circadian clock metabolism, also functions as a cofactor of NR2E3 and regulates expression of a number of genes in the retina (Cheng et al., 2004 Hum Mol Genet, 13: 1563-1575; Mollema et al. 2011 PLoS One 6, el7494). The expression profile of the retinas of rd7 animals was previously characterized, and 30 genes that are misregulated in Nr2e3 deficient retinas were identified, 24 of which are directly regulated by NR2E3, NR1D1 or co-regulated by both receptors (Haider et al, 2009 Exp Eye Res, 89: 365- 372). As described in detail below, it was determined that NR1D1 is able to suppress rd7 associated retinal degeneration through molecular rescue of key developmental and functional pathways that are misregulated in the rd7 retina.

As the level of misregulation varied greatly for all 30 genes, the expression of Opnlsw (the S-cone specific opsin) and Gnatl (cone photoreceptor specific transducin) (key components of the cone phototransducion cascade that are significantly up-regulated in rd7) was evaluated. Quantitative RT-PCR was performed to assay expression of Opnlsw and

Gnat2 in the retina of rd7 animals 30 days after GFP.Nrldl AKR/J delivery. Expression of Opnlsw was 1.5 fold increased in the retina of untreated rd7 animals (left eye, n=3) compared to C57BL/6J, consistent with previous reports (p=0.004, Figure 4).

GFP.Nrldr ^J delivery to the right eye of the same rd.7 animals resulted in a significant reduction in Opnlsw expression (p=0.035, Figure 4). Furthermore, Opnlsw levels in

GFP.Nrldl injected eyes were not significantly different from those present in

C57BL/6J retinas (p=0.86), indicating that Nrldl delivery rescues Opnlsw expression to near normal levels. Gnat2 was also significantly decreased in eyes injected with GFP.Nrldl AKR/J , compared to uninjected eyes from the same animals (p=0.005, Figure 4). These results indicate that Nrldl up-regulation is able to suppress Nr2e3 associated retinal degeneration by compensating for Nr2e3 loss in the retina and by redirecting the biological networks that modulate photoreceptor development and function.

Example 2: Nrldl rescues retinal degeneration 1 (rdl), a mouse model for severe autosomal dominant retinitis pigmentosa

Retinitis pigmentosa (RP) is an inherited, degenerative eye disease that causes severe vision impairment and often blindness in an estimated 1 in 1,000 individuals in the U.S. population. Mutations in the human pde6b gene cause autosomal dominant RP, an Nr2e3- associated retinal disease. The retinal degeneration 1 (rdl) mouse is a model for a severe form of RP and comprises a mutation in the mouse pde6b gene.

As show in in Figure 5, Nrldl rescues phenotype in the rdl mouse. Specifically, as compared to the B6 control eye, the retina of the left eye (no gene therapy) of the rdl mouse does not have any photoreceptor cells in the outer nuclear layer (ONL). By contrast, the retina of the right eye of the rdl mouse after rescue with Nrldl comprises about 40-50% more photoreceptor cells in the ONL, as compared to the left eye (no gene therapy).

The sequence alignment below shows an Nrldl variant observed in a human patient with an Nr2e3 mutation. The patient had a milder phenotype (i.e., retinal degeneration) to begin with. The Nrldl variant illustrated below is located in the promoter region of Nrldl, similar to the mouse variant that confers resistance to Nr2e3 associated retinal degeneration.

Example 3: Nanoparticle delivery and rescue in rd7 mouse model

Preparation of cDNA loaded nanoparticles

The nanoparticles were formulated using the previously described double emulsion solvent evaporation method (Aukunuru JV 2003, Singh SR 2009). The biodegradable polymer used was Poly(L-lactide-co-glycolide)(PLGA) Resomer 503H (50:50; i.v. 0.44 dl/g; Boehringer Ingelheim, Petersburg, VA). After preparing nanoparticles, drug loading, encapsulation efficiency and particle size were determined.

Administration of cDNA loaded nanoparticles

cDNA loaded Nile red PLGA nanoparticles are introduced into postnatal day 0 (P0) eyes by intravitreal or subretinal injection, and in some instances the injection was followed by electroporation. In brief, particles were reconstituted in IX PBS and animals received 1.5 ug of modifier cDNA into the right eyes and the same dose of control (empty) nanoparticles into the left eyes at P0. The total volume injected was 0.5 ul. The efficacy of delivery was assessed using Nile red as a nanoparticle tracking dye, and green fluorescent protein expression as a marker of transfected cells. Mice were aged to P30.5 and phenotyped by indirect ophthalmoscopy, electroretinogram (ERG), and immunohistochemistry (Haider et al 2006). Half of the animals injected with nanoparticles were then subjected to electroporation of the eye to test for enhancement of nanoparticles uptake based on the degree of the rd7 phenotype recovery.

Results

The phenotype of the rd7 mouse model includes: appearance of white spots in the retina (Figure 7B, right panel), retinal whorls and rosettes in the outer nuclear layer (Figure 7B, left panel), electroretinograms show reduction in cone and rod signals after 5 months, cone and rod cell loss causes reduction of ONL thickness by half, and significant increase in the percentage of cone cells expressing blue opsin.

Mutant rd7 retinas treated with Nr2e3 nanoparticles exhibited clinical improvements with the disappearance of the characteristic rd7 spotted retina and morphologically with rescue of rd7 associated whorls and rosettes. Specifically, fundus examinations were performed to determine gross phenotypic differences between treated and untreated retinas. While spotting of the fundus was clearly observable when rd7 mice were injected with blank vector, administration of Nr2e3-containing nanoparticles reduced the severity of spotting and provided a partial recovery of the phenotype (Figure 8).

Electroretinograms were performed under both photopic and scotopic conditions to further assess nanoparticle effectiveness. These assessments were performed in animals of 2 months of age and 6 months of age. ERG analysis showed a significantly improved amplification of both a and b wave of the treated eyes compared to the reduced function observed in untreated rd7 eyes.

These results demonstrate that rescue of rd7 associated retinal degeneration was attained in both electroporated and nonelectroporated Nr2e3 nanoparticle treated mutant eyes. Nanoparticle delivery of Nr2e3 in the rd7 mice efficiently ameliorated clinical,

morphological, and functional defects associated with rd7 retinal degeneration. Further, evaluation of the efficacy of delivery with and without electroporation demonstrated that particles are able to enter and function in the retina effectively without electroporation. OTHER EMBODIMENTS

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.