WO2025015328A2

WO2025015328A2 - Increasing nfe2l1 activity or expression as therapy for eye disorders

Info

Publication number: WO2025015328A2
Application number: PCT/US2024/038006
Authority: WO
Inventors: Ekaterina S. LOBANOVA
Original assignee: University Of Florida Research Foundation, Incorporated
Priority date: 2023-07-13
Filing date: 2024-07-15
Publication date: 2025-01-16

Description

INCREASING NFE2L1 ACTIVITY OR EXPRESSION AS THERAPY FOR EYE DISORDERS

GOVERNMENT SUPPORT CLAUSE

[0001] This invention was made with government support under grant EY030043 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0002] Vision deterioration and blindness are commonly driven by the loss of retinal neurons or molecular defects in cells supporting retinal function, such as retinal pigment epithelium (RPE), vasculature, choroidal, microglial cells, or blood-derived monocyte recruits. Thousands of mutations in more than two hundred genes have been reported to cause inherited blinding retinal degeneration. Other retinal degenerative disorders leading to irreversible vision loss include complex multifactorial diseases such as age-related macular degeneration (AMD) and glaucoma. Dysregulated proteostasis is a hallmark of many inherited and age-related human diseases, including eye disorders. The approaches allowing improved efficiency to clear misfolded proteins could be applied to treat a broad range of human diseases. Mouse models of photoreceptor degeneration stressed by misfolded proteins and accumulating UPS reporter could serve as effective tools for proof-of-principle studies and testing approaches to manipulate proteostasis.

[0003] Proteasomes are the central proteolytic machines that are critical for breaking down the majority of damaged and abnormal proteins in human cells. Although universally applicable drugs are not yet available, the stimulation of proteasomal activity is being analyzed as a proof-of-principle strategy to increase cellular' resistance to a broad range of proteotoxic stressors. These approaches have included the stimulation of proteasomes through the overexpression of individual proteasome subunits, phosphorylation, or conformational changes induced by small molecules or peptides.

SUMMARY

[0004] In contrast to conventional methods, certain approaches disclosed herein augment the proteolytic capacity of retinal cells by increasing total pool of proteasomes on the transcriptional level via stimulation of the Nfe211 pathway. [0005] Increasing levels of Nfe211 in the target cells leads to the elevation of proteasomal levels and activity. In an aspect of the invention, provided is a nucleic acid for use in a method of treatment or prophylaxis of eye disorders in a subject. The nucleic acid may be capable of driving the expression of Nfe211 in a target cell. Proteasome activity may be maintained or increased in the target cell comprising the nucleic acid compared to an equivalent cell not comprising the nucleic acid.

[0006] The eye disorders may include disease leading to retinal degeneration and may be age-related macular degeneration (AMD), glaucoma, or inherited diseases affecting retinal neurons and RPE.

[0007] In some embodiments, the nucleic acid sequence encodes a polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2 or 4. The nucleic acid sequence may encode the Nfe2l1 protein, its portion, or variant thereof. In some embodiments, the nucleic acid codes for Nfe211 polypeptide fused with cell-penetrating peptide (CPP), peptide-based cleavable linker (PCL), destabilization domains, epitope-binding sequence, or cellular localization signals (e.g. nuclear, endoplasmic reticulum, plasma membrane targeting signal), DNA-binding domains. The epitope-binding molecule may be an antibody or antibody-like molecule.

[0008] In some embodiments, the nucleic acid is DNA. The nucleic acid may be an episome. In some embodiments, the nucleic acid is a plasmid or a minicircle. In some embodiments, the nucleic acid is RNA. The nucleic acid may be messenger RNA or circular RNA.

[0009] The nucleic acid may be suitable for integration into the genome of the target cell by an RNA- guided endonuclease system. The RNA-guided endonuclease may be a CRISPR-Cas system.

[0010] In some embodiments, the nucleic acid is delivered to a target cell via a non-viral carrier. The non- viral carrier may be selected from the group consisting of nanoparticles, liposomes, cationic polymer, and calcium phosphate particles.

[0011] In some embodiments, a vector virion system comprises nucleic acid and is used as a method of treatment or prophylaxis of eye disorders in a subject. The vector virion may be selected from the group consisting of adeno- associated virus, adenovirus, retrovirus, orthomyxovirus, paramyxovirus, papovavirus, picornavirus, lentivirus, herpes simplex virus, vaccinia virus, pox virus, anellovirus, and alphavirus. In some embodiments, the vector virion is an adeno-associated virus (AAV). In some embodiments, Nfe211 expression is increased in the target cell comprising the vector virion compared to an equivalent cell not comprising the vector virion. Proteasome activity may be maintained or increased in the target cell comprising the vector virion compared to an equivalent cell not comprising the vector virion. [0012] The target cell may be a cell of the retina or other cell in the eye. The target cell may be a cell of the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL), the outer plexiform layer (OPL), or the retinal pigmental epithelium (RPE), choroid. In some embodiments, multiple cells in the retina and eye could be targeted for eye prophylaxis and treatment.

[0013] The nucleic acid or the viral vector system may be administered intraocularly, intravitreally, subretinally, periocularly, or systemically to a subject by injection or infusion. In some embodiments, the subject is human. The subject may be affected by or at risk of developing eye disorders.

[0014] In some embodiments, methods are provided such that the endogenous Nfe211 expression is increased. For example, an agent such as a polypeptide or nucleic acid binding molecule (e.g., nucleic acid binding portion) capable of binding to a target sequence is delivered to a cell and drives Nfe211 expression, or alternatively a small molecule is delivered to the cell that causes Nfe211 to translocate to the nucleus.

[0015] The nucleic acid may be capable of driving the expression of Nfe2l1 in a target cell. Proteasome activity may be maintained or increased in the target cell comprising the nucleic acid compared to an equivalent cell not comprising the nucleic acid. In a specific example, provided is a method that involves administering a therapeutically effective amount of a composition comprising a nucleic acid encoding a polypeptide wherein the nucleic acid comprises a nucleic acid sequence encoding a polypeptide comprising at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NOs 2 or 4, and wherein expression of the nucleic acid increases proteosome activity in a target cell.

[0016] In some embodiments, a promoter is operably linked to the nucleic acid sequence. The promoter may be an RPE-specific promoter. The RPE-specific promoter may be selected from the group consisting of a RPE65 promoter, a NA65 promoter, a VMD2 promoter (also known as Bestl promoter), and a Synpiii promoter. Promoter might be photoreceptor-specific - promoter (Rhodopsin or Rhodopsin Kinase 1 or similar), RPE-specific (a Bestl promoter or similar), neuron-specific promoter (synapsin (SYN)). In alternative embodiments, the promoter is a ubiquitous promoter. The ubiquitous promoter may be selected from the group consisting of a CMV promoter, a CAG promoter, a GAPDH promoter, a UbiC promoter, and an EF-la promoter. In other embodiments, the promoter is the native promoter for Nfe2Ll or a functional fragment thereof.

[0017] In some embodiments, the nucleic acid sequence encodes a polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2 . In some embodiments, the nucleic acid sequence encodes a functional polypeptide. The nucleic acid sequence may encode an Nfe211 protein or variant thereof.

[0018] In some embodiments, the vector virion comprises a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2 .

[0019] A further aspect provides an Nfe211 polypeptide for use in a method of treatment or prophylaxis of eye disorders in a subject. In some embodiments, the polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0020] In some embodiments, the polypeptide further comprises a cell penetrating peptide (CPP). In some embodiments, the polypeptide further comprises a peptide-based cleavable linker (PCL). The CPP may be conjugated to the N-terminus of the PCL. The amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2 may be conjugated to the C-tcrminus of the PCL. In some embodiments, the PCL is a peptide sequence that is cleavable by cathepsin D.

[0021] Proteosome activity may be maintained or increased in the target cell comprising the polypeptide compared to an equivalent cell not comprising the polypeptide.

[0022] In an aspect of the invention, a nucleic acid system is provided comprising one or more nucleic acids, comprising: a) a nucleic acid sequence encoding an RNA-guided endonuclease: b) a nucleic acid sequence encoding a guide RNA complementary to a target sequence associated with an insertion site in the genome of the target cell and capable of directing said RNA- guided endonuclease to said target sequence; and c) a nucleic acid sequence encoding Nfe211, for use in a method of treatment or prophylaxis of eye disorders in a subject. The nucleic acid sequence encoding Nfe211 is capable of driving expression of Nfe211 in a target cell of the subject and the nucleic acid system is suitable for directed insertion of the nucleic acid sequence encoding Nfe211 at the insertion site in the genome of the target cell.

[0023] The nucleic acid sequence encoding Nfe211 may be flanked by 5’ homology arm and a 3’ homology arm. In some embodiments, the 5’ homology arm is homologous to a DNA sequence 5’ of the target sequence from the insertion site and the 3’ homology arm is homologous to a DNA sequence 3’ of the target sequence from the insertion site. The nucleic acid sequence encoding Nfe211 may further comprise a 5’ flanking sequence comprising a target sequence and a 3’ flanking sequence comprising a target sequence. In some embodiments, the 5’ flanking sequence is 5’ of the 5’ homology arm and the 3’ flanking sequence is 3’ of the 3’ homology arm.

[0024] In alternative embodiments, the nucleic acid sequence encoding Nfe211 is flanked by a 5’ target sequence and a 3’ target sequence. The 5’ target sequence and the 3’ target sequence may be identical to target sequence from an insertion site in the genome. In some embodiments, the one or more nucleic acids are one or more viral vector genomes. The one or more viral vector genomes may be one or more adeno- associated virus vector genomes.

[0025] An alternative targeted approach for increasing endogenous expression of Nfe211 in a target cell may use a nucleic acid binding molecule (e.g., nucleic acid binding portion) capable of binding to a target sequence.

[0026] Thus, an aspect provides a nucleic acid comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising: a) a nucleic acid binding molecule capable of binding to a target sequence in the promoter or regulatory sequences of the Nfe2Ll gene; and b) one or more transcriptional activators, for use in a method of treatment or prophylaxis of eye disorders in a subject, wherein the fusion protein is capable of increasing Nfc211 expression in a target cell of the subject.

[0027] A further aspect provides a pharmaceutical composition comprising one or more of the agents as described herein. In some embodiments, the pharmaceutical composition is formulated for ocular delivery. The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Fig. 1. Nfe211 levels establish the size of the proteasome pool in retinas. (A) Chymotrypsin- like peptidase activity measured in the retinal extracts prepared from Nfe211^OE and Nfe211^{Retina K0} mice and liver extracts prepared from Nfe211^0E mice. (B) Transcriptional analysis of representative proteasome subunits in retinas or livers of the indicated mice performed via RT-qPCR. (C) Quantification graphs and (D, E, F) western blots showing proteasomal components detected in (D) livers and (E, F) extracts prepared from the retinas of the indicated mice. Changes in proteasome activity, tianscript levels, and protein levels were expressed as the percentage of difference (AWT) from the average value obtained with wild-type mice. (G) Comparative western blots showing the levels of Nfe211 in the lysates prepared from the livers and retinas of indicated mice. Retinal lysates of Nfe211^{Retina KO} mice were used to control for antibody specificity. (H) Detection of Nfe211 in whole lysates and subcellular fractions prepared from the livers of Nfe211 -overexpressing mice and their wild-type littermates. All mice were one month old. The data are presented as the mean ± SD. All experiments were repeated at least three times. Color- stained protein markers (M) were either detected as non-specific bands together with proteins of interest during ECL and infrared imaging or were added from blot photographs (separated with a vertical gray line).

[0029] Fig. 2. Nfe211 overexpression increases the levels of proteasome transcripts in rod photoreceptors. (A) Nfe211 transcripts detected in the retinas of the indicated mice via RNA in situ hybridization (RNA ISH). ONL: outer nuclear layer (containing photoreceptor nuclei); INL: inner nuclear layer; and GCL: ganglion cell layer. Tire scale bar is 25 pm. (B) Uniform manifold approximation and projection (UMAP) plot of cells prepared from Nfe211^OE and WT mouse retinas. (C) Expression levels of Nfe211 and proteasome transcripts in the rod photoreceptor fractions of the indicated mice. Rhodopsin (Rho) transcripts serve as control markers for rods. (D, E) Volcano plots showing differentially expressed genes in (D) retinas and (E) livers of Nfe211^0E mice as detected with bulk RNA-seq. Black dots represent genes with a false discovery rate less than 0.05. (F) Top pathways impacted by Nfe211 overexpression in livers and identified with Ingenuity Pathway Analysis Software (QIAGEN, Hilden, Germany). (G) Heatmap of changes in proteasome transcripts in livers of Nfe211^0E and WT mice calculated from raw counts and presented as z-scores. FDR: false discovery rate.

[0030] Fig. 3. AAV-based delivery of NFE2L1 stimulates proteasomal activity and biogenesis in the retina. (A) Western blots and (B) quantification plots showing proteasomal components detected in the retinas of C57BL6/J mice transduced with either AAV5-CBA-Nfe211-HA or AAV5-CBA-eGFP constructs. (C) Chymotrypsin-like peptidase activity was measured in the extracts prepared from the retinas of C57BL6/J mice transduced with either AAV5-CBA-Nfe211 -HA or AAV5-CBA-eGFP constructs. Changes in protein levels and proteasome activity were expressed as the percentage of the average values obtained for mice transduced with AAV5-CBA-eGFP constructs. Analysis was performed by individuals unaware of treatment.

[0031] Fig. 4. Nfe211 overexpression counteracts UPS insufficiency in a Rho^P23H/WT mouse model of human blindness. (A) Ub^G76V-GFP reporter (green) in retinal cross sections of Rho^p23H/WT and Rho^p23H/WT/Nfe211^OE littermates, and Ub^G76v-GFP/WT control mice. Rod outer segments (red) were stained with wheat germ agglutinin (WGA). The scale bar is 25 pm. (B) Quantification plot and (C) representative western blot of the Ub^G76v-GFP reporter in retinas of mice with the indicated genotypes detected with an anti-GFP antibody. Extracts prepared from littermates negative for the Ub^G76v-GFP transgene were used to control for antibody specificity. The results are shown as a percentage of the average signal in Rho^p23H/WT/Ub^G76V-GFP mice. (D) Chymotrypsin-like peptidase activity was measured in the extracts prepared from retinas of Rho^p23H/WT and Rho^p23H/WT/Nfe211^OE littermate mice. (E) Transcription analysis of the representative proteasome subunits in indicated mice was performed with RT-qPCR. Quantification graphs of the western blot bands for (F) proteasome components and (G) autophagy markers detected in the extracts prepared with retinas from the indicated mice. Images of western blots quantified to generate the plots are shown in Fig. S2. (H, I, J, K) Polyubiquitin chains in the extracts prepared from retinas of the indicated mice as detected by western blotting with (H) FK2 and (J) P4D1 antibodies. (I, K) Averaged density profiles of the polyubiquitin staining shown in (H) and (J). All animals were 28 days old. The data are presented as the mean ± SD (B, D, E, F, G) or the mean ± 95% CI (I, K).

[0032] Fig. 5. Nfe211 overexpression delays retinal degeneration in a Rlio^P23H/WT mouse model of human blindness. (A) Comparative analysis of age-related thinning of the outer nuclear layer (ONL) in Rh_oP23H/wr _anj pho^{P2 ,H/w l}/Nfc2l 1^0H mice. To generate the plot, the measurements from horizontal optical coherence tomography (OCT)-based spider diagrams built around optic nerve head (ONH) at the indicated ages were summed, normalized to average values of 30-day-old Rho^p23H/WT mice, and fitted with an exponent. (B, C) Representative horizontal SD-OCT scans and (D) OCT-based spider diagrams showing ONL thickness at indicated ages. The ONL is marked with a blue quadrilateral. The scale bar for the OCT images is 100 pm. The numbers of eyes analyzed at P30 were as follows: Rho^p23H/WT - 8, _RhoP23H/wT_/Nfe211OE __{5 > WT} . ₄₎ Nfc2l l ^0H- 3; at P45: Rho^K3H/WT - 14, Rho^p23H/WT/Nfe211^OE - 12, WT - 8, Nfe211^0E- 8; at P90 : Rho^p23H/WT - 14, Rho^p23H/WT/Nfe211^OE - 12, WT - 10, Nfe211^0E- 22; at P180 : Rho^M3H/WT - 14, Rho^p23H/WT/Nfe211^OE - 12, WT - 11, Nfe211^OE- 13. The data are presented as the mean ± SD. Quantification was performed by individuals not aware of genotype.

[0033] Fig. 6. Nfe211 overexpression improves photoreceptor survival in a Rho^P23H/WT mouse model of human blindness. Morphometric analysis of retinas obtained from 6-month-old mice of the indicated genotypes. (A, D) Images of the representative regions of H&E-stained retinal cross sections from (A) inferior and (D) superior parts of the retinas -750 pm from the center of the optic nerve head (ONH). The scale bar is 25 pm. (B) Spider diagrams show the number of photoreceptor nuclei in 100-pm segments counted along the inferior-superior axis of the mouse eyes and (C) length of the distance from the outer limiting membrane to the tip of the outer segments (IS/OS length) measured at the indicated distances from the center of the ONH. The number of eyes analyzed was as follows: Rho^p23II/WT - 10 and Rho^p23H/WT/Nfe211^OE > | 'rhe data are presented as the mean + SD. Quantification was performed by individuals not aware of genotype.

[0034] Fig. 7. Nfe211 overexpression delays vision loss in a Rho^P23H/WT mouse model of human blindness. Response amplitudes of electroretinography (ERG) a- and b-waves evoked by light flashes of increasing intensity in the mice with the indicated genotypes as determined at (A-D) 3 and (E-H) 6 months of age. The number of eyes analyzed at 3 months was as follows: Rho^p23H/WT - 13, Rho^p23H/WT/Nfe211^OE > j2. WT - 7 eyes, and Nfe211^0E - 7. The number of eyes analyzed at 6 months was as follows: Rho^p23H/WT - 12, Rho^p23H/WT7Nfe211^OE - 12, WT - 6, Nfe211^0E - 12. (C, D, G, H) The representative ERG recordings evoked by flashes of indicated light intensities. The data are presented as the mean ± SEM.

[0035] Fig. 8. Nfe211 overexpression and Tsc2 knockout counteract UPS insufficiency in a Gyi⁷' mouse model of photoreceptor degeneration. (A, B, C) The fluorescence signal of Ub^G76v-GFP reporter (green) in retinal cross-sections of (A) Gyi ^/7Nfe211^OEand (B) Gyf^/7Tsc2^{Rod K0} mice shown along with their Gyi ² littermates and (C) Ub^G76v-GFP/WT control mice. The outer rod segments (red) are stained with wheat germ agglutinin (WGA). The scale bar- is 25 pm. (D) The quantification plot and (E) representative western blot of the Ub^G76v-GFP reporter in lysates prepared from the retinas of indicated mice as detected with an anti-GFP antibody. (F) Chymotrypsin-like proteasome activity was measured in retinal extracts with or without lambda protein phosphatase treatment ( PP). (G) Quantification plot and (H) western blots showing representative proteasome subunits in the retinal extracts of the indicated mice. The protein markers (M) were detected as non-specific bands together with proteins of interest or added from blot photographs and separated with a vertical gray line. (I) Transcript analysis of Nfe211 and representative proteasome subunits in retinas from the indicated mice performed with RT-qPCR and shown as a percentage of the average values for Gyf^A or WT littermates. (J) Nfe211 transcripts in the retinas of indicated mice as detected with RNA in situ hybridization (RNA ISH). ONL: outer nuclear layer (containing photoreceptor nuclei); INL: inner nuclear layer; and GCL: ganglion cell layer. The scale bar is 25 pm. All animals were one month old. The data are shown as the mean ± SD.

DETAILED DESCRIPTION

[0036] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the ait. All documents mentioned in this text are incorporated herein by reference.

[0037] In some embodiments, methods described by the disclosure are useful for the treatment of certain eye diseases or disorders (for example, retinal degeneration, retinitis pigmentosa (RP), age-related macular degeneration (AMD), glaucoma, etc).

[0038] The present disclosure is based on finding of a critical role of Nfe211 in the control of proteasomal levels in the retina: its overexpression increases, and knockout reduces the proteasomal pool and activity. It was found that overexpression of Nfe2l1 is not toxic to the retina and improved clearance of in vivo UPS reporter in photoreceptors of mice struggling with misfolded proteins, supporting an augmentation of Nfe211 pathway as a potent approach to stimulate degradation of ubiquitinated proteins. In addition, it was shown that Nfe211 overexpression delayed visual loss in a preclinical model of human blindness. These findings position the Nfe211 pathway as an emerging target for drug development and focus on enhancing this pathway to treat eye diseases. Accordingly, the present disclosure relates to increasing Nfe211 expression for the prophylaxis of and treatment of eye disorders.

[0039] Several strategies can be used to achieve increased Nfe211 expression in a subject. These are discussed in detail below.

Definitions

[0040] The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides having modified peptide backbones. The term “domain” refers to any part of a protein or polypeptide having a particular function or structure.

[0041] Proteins are said to have an “N-terminus” and a “C-terminus.” The term “N- terminus” relates to the start of a protein or polypeptide, terminated by an amino acid with a free amine group (-NH2). The term “C-terminus” relates to the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH).

[0042] The terms “nucleic acid” and “polynucleotide,” used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0043] Nucleic acids are said to have “5’ ends” and “3" ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5’ phosphate of one mononucleotide pentose ring is attached to the 3’ oxygen of its neighbor in one direction via a phosphodiester linkage. An end of an oligonucleotide is referred to as the “5’ end” if its 5’ phosphate is not linked to the 3’ oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the “3’ end” if its 3’ oxygen is not linked to a 5’ phosphate of another mononucleotide pentose ring. A nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5’ and 3’ ends. In either a linear or circular DNA molecule, discrete elements are referred to as being “upstream” or 5’ of the “downstream” or 3’ elements. [0044] The term “genomically integrated” refers to a nucleic acid that has been introduced into a cell such that the nucleotide sequence integrates into the genome of the cell. Any protocol may be used for the stable incorporation of a nucleic acid into the genome of a cell.

[0045] The term “viral vector” refers to a recombinant nucleic acid that includes at least one element of viral origin and includes elements sufficient for or permissive of packaging into a viral vector particle. The vector and/or particle can be utilized for the purpose of transferring DNA, RNA, or other nucleic acids into cells in vitro, ex vivo, or in vivo. Numerous forms of viral vectors are known.

[0046] The term “isolated” with respect to cells, tissues, proteins, and nucleic acids includes cells, tissues, proteins, and nucleic acids that are relatively purified with respect to other bacterial, viral, cellular, or other components that may normally be present in situ, up to and including a substantially pure preparation of the cells, tissues, proteins, and nucleic acids. The term “isolated” also includes cells, tissues, proteins, and nucleic acids that have no naturally occurring counterpart, have been chemically synthesized and are thus substantially uncontaminated by other cells, tissues, proteins, and nucleic acids, or has been separated or purified from most other components (e.g., cellular components) with which they are naturally accompanied (e.g., other cellular proteins, polynucleotides, or cellular components).

[0047] The term “wild type” includes entities having a structure and/or activity as found in a normal (as contrasted with mutant, diseased, altered, or so forth) state or context. Wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0048] The term “endogenous sequence” refers to a nucleic acid sequence that occurs naturally within a cell or animal. For example, an endogenous ALB sequence of a human refers to a native ALB sequence that naturally occurs at the ALB locus in the human.

[0049] “Exogenous” molecules or sequences include molecules or sequences that are not normally present in a cell in that form. Normal presence includes presence with respect to the particular developmental stage and environmental conditions of the cell. An exogenous molecule or sequence, for example, can include a mutated version of a corresponding endogenous sequence within the cell, such as a humanized version of the endogenous sequence, or can include a sequence corresponding to an endogenous sequence within the cell but in a different form (i.e., not within a chromosome). In contrast, endogenous molecules or sequences include molecules or sequences that are normally present in that form in a particular cell at a particular developmental stage under particular environmental conditions.

[0050] The term “heterologous” when used in the context of a nucleic acid or a protein indicates that the nucleic acid or protein comprises at least two segments that do not naturally occur together in the same molecule. For example, the term “heterologous,” when used with reference to segments of a nucleic acid or segments of a protein, indicates that the nucleic acid or protein comprises two or more sub-sequences that are not found in the same relationship to each other (e.g., joined together) in nature. As one example, a “heterologous” region of a nucleic acid vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid vector could include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Likewise, a “heterologous” region of a protein is a segment of amino acids within or attached to another peptide molecule that is not found in association with the other peptide molecule in nature (e.g., a fusion protein, or a protein with a tag). Similarly, a nucleic acid or protein can comprise a heterologous label or a heterologous secretion or localization sequence.

[0051] “ Codon optimization” (i.e., “codon optimized” sequences) takes advantage of the degeneracy of codons, as exhibited by the multiplicity of three-base pair codon combinations that specify an amino acid, and generally includes a process of modifying a nucleic acid sequence for enhanced expression in particular host cells by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. For example, a nucleic acid encoding a factor IX protein can be modified to substitute codons having a higher frequency of usage in a given prokaryotic or eukaryotic cell, including a bacterial cell, a yeast cell, a human cell, a non- human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, a hamster cell, or any other host cell, as compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, at the “Codon Usage Database.” These tables can be adapted in a number of ways. See Nakamura et al. (2000) Nucleic Acids Res. 28(1 ):292, herein incorporated by reference in its entirety for all purposes. Computer algorithms for codon optimization of a particular sequence for expression in a particular host are also available (see, e.g., Gene Forge). [00234] The term “locus” refers to a specific location of a gene (or significant sequence), DNA sequence, polypeptide-encoding sequence, or position on a chromosome of the genome of an organism. For example, an "ALB locus” may refer to the specific location of an ALB gene, ALB DNA sequence, albumin-encoding sequence, or ALB position on a chromosome of the genome of an organism that has been identified as to where such a sequence resides. An “ALB locus” may comprise a regulatory element of an ALB gene, including, for example, an enhancer, a promoter, 5' and/or 3’ untranslated region (UTR), or a combination thereof.

[0052] The term “gene” refers to DNA sequences in a chromosome that may contain, if naturally present, at least one coding and at least one non-coding region. The DNA sequence in a chromosome that codes for a product (e.g., but not limited to, an RNA product and/or a polypeptide product) can include the coding region interrupted with non-coding introns and sequence located adjacent to the coding region on both the 5’ and 3’ ends such that the gene corresponds to the full-length mRNA (including the 5’ and 3’ untranslated sequences). Additionally, other non-coding sequences including regulatory sequences (e.g., but not limited to, promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequence, and matrix attachment regions may be present in a gene. These sequences may be close to the coding region of the gene (e.g., but not limited to, within 10 kb) or at distant sites, and they influence the level or rate of transcription and translation of the gene.

[0053] The term "administering" or “administration” of an agent as used herein means providing the agent to a subject using any of the various methods or delivery systems for administering agents or pharmaceutical compositions known to those skilled in the art. Agents described herein may be administered by oral, intradermal, intravenous, intramuscular', intraocular, intranasal, intrapulmonary, epidermal, subcutaneous, mucosal, or transcutaneous administration. In typical embodiments, the agent is administered by intraocular administration.

[0054] The term "co- administration" or "co-administering" as used herein refers to the administration of an active agent before, concurrently, or after the administration of another active agent such that the biological effects of either agents overlap.

[0055] A “therapeutically effective amount” refers to an amount which, when administered in a proper dosing regimen, is sufficient to reduce or ameliorate the severity, duration, or progression of the disorder being treated (e.g., eye disorders), prevent the advancement of the disorder being treated (e.g., eye disorders), cause the regression of the disorder being treated (e.g., eye disorders), or enhance or improve the prophylactic or therapeutic effects(s) of another therapy. The full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations per day for successive days.

[0056] The term “allele” refers to a variant form of a gene. Some genes have a variety of different forms, which are located at the same position, or genetic locus, on a chromosome. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. Genotypes are described as homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ.

[0057] A “promoter” is a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter may additionally comprise other regions which influence the transcription initiation rate. The promoter sequences disclosed herein modulate transcription of an operably linked polynucleotide. A promoter can be active in one or more of the cell types disclosed herein (e.g., a mouse cell, a rat cell, a pluripotent cell, a one-cell stage embryo, a differentiated cell, or a combination thereof). A promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO 2013/176772, herein incorporated by reference in its entirety for all purposes.

[0058] “Operable linkage” or being “operably linked” includes juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include such sequences being contiguous with each other or acting in trans (e.g., a regulatory sequence can act at a distance to control transcription of the coding sequence). [00239] The methods and compositions provided herein employ a variety of different components. Some components throughout the description can have active variants and fragments. The term “functional” refers to the innate ability of a protein or nucleic acid (or a fragment or variant thereof) to exhibit a biological activity or function. The biological functions of functional fragments or variants may be the same or may in fact be changed (e.g., with respect to their specificity or selectivity or efficacy) in comparison to the original molecule, but with retention of the molecule’s basic biological function.

[0059] The term “variant” refers to a nucleotide sequence differing from the sequence most prevalent in a population (e.g., by one nucleotide) or a protein sequence different from the sequence most prevalent in a population (e.g., by one amino acid).

[0060] The term “fragment,” when referring to a protein, means a protein that is shorter or has fewer amino acids than the full-length protein. The term “fragment,” when referring to a nucleic acid, means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. A fragment can be, for example, when referring to a protein fragment, an N- terminal fragment (i.e., removal of a portion of the C-terminal end of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminal end of the protein), or an internal fragment (i.e., removal of a portion of each of the N-terminal and C-terminal ends of the protein). A fragment can be, for example, when referring to a nucleic acid fragment, a 5' fragment (i.e., removal of a portion of the 3’ end of the nucleic acid), a 3’ fragment (i.e., removal of a portion of the 5’ end of the nucleic acid), or an internal fragment (i.e., removal of a portion each of the 5’ and 3’ ends of the nucleic acid).

[0061] “Sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).

[0062] “Percentage of sequence identity” includes the value determined by comparing two optimally aligned sequences (greatest number of perfectly matched residues) over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0063] Unless otherwise stated, sequence identity/ similarity values include the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. “Equivalent program” includes any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

[0064] The term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, or leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a nonpolar- (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.

[0065] The term “eye disorders” as used herein refers to eye condition selected from (i) inherited diseases causing rod and/or cone death, (ii) age-related Macular Degeneration and/or (iii) glaucoma.

[0066] A “homologous” sequence (e.g., nucleic acid sequence) includes a sequence that is either identical or substantially similar to a known reference sequence, such that it is, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence. Homologous sequences can include, for example, orthologous sequence and paralogous sequences. Homologous genes, for example, typically descend from a common ancestral DNA sequence, either through a speciation event (orthologous genes) or a genetic duplication event (paralogous genes). “Orthologous” genes include genes in different species that evolved from a common ancestral gene by speciation. Orthologs typically retain the same function in the course of evolution. “Paralogous” genes include genes related by duplication within a genome. Paralogs can evolve new functions in the course of evolution.

[0067] As used herein, a “control” as in a control sample or a control subject is a comparator for a measurement, e.g., a diagnostic measurement of a sign or symptom of a disease. In certain embodiments, a control can be a subject sample from the same subject an earlier time point, e.g., before a treatment intervention. In certain embodiments, a control can be a measurement from a normal subject, i.e., a subject not having the disease of the treated subject, to provide a normal control, e.g., FIX concentration or activity in a subject sample. In certain embodiments, a normal control can be a population control, i.e., the average of subjects in the general population. In certain embodiments, a control can be an untreated subject with the same disease. In certain embodiments, a control can be a subject treated with a different therapy, e.g., the standard of care. In certain embodiments, a control can be a subject or a population of subjects from a natural history study of subjects with the disease of the subject being compared. In certain embodiments, the control is matched for certain factors to the subject being tested, e.g., age, gender. In certain embodiments, a control may be a control level for a particular lab, e.g., a clinical lab. Selection of an appropriate control is within the ability of those of skill in the art.

[0068] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients. The transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified elements recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”

[0069] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur and that the description includes instances in which the event or circumstance occurs and instances in which the event or circumstance does not.

[0070] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range. For example, 5-10 nucleotides is understood as 5, 6, 7, 8, 9, or 10 nucleotides, whereas 5-10% is understood to contain 5% and all possible values through 10%. [00254] At least 17 nucleotides of a 20 nucleotide sequence is understood to include 17, 18, 19, or 20 nucleotides of the sequence provided, thereby providing a upper limit even if one is not specifically provided as it would be clearly understood. Similarly, up to 3 nucleotides would be understood to encompass 0, 1, 2, or 3 nucleotides, providing a lower limit even if one is not specifically provided. When “at least”, “up to”, or other similar language modifies a number, it can be understood to modify each number in the series.

[0071] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex region of “no more than 2 nucleotide base pairs” has a 2, 1, or 0 nucleotide base pairs. When “no more than” or “less than” is present before a series of numbers or a range, it is understood that each of the numbers in the series or range is modified. [0072] As used herein, it is understood that when the maximum amount of a value is represented by 100% (e.g., 100% inhibition or 100% encapsulation) that the value is limited by the method of detection. For example, 100% inhibition is understood as inhibition to a level below the level of detection of the assay, and 100% encapsulation is understood as no material intended for encapsulation can be detected outside the vesicles.

[0073] Unless otherwise apparent from the context, the term “about” encompasses values ± 10% of a stated value. In certain embodiments, the term “about” is understood to encompass tolerated variation or error within the art, e.g., 2 standard deviations from the mean, or the sensitivity of the method used to take a measurement, or a percent of a value as tolerated in the art, e.g., with age. When “about” is present before the first value of a series, it can be understood to modify each value in the series.

[0074] The term “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0075] The term “or” refers to any one member of a particular list and also includes any combination of members of that list.

[0076] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a protein” or “at least one protein” can include a plurality of proteins, including mixtures thereof.

[0077] Statistically significant means p <0.05.

[0078] In the event of a conflict between a sequence in the application and an indicated accession number or position in an accession number, the sequence in the application predominates.

[0079] The foregoing terms and additional related terms are described and defined herein.

Overview

[0080] Alterations in the ability of cells to maintain a healthy proteome arc thought to contribute to the pathogenesis of multiple human diseases and aging (/-.?). The majority of proteins in human cells are degraded by the ubiquitin-proteasome system (UPS), with proteasomes being essential proteolytic machines cleaving proteins into smaller polypeptides (4). A protcasomc consists of several principal components. The core 20S particle of a proteasome contains proteolytic sites, which become accessible after association with the 19S cap, facilitating the degradation of polyubiquitinated proteins or 11 S and PA200 regulators, allowing the degradation of polypeptides and unstructured proteins not modified with ubiquitin (5-9). An increasing number of studies have demonstrated that stimulation of proteasomes increases cell resistance to various types of proteotoxic stressors and delays aging 10-16). Some of the investigated approaches to stimulating proteasomes include overexpression of individual proteasome subunits (e.g., PSMD11, P5, USa, a3AN), modulation of proteasome activity through phosphorylation, and the development of small compounds and peptides capable of opening 20S particles to allow access to their proteolytic sites by certain protein substrates (4, 15-21).

[0081] An alternative approach to increase the proteolytic capacity of cells and treat human diseases caused by protein misfolding might include increasing the total proteasome pool. Several studies in diverse experimental systems, including mouse fibroblasts, the brain, the retina, and muscles, reported higher levels of proteasomes under genetic activation of the (mechanistic target of rapamycin complex 1) mTORCl pathway (22-26). The mechanisms driving this transcriptional program have not been fully characterized but are proposed to be triggered by sterol-regulatory element binding protein 1 (Srebpl)- mediated transcriptional upregulation of the nuclear factor, erythroid-2, like 1 (Nfe211) transcription factor (22, 27). Drugs that increase proteasome activity through Nfe211 -mediated proteasomal transcription have not yet been developed. Building on these findings, we previously examined the genetic activation of the mTORCl pathway to increase proteasome levels and treat retinal diseases (24). Although we observed elevated levels of Nfe21 transcripts and proteasomes in rod photoreceptor-specific Tsc2-knockout mice (Tsc2^{Rod KO}), this transcriptional response was suppressed in degenerating rods stressed by misfolded proteins (24). Recent studies indicated that overexpression of Nfe211 can raise proteasomal levels in vivo in brown fat and cardiomyocytes in mice (28, 29). Therefore, we examined direct Nfe211 overexpression as an alternative approach to increase the pool of proteasomes in neurodegenerative diseases linked to impaired proteostasis in the retina.

[0082] The studies provided herein show that overexpression of Nfe211 does not have adverse effects on retinal function or structure, drives proteasomal activity and expression, improves clearance of UPS reporter in photoreceptors struggling with misfolded proteins, and delays vision loss in a mouse model of human blindness. The findings pave the way to consider the relatively poorly investigated Nfc211 pathway as a therapeutic target for treating neurodegenerative diseases linked to protein misfolding and promoting drug development to enhance its activity.

[0083] An overexpression of transcriptional factor might have pleiotropic effects making it difficult to pinpoint with certainty mechanisms leading to the improved survival of photoreceptors. Nfe211 has become a topic of increasing interest in recent years. It was proposed to play a role in controlling proteasomal levels and other genes involved in proteostasis regulation, protection against oxidative stress, serve as a cholesterol and metabolic sensor, and regulator of ferroptosis (29, 33, 43-48). We cannot exclude the possibility that observed improvement in model of human blindness is not exclusively attributed to the changes in proteasome amounts but is also caused by slight transcriptional changes impacting other genes and Nfe211 -mediated signaling pathways. Still, to date, the most clearly documented and understood function of Nfe211 is to control the levels of proteasomes and drive an increase in proteasome transcripts in response to sublethal doses of proteasome inhibitors (49, 50). Our findings warrant studies of the Nfe211 pathway in the retina and focus on its enhanced activation for the treatment of diseases associated with protein misfolding, including drug development.

[0084] Intriguingly, a side-by-side comparison of proteasomal biogenesis in the retinas of Gyi ⁷ mice with activated mTORCl pathway and mice overexpressing Nfe211 clearly showed that in vivo mTORCl activation is as efficient, if not more efficient, in driving proteasome biogenesis without substantial Nfe211 increase (at least in comparison to Nfe211 overexpressing mice). This observation highlights and encour ages molecular studies of poorly defined mechanisms underlying mTORCl -driven Nfe211 activation, which could lead to the identification of more targeted and potent approaches to stimulate the Nfe211 pathway (52).

[0085] Finally, our findings add Nfe211 to the growing list of transcriptional master regulators of proteostasis that are being investigated as potential targets to delay vision loss. Indeed, a series of studies solidified the critical role of basal activity of ubiquitously expressed activating transcription factor 6 (ATF6) in regulating stress responses of rods in retinitis pigmentosa and the preservation of cones in humans (53-56). Recently, modulation of autophagy through selective manipulation of the retinoic acid receptor alpha (RARa) transcriptional program has been shown to increase photoreceptor survival in the rdlO model of retinitis pigmentosa (57). Together with our findings, these previous studies support the mapping and analysis of transcription networks controlling proteostasis in the retina to harness the potential of these factors to treat age-related and inherited retinal degeneration in a gene- and mutationindependent manner.

Medical uses

[0086] Accordingly, the invention relates to agents as described herein for increasing Nfe211 expression in a target cell and/or increasing Nfe211 activity in a target cell, for use in a method of treatment or prophylaxis of eye disorders in a subject. An option for increasing Nfe211 expression in a target cell may involve introducing exogenous Nfe211 to a target cell. Thus, the agent may be an Nfe211 polypeptide or a nucleic acid encoding Nfe211. In some embodiments, the agent is heterologous. Another option for increasing Nfe211 expression involves increasing endogenous Nfe211 expression in a target cell. Thus, the agent may be capable of increasing endogenous Nfe211 expression. Alternatively, the agent may be capable of increasing Nfe211 activity in a target cell. [0087] Further provided is a method of treatment or prophylaxis of eye disorders in a subject comprising administering an agent for increasing Nfe211 expression and/or activity as described herein to the subject. Additionally provided is the use of such agent as described herein for the manufacture of a medicament for the treatment or prophylaxis of eye disorders in a subject. The present invention also relates to a nucleic acid, a vector virion, a polypeptide, a nucleic acid system, a viral vector system, or a pharmaceutical composition, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the nucleic acid increases Nfe211 expression in a target cell of the subject.

[0088] Also provided is a method of treatment or prophylaxis of eye disorders in a subject comprising administering a nucleic acid, a vector virion, a polypeptide, a nucleic acid system, a viral vector system, or a pharmaceutical composition as described herein to the subject. In another aspect, provided is the use of a nucleic acid, a vector virion, a polypeptide, a nucleic acid system, a viral vector system, or a pharmaceutical composition as described herein, for the manufacture of a medicament for the treatment or prophylaxis of eye disorders in a subject.

Eye disorders

[0089] Eye disorders is a medical condition which may result in deterioration of vision, resulting in blurred or no vision in the center of the visual field. The term “eye disorders” refers to any of a number of conditions in which the retinal macula degenerates or becomes dysfunctional, e.g. as a result of decreased growth of cells of the macula, increased death or rearrangement of the cells of the macula (e.g. RPE cells), loss of normal biological function, or a combination of these events. Eye disorders also involves inherited conditions in which retinal cells die, and/or glaucoma in which the optic nerve atrophies.

[0090] In particular, the present invention relates to age-related eye disorders. As used herein, "age- related eye disorders" or "AMD" includes early, intermediate, and advanced/late AMD and includes both dry AMD such as geographic atrophy and wet AMD, also known as neovascular or exudative AMD. Degeneration/dysregulation of the retinal pigment epithelium (RPE), a supportive monolayer of cells underlying the photoreceptors, is commonly seen in patients with AMD. The retinal pigment epithelium (RPE) is a multifunctional monolayer of neuroepithelium-derived cells, flanked by photoreceptor (PR) cells and the choroid complex. The RPE is typically composed of a single layer of hexagonal cells that are densely packed with pigment granules.

[0091] Typically, in AMD there is a progressive accumulation of characteristic yellow deposits, called drusen in the macula (a part of the retina) between the RPE and the underlying choroid. Drusen are formed of extracellular proteins and lipids. The accumulation of drusen damages the retina over time. AMD can be divided into 3 stages: early, intermediate, and late, based partially on the extent (size and number) of drusen. In some embodiments, administration of the therapy of the invention results in a reduction of drusen in a target cell compared to a cell not comprising the therapy of the invention. In some embodiments, administration of therapy of the invention to the target cell prevents the formation of drusen in a target cell compared to a cell not comprising the therapy of the invention. In some embodiments, the age-related eye disorders (AMD) is early AMD. Early AMD is typically diagnosed based on the presence of medium-sized drusen. Early AMD tends to be asymptomatic. In some embodiments, the age-related eye disorders (AMD) is intermediate AMD. Intermediate AMD is typically diagnosed by large drusen and/or any retinal pigment abnormalities. Intermediate AMD can lead to some vision loss, but generally is asymptomatic. In some embodiments, the age-related eye disorders (AMD) is late AMD (also known as advanced AMD). Typically, in late AMD, patients experience symptomatic central vision loss caused by retinal damage. This damage can be caused by atrophy or by the onset of neovascular disease. Late AMD is further divided into two subtypes based on the type of damage. These are called geographic atrophy/dry AMD and wet AMD/neovascular AMD. In some embodiments, the AMD is selected from the group consisting of early AMD, intermediate AMD, and late AMD.

[0092] In some embodiments, the age-related eye disorders (AMD) is dry AMD. Dry AMD encompasses all forms of AMD that are not wet AMD, including early and intermediate forms of AMD as well as the advanced form of dry AMD, called geographic atrophy. In some embodiments, the age-related eye disorders (AMD) is geographic atrophy. Geographic atrophy, also known as atrophic AMD, is an advanced form of dry AMD. It is characterised by progressive and irreversible loss of retinal cells leading to a loss of visual function. Typically, in geographic atrophy, three areas of the retina undergo atrophy. These are the choriocapillaris, retinal pigment epithelium, and the overlying photoreceptors.

[0093] In contrast, wet AMD (also called neovascular or exudative AMD) is the wet form of advanced AMD. It is characterised as vision loss due to abnormal blood vessel growth (choroidal neovascularization) in the choriocapillaris, through Bruch’s membrane. It is usually, but not always, preceded by the dry form of AMD. The proliferation of abnormal blood vessels in the retina is stimulated by vascular endothelial growth factor (VEGF). These abnormal blood vessels are more fragile than typical blood vessels, and so lead to blood and protein leakage below the macula. Bleeding, leaking, and scarring from these blood vessels eventually cause irreversible damage to the photoreceptors and rapid vision loss if left untreated. In some embodiments, the age-related eye disorders (AMD) is not wet AMD. In some embodiments, the age-related eye disorders (AMD) is dry AMD and excludes wet AMD.

[0094] Typically, in patients with eye disorders the retina and the choroid are affected. Thus, in some embodiments, the target cell is a cell of the retina or the choroid. In some embodiments, the target cell is a cell of the retina. The retina is the innermost, light-sensitive tissue of the eye. The retina comprises several layers, including a layer comprising photoreceptors. The principal functional layers of the retina comprise the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL), the outer plexiform layer (OPL), the outer nuclear layer (ONL), the photoreceptor outer segment (POS), and supporting the retina, the retinal pigmental epithelium (RPE). In some embodiments, the target cell is a cell of the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL), the outer plexiform layer (OPL), the outer nuclear layer (ONL), the photoreceptor outer segment (POS ), or the retinal pigmental epithelium (RPE). In some embodiments, the target cell is a cell of the retinal pigmental epithelium (RPE).

[0095] In some embodiments, the method of treatment or prophylaxis of eye disorders in a subject includes the step of contacting a target cell or tissue with the agent as described herein. In some embodiments, the method of treatment or prophylaxis of eye disorders in a subject includes the step of contacting a target cell or tissue with the nucleic acid, a vector virion, a polypeptide, a nucleic acid system, a viral vector system, or a pharmaceutical composition described herein.

Nfe211

[0096] Exogenous Nfc211 polypeptide and/or peptide may be directly delivered into the cytoplasm of ocular cells. Accordingly, in an aspect of the invention provides an Nfe211 polypeptide and/or peptide for use in a method of treatment or prophylaxis of eye disorders in a subject. The human amino acid sequence of Nfe211 is provided below as SEQ ID NO: 2.

[0097] An aspect provides a polypeptide for use in a method of treatment or prophylaxis of eye disorders in a subject. The method may comprise administering a therapeutically effective amount of a polypeptide comprising or is an amino acid sequence having at at least 50%, least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0098] In some embodiments, the polypeptide has an amino acid sequence having at least 50%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the polypeptide is a fragment of SEQ ID NO: 2. In other embodiments, the amino acid sequence of polypeptide consists of SEQ ID NO: 2.

[0099] In some embodiments, the nucleic acid or vector virion comprising said nucleic acid comprises a nucleic acid sequence encoding a polypeptide having at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid or vector virion comprising said nucleic acid comprises a nucleic acid sequence encoding a polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid or vector virion comprising said nucleic acid comprises a nucleic acid sequence consisting of SEQ ID NO: 1.

[0100] Percent (%) amino acid sequence identity with respect to a reference sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Various known tools can be used to measure sequence identity, including but not limited to Clustal Omega, Multiple Sequence Alignment (EMBL- EBI).

[0101] In some embodiments, the polypeptide of the invention is formulated for ocular delivery. In some embodiments, the polypeptide according to the invention is functional. In some embodiments, the polypeptide is capable of activating proteosomal activity in a target cell. In some embodiments, the polypeptide is capable of causing proteosomes to degrade misfolded proteins in a target cells.

[0102] It has been found that augmentation of Nfe211 expression provided protection to the RPE and retina more generally. By introducing human and mouse Nfc211 transgcnc to retinal cells, proteosomal activity was increased.

[0103] In some embodiments, proteosome activity is increased in the target cell relative to a cell that is not modified with the agent described herein (e.g., the small molecule, nucleic acid, vector virion, polypeptide, nucleic acid system, viral vector system, or pharmaceutical composition described herein). Proteosome activity can be used to determine proteolytic capacity of the cell. By “increase(s)(d) proteosome activity” is meant that the proteosome activity is higher in the target cell into which a nucleic acid or polypeptide has been introduced compared to an equivalent cell not having a nucleic acid or polypeptide introduced.

[0104] In some embodiments, the polypeptide is a recombinant polypeptide modified for delivery to a target cell. In an example, Nfe211 may be conjugated to peptides that are described in Bhattacharya et al. to mediate delivery into RPE cells. Bhattacharya 2017, Journal of Controlled Release 251 , 37-48 describes a peptide-based delivery system that allows for controlled cargo release in RPE cells. The described system is typically used for intravitreal administration. Other possible routes of delivery are described herein. The peptide-based delivery system comprises a peptide-based cleavable linker (PCL) with a cell penetrating peptide (CPP) conjugated to the N-terminus and the cargo (e.g. Nfe211) is conjugated to the C-terminus. Example PCLs include peptide sequences sensitive to cathepsin D. Cathepsin D, a lysosomal enzyme has relatively high expression in RPE cells. CPPs are charged peptide sequences capable of intracellular delivery of molecular cargo.

[0105] A cell penetrating peptide (CPP) is typically a short peptide that facilitates cellular intake and uptake of molecules (e.g. polypeptides). CPPs typically deliver cargo into cells via endocytosis. CPPs generally have an amino acid composition comprising a high abundance of positively charged amino acids (e.g. lysine or arginine) or comprising sequence containing an alternating pattern of polar, charged amino acids and non-polar, hydrophobic amino acids. Example CPPs include but are not limited to, penetratin peptide, Tat peptide (48-60), VP22 peptide, Mouse PrP peptide, pVEC peptide, Transportan peptide, TP10 peptide, Polyarginine peptide, etc. Example CPPs for RPE cells are provided in Bhattacharya 2017, Journal of Controlled Release 251 , 37-48. Non-limiting examples include GRKKRRQRRPPQ (SEQ ID NO: 5), rrrrmrr (SEQ ID NO: 6), RLVSYNGIIFFLK (SEQ ID NO: 7), FNLPLPSRPLLR (SEQ ID NO: 8), where “r” is D-Arg. In some embodiments, the CPP further comprises a short flexible linker in between the CPP and PCL. In some embodiments, the short flexible linker has the amino acid sequence GGS. A PCL is a peptide-based cleavable linker. In the context of the invention other cleavage linkers may be used. Example PCLs cleavable by cathepsin D are described in Bhattacharya 2017, Journal of Controlled Release 251 , 37-48. Non-limiting examples include KGKPILFFRLKr (SEQ ID NO: 6), KPILFFRLGK (SEQ ID NO: 9), and KGSALISWIKR (SEQ ID NO: 10), where “r” is D-Arg.

[0106] Accordingly, example CPPs conjugated to PCLs include but are not limited to GRKKRRQRRPPQGGSKGKPILFFRLKr (SEQ ID NO: 11), GRKKRRQRRPPQGGS KPILFFRLGK (SEQ ID NO: 12), GRKKRRQRRPPQGGSKGSALISWIKR (SEQ ID NO: 13), rrrrrrrrrGGSKGKPILFFRLKr (SEQ ID NO: 14), rrrrrrrrrGGS KPILFFRLGK (SEQ ID NO: 15), rrrrrrrrrGGSKGSALISWIKR (SEQ ID NO: 16), RLVSYNGIIFFLKGGSKGKPILFFRLKr (SEQ ID NO: 17), RLVSYNGIIFFLKGGS KPILFFRLGK (SEQ ID NO: 18), RLVSYNGIIFFLKGGSKGSALISWIKR (SEQ ID NO: 19), FNLPLPSRPLLRGGSKGKPILFFRLKr (SEQ ID NO: 20), FNLPLPSRPLLRGGSKPILFFRLGK (SEQ ID NO: 21), FNLPLPSRPLLRGGSKGSALISWIKR (SEQ ID NO: 22), GRKKRRQRRPPQKGKPILFFRLKr (SEQ ID NO: 23), GRKKRRQRRPPQKPILFFRLGK (SEQ ID NO: 24), GRKKRRQRRPPQKGSALISWIKR (SEQ ID NO: 25), rrrrrrrrrKGKPILFFRLKr (SEQ ID NO: 26), rrrrrrrrrKPILFFRLGK (SEQ ID NO: 27), rrrrrrrrrKGSALISWIKR (SEQ ID NO: 28), RLVSYNGIIFFLKKGKPILFFRLKr (SEQ ID NO: 29), RLVSYNGIIFFLKKPILFFRLGK (SEQ ID NO: 30), RLVSYNGIIFFLKKGSALISWIKR (SEQ ID NO: 31), FNLPLPSRPLLRKGKPILFFRLKr (SEQ ID NO: 32), FNLPLPSRPLLRKPILFFRLGK (SEQ ID NO: 33), and FNLPLPSRPLLRKGSALISWIKR (SEQ ID NO: 34). [0107] Any one of the above peptides may be conjugated to the Nfe211 polypeptide, directly or indirectly.

[0108] In some embodiments, provided is a molecule comprising a PCL with a CPP conjugated to the N- terminus and an Nfe211 polypeptide or peptide conjugated to the C-terminus of the PCL (e.g. CPP- PCL- Nfe211). In some embodiments, provided is a molecule comprising a PCL with a CPP conjugated to the N-terminus and an Nfe211 polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% sequence identity to the amino acid sequence of SEQ ID NO:2 conjugated to the C-terminus of the PCL (e.g. CPP-PCL-Nfe211).

Gene therapy

[0109] Gene therapy involves introducing genetic material into target cells for the purpose of modulating the expression of specific proteins which are altered, thus reversing the biological disorder causing the alteration thereof. The present invention contemplates a nucleic acid sequence encoding Nfe211 protein for use in a method of treatment or prophylaxis of eye disorders in a subject.

[0110] In human cells, Nfe211 is encoded by the Nfe2Ll gene. In the context of the present invention “Nfe2Ll gene” refers to the DNA sequence encoding Nfe211 (e.g., found in the genome). The Nfe2Ll gene may be operably linked to any suitable transcriptional and/or translational regulatory sequences in the nucleic acid and vector systems described herein.

[0111] The term "nucleic acid" herein is meant either DNA or RNA, or molecules which contain both ribo- and deoxyribonucleotides. The nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids. The nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence. In some embodiments, the nucleic acid is a recombinant nucleic acid.

[0112] In some embodiments, the nucleic acid sequence encoding Nfe211 is exogenous. In some embodiments, the nucleic acid sequence encoding Nfe211 is heterologous. The term "exogeneous” herein is meant nucleic acid which encodes proteins not ordinarily made in appreciable or therapeutic amounts in ocular cells. Exogeneous nucleic acid also includes nucleic acid which is ordinarily found within the genome of the ocular cell, but which is no longer being expressed or is being expressed at a reduced amount compared to non-diseased tissue. Thus, the genetically engineered ocular cell may contain extra copies of a gene ordinarily found within its genome. The term “heterologous” with reference to a nucleic acid refers to a nucleic acid that does not naturally occur in the target cell. In some embodiments, the nucleic acid is an episome. An episome is a genetic element that can replicate independently of the target cell and also in association with a chromosome with which it becomes integrated. The nucleic acid may be a plasmid or a minicircle. A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. A minicircle is a small (~4kb) circular replicon. In some embodiments, the nucleic acid is messenger RNA or circular' RNA.

[0113] In some embodiments, the nucleic acid can be integrated into the host’s genome. In alternative embodiments, the nucleic acid is not inserted into the host’s genome. A nucleic acid randomly integrating into the host’s genome can cause adverse events following insertional mutagenesis. A nucleic acid that is not randomly inserted into the host’s genome advantageously avoids any insertional mutagenesis.

[0114] As will be understood by those of skill in the art, nucleic acids for gene therapy contain the necessary elements for the transcription and translation of the inserted coding sequence (and may include, for example, a promoter, an enhancer, and other regulatory elements). Promoters can be constitutive or inducible. Promoters can be selected to target preferential gene expression in a target tissue, such as the RPE (Sutanto et al., 2005, "Development and evaluation of the specificity of a cathepsin D proximal promoter in the eye" Curr Eye Res. 30:53-61 ; Zhang et al., 2004, "Concurrent enhancement of transcriptional activity and specificity of a retinal pigment epithelial cell-preferential promoter" Mol Vis. 10:208-14; Esumi et al., 2004. "Analysis of the VMD2 promoter and implication of E-box binding factors in its regulation" J Biol Chem 279:19064-73; Camacho-Hubner et al., 2000, "The Fugu rubripes tyrosinase gene promoter targets transgene expression to pigment cells in the mouse" Genesis. 28:99-105; and references therein). Promoters can also be active in any cell or tissue type.

[0115] The nucleic acid encoding Nfe211 is typically operably linked to regulatory elements, such as promoters and enhancers, which drive transcription of the DNA in the target cells of an individual. The promoter may drive expression of Nfc211 in all cell types. Alternatively, the promoter may drive expression of the Nfe211 only in specific cell types, for example, in cells of the retina, e.g. RPE. In some embodiments, the promoter is a ubiquitous promoter. The term “ubiquitous promoter” means a promoter that is active in any cell, tissue, and/or cell cycle stage. Typically, the ubiquitous promoter is strongly active in a wide range of cells, tissues, and/or cell cycle stages. In further embodiments, the ubiquitous promoter is selected from the group consisting of CMV promoter, CAGGS promoter (aka CBA or CAG), mini CAG (SV40 Intron) promoter, SV40 promoter, CBA/CB7 promoter, smCB A promoter, CBh promoter, MeCP2 promoter, shCMV promoter, CMVd2 promoter, core CMV promoter, SV40mini promoter, SCP3 promoter, EFl -a promoter, PGK promoter, GAPDH promoter, and UbC promoter. In some embodiments, the promoter is an RPE-specific promoter. In further embodiments, the RPE- specific promoter is selected from the group consisting of a RPE65 promoter, NA65 promoter, VMD2 promoter (also known as Bestl promoter) and Synpiii promoter. Suitable promoters, in particular retina-specific promoters are described in Buck et al. Int. J. Mol. Sci. 2020, 21 , 4197. Synthetic promoters for RPE are also described in Johari et al. 2021 “Design of synthetic promoters for controlled expression of therapeutic genes in retinal pigment epithelial cells”. Biotechnology and Bioengineering.

[0116] In yet another embodiment, the promoter is the native promoter for Nfe2Ll or a functional fragment thereof.

[0117] In this specification the term “operably linked” may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence, such as a promoter sequence are covalently linked in such a way as to place the expression of a nucleotide coding sequence under the influence or control of the regulatory sequence. Thus, a regulatory sequence is operably linked to a selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of a nucleotide coding sequence which forms part or all of the selected nucleotide sequence. Where appropriate, the resulting transcript may then be translated into a desired protein or polypeptide.

[0118] In some embodiments, introduction of a nucleic acid encoding Nfe211 results in a genetically engineered target cell or tissue. By the term "genetically engineered" herein is meant a cell or tissue that has been subjected to recombinant DNA manipulations, such as the introduction of exogeneous nucleic acid. For example, the cell contains exogeneous nucleic acid. Generally, the exogeneous nucleic acid is made using recombinant DNA techniques.

[0119] Therapeutic nucleic acid can be delivered in vivo. Alternatively, therapeutic nucleic acid can be delivered ex vivo, whereby cells of a patient are extracted and cultured outside of the body. The cells are then genetically modified by introduction of a therapeutic nucleic acid and then re-introduced back into the patient. In preferred embodiments, the nucleic acid is delivered in vivo. In the context of the invention, it is preferable that expression of the nucleic acid encoding Nfe211 lasts for as long as possible. It is also preferable that there is low immunogenicity since the host’s immune response can determine transgenic expression.

[0120] Gene delivery into target tissue/cells is a key step in gene therapy. This step may be earned out by gene delivery vehicles called vectors. Vectors for gene therapy are vehicles that carry the gene of interest to the target cell. There are two types of vector, viral and non-viral. In some embodiments, the vector is a viral vector. In alternative embodiments, the vector is a non-viral vector.

Viral vector gene delivery systems

[0121] Recombinant viral vectors that are preferably replication deficient have been used as vehicles to deliver transgenes into target cells. [0122] In some embodiments, the nucleic acid is delivered to the target cell via a viral vector. Viral gene delivery vectors include, but are not limited to nucleic acid sequences from the following viruses: RNA viruses such as a retrovirus, adenovirus, adeno-associated virus, SV40-type viruses, polyoma viruses, Epstein-Barr viruses, papilloma viruses, herpes virus, vaccinia virus, polio virus, orthomyxovirus, paramyxovirus, papovavirus, picornavirus, lentivirus, pox virus, anellovirus, and alphavirus. In some embodiments, the viral vector is selected from the group consisting of adeno- associated virus vector, adenovirus vector, retrovirus vector, orthomyxovirus vector, paramyxovirus vector, papovavirus vector, picornavirus vector, lentivirus vector, herpes simplex virus vector, vaccinia virus vector, pox virus vector, anellovirus virus vector, and alphavirus vector.

[0123] In some embodiments, the viral vector is an adeno-associated virus vector. In some embodiments, the nucleic acid is a viral vector genome. An aspect of the invention provides a vector virion for use in a method of treatment or prophylaxis of eye disorders in a subject. The vector virion comprises a nucleic acid comprising a nucleic acid sequence encoding Nfe211 and is capable of driving expression of Nfe211 in a target cell. In some embodiments, the vector virion is a recombination vector virion.

[0124] Virion particles comprising vector genomes of the invention are typically generated in packing cells capable of replicating viral genomes, expressing viral proteins (e.g. structural virion proteins and associated enzymes), and assembling virion particles. Also provided is a packaging cell comprising a nucleic acid construct encoding a vector genome described herein. Packing cells may also require helper virus functions, e.g. from adenovirus, El-deleted adenovirus or herpes virus. Techniques for producing virion particles are well known in art. The packaging cell is typically a eukaryotic cell, such as a mammalian cell, e.g., a primate cell, e.g. a human cell. In some embodiments, a cell line is used. In some embodiments, the packaging cells may be stably transformed cells such as HeLa cells, 293 cells (HEK293, HEK293T or HEK293ET cells) and PerC.6 cells. Other cell lines include MRC-5 cells, WI-38 cells, Vero cells and FRhL-2 cells. The invention also provides method of producing a vector virion.

[0125] The size of the transgene which can be incoiporated into the viral vector will depend on various factors, such as the specific virus on which the vector is based, the packaging capacity of the virion and which (if any) of the native viral genes have been deleted from the vector.

[0126] Non-limiting examples of viral vectors are provided below.

[0127] Adenovirus:

[0128] Adenoviruses are commonly used in gene therapy because of their ability to be successfully transduced into a large number of cell types. They generally have a packaging ability of about packaging ability of 30 to 40 kb nucleic acid. [0129] To improve safety different generations of adenoviral vectors have been generated. First generation adenoviral vectors were engineered by removing the El region making them replication defective and removing the E3 region. Newer second-generation adenoviruses have been engineered with additional deletions or mutations in the viral E2 and E4 regions, preventing transcriptional control of viral gene expression and viral genome replication, respectively. Further improvement in the safety and efficacy of adenoviral vectors has come with the development of “gutless” or “helper-dependent” adenoviral vectors that have all viral sequences deleted except for the inverted terminal repeat (ITRs) and the packing signal allowing for around 36kb of space for cargo genes. This third-generation virus requires an additional adenoviral helper virus that is similar in composition to the first general virus (except they contain loxP sites inserted to flank the packing signal) to help with replication and packaging. These third-generation vectors retain the advantages of the first-generation adenoviral vectors in terms of high efficiency in in vivo transduction and transgene expression, and can mediate high-level, long-term transgene expression in the absence of toxicity.

[0130] In some embodiments, the viral vector is an adenoviral vector. In some embodiments, the viral vector is a first-generation adenoviral vector, a second-generation adenoviral vector, or a third-generation adenoviral vector.

[0131] Lenti virus:

[0132] Lentiviruses are RNA viruses of the retrovirus family. The packaging capacity of this viral vector ranges from 8-9 kb nucleic acid. They possess a reverse transcriptase through which they can integrate their retrotranscribed proviral DNA into the chromosomes of host cells. Lentiviruses are able to integrate their genome into the host cell, resulting in stable expression. However, genome integration can result in insertional mutagenesis. Accordingly, non-integral lentiviral vectors have been developed, typically by making them deficient in integrases. These persist as episomal dsDNA circles capable of transducing nondividing cells. These non-integral vectors allow for efficient and sustained transgenic expression in post-mitotic tissues.

[0133] In some embodiments, the viral vector is a lentiviral vector. In some embodiment, the viral vector is a non-integral lentiviral vector.

[0134] Adeno-associated viruses:

[0135] Adeno-associated virus is a replication-deficient parvovirus having a single stranded DNA genome of which is about 4.7kb in length, including 145 nucleotide ITRs. Several features render them suitable for retinal gene therapy, such as lack of pathogenicity, minimal immunogenicity, ability to transduce nondividing cells, and capacity to mediate sustained levels of therapeutic gene expression. Adeno- associated viruses are among the smallest viruses, with an uncoiled icosahedral capsid of about 22 nm. Since they require the presence of a helper virus for replication to occur, adeno-associated viruses are classified as dependo viruses that are naturally deficient in replication and nonpa thogenic. Importantly, AAV recombinant genomes persist as episomes in transduced cells, leading to long- lasting expression of the transgene in nondividing retinal cells (Bordet T et al. Drug Discovery Today, Volume 24, Number 8, August 2019). AAVs have also been routinely used in ocular gene therapy (Buck T.M. Int. J. Mol. Sci. 2020, 21 , 4197).

[0136] Preferred viral gene delivery vectors are AAV vectors. “AAV" is an abbreviation for adeno- associated virus and may be used to refer to the virus itself or derivatives thereof. The term covers all serotypes and variants both naturally occurring and engineered forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector"). The rAAV may comprise the polynucleotide of interest (e.g. the nucleic acid sequence encoding Nfe211). In general, the rAAV vectors contain 5’ and 3’ adeno-associated virus inverted terminal repeats (ITRs), and the polynucleotide of interest operatively linked to sequences which regulate its expression in a target cell.

[0137] The term "AAV" includes but is not limited to AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV- 8), and AAV type 9 (AAV9). The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the ait. Such sequences may be found in the literature or in public databases, for example, such as GenBank. See, e.g., GenBank Accession Numbers NC_002077 (AAV-1), AF063497 (AAV-1), NC_001401 (AAV-2), AF043303 (AAV-2), NC_001729 (AAV-3), NC_001829 (AAV- 4), U89790 (AAV- 4), NC_006152 (AAV-5), AF513851 (AAV-7), AF513852 (AAV-8), and NC_006261 (AAV-8).

[0138] An AAV is able to infect both dividing and nondividing cells and has a broad tropism that allows it to infect many cell types depending on the particular serotype. The recombinant vectors of AAV (rAAV) used for gene therapy are mainly based on serotype 2 (AAV2); this was the first human serotype described and the best characterized AAV serotype. Since the AAV capsid protein is responsible for its tropism and, therefore, for its efficacy, a pseudotyped strategy has previously been developed in which pseudotyped or hybrid AAV vectors encode a serotype rep, usually AAV2, and the cap gene of a different serotype.

[0139] The vector may be a pseudotyped AAV vector. The phrase "pseudotyped AAV vector", herein designates a vector particle comprising a native AAV capsid including an rAAV vector genome and AAV Rep proteins, wherein Cap, Rep and the ITRs of the vector genome come from at least 2 different AAV serotypes. Examples of AAV chimeric vectors include but are not limited to AAV2/5, AAV2/6, and AAV2/8.

[0140] As the signals directing AAV replication, genome encapsulation and integration are contained within the ITRs of the AAV genome, some or all of the internal sequence of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA such as an expression cassette, with the rep and cap proteins provided in trans. The sequence located between the ITRs of an AAV vector may be referred to as a “payload”. In some embodiments, the payload is a nucleic acid comprising a nucleic acid sequence encoding Nfe211. The actual capacity of any particular AAV particle may vary depending on the viral proteins employed.

[0141] The vector may be an engineered AAV vector. For example, the engineered AAV vector is the SH10 vector as described in Klimczak RR, et al. 2009. PLoS One 4(10):e7467. The AAV engineered vector may have a mutated capsid, in particular a tyrosine mutated capsid. Other known suitable engineered capsids include AAV2tYF, AAV2.7m8, R100, AAV2.GL, AAV2.NN, AAV44.9, and AAV44.9(E531 D). Techniques to produce AAV vector particles in packaging cells are standard in the ait. For example, production of pseudotyped AAV is disclosed in WO 01/83692. In various embodiments, AAV capsid proteins may be modified to enhance delivery of the recombinant vector. Modifications to capsid proteins are generally known in the art. See, for example, US 2005/0053922 and US 2009/0202490.

[0142] A non-limiting example method of generating a packaging cell is to create a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising an AAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the AAV genome, and a selectable marker, such a neomycin resistance gene, are integrated into the genome of the cell. The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selected and are suitable for large-scale production of AAV. This can also be achieved using an adenovirus or baculovirus instead of plasmids for introducing AAV genomes and/or rep and cap genes into packaging cells.

[0143] In some embodiments, the viral vector is an adeno-associated virus vector (AAV). In some embodiments, the AAV is selected from the group consisting of AAV type 1 (AAV-1), AAV type 2 (AAV- 2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV- 5), AAV type 6 (AAV6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), and AAV type 9 (AAV9). In some embodiments, the AAV is AAV2. In some embodiments, the AAV is AAV8. In some embodiments, the AAV is Anc80. In some embodiments, the AAV is AAV44.9. In some embodiments, the AAV is AAV44.9(E531 D). [01441 Nonviral gene transfer:

[0145] Nonviral systems typically comprise all the physical and chemical systems except viral systems and generally include either chemical methods, such as cationic liposomes and polymers, or physical methods such as gene gun, electroporation, particle bombardment, ultrasound utilisation, and magnetofection.

[0146] Nonviral gene transfer has the benefit that it is typically more cost-effective, has reduced induction of the immune system and has no limitation in the size of the transgenic DNA. Nonviral DNA vectors can include a plasmid or minicircle. Nonviral RNA vectors can include a messenger RNA or circular RNA.

[0147] In some embodiments, the non- viral carrier is selected from the group consisting of nanoparticles, liposomes, cationic polymer, and calcium phosphate particles.

[0148] In some embodiments, the nucleic acid is delivered to the target cell via a non- viral delivery system. In some embodiments, the non- viral delivery system is selected from the group consisting of nanoparticles, liposomes, cationic polymer, calcium phosphate particles, gene gun, electroporation, particle bombardment, ultrasound utilisation, and magnetofection. Nanoparticles (NPs) can be used to provide plasmid DNA containing a functional copy of a gene into target tissues, for example, the retina. NPs are usually engulfed by the target cells via phagocytosis or endocytosis. Typically, nanoparticle compositions can pass through the plasma membrane, escape endosomes, and transport the plasmid DNA to the nucleus (Sahu B et al. Biomolecules 2021 , 11 , 1135).

[0149] Generally, nanoparticles wrap or adsorb DNA or RNA on the surface. Nanoparticle uptake by target cells depends on their composition and net charge. There are many different types of nanoparticle, including but not limited to, lipid-based NPs, peptide-based NPs, polymer-based NPs, and metal-NPs.

[0150] Lipidic nanoparticles are stable and biocompatible, and do not cause immune responses after administration (e.g. to the eye). Typically, lipid-based NPs are composed of a cationic lipid (having a positive charge, a hydrophilic head, and a hydrophobic tail, such as DOTAP) and a helper lipid (such as cholesterol). The positively charged head binds to a negatively charged phosphate group in the DNA to form a compact structure of lipoplexes. When DNA is enclosed in lipoplexes, it is protected from degradation. The lipid-DNA complex enters the cell by endocytosis.

[0151] Peptide-based NPs generally comprise a cationic peptide, enriched in lysine/arginine forming a tight compact structure with the DNA. Polymer-based NPs generally comprise a cationic polymer mixed with DNA to form nanosized polyplexes. Some examples of polymer-based vectors are polyethylene (PEI), dendrimers, and polyphosphoesters. Example synthetic polymers include but are not limited to Poly (L-ornithine), polyethyleneimine, and poly(amidoamine) dendrimers. Some example natural polymers include but are not limited to chitosan, dextran, and gelatin. An example of a metal NP is a gold NP (AuNP). DNA-gold nanoparticles are easy to generate and have high tolerability and low toxicity. Other nanoparticles considered are calcium-phosphorus silicate nanoparticles, calcium phosphate nanoparticles, silicon dioxide nanoparticles.

[0152] In some embodiments, the nucleic acid according to the invention is delivered to a target cell using nanoparticles. In some embodiments, the nanoparticle is a lipid-based nanoparticle. In some embodiments, the nanoparticle is a peptide-based nanoparticle. In some embodiments, the nanoparticle is a polymer-based nanoparticle. In some embodiments, the nanoparticle is a metal nanoparticle, optionally a gold nanoparticle.

[0153] The positive charge on the surface of the cationic polymer can form a positive complex with the negatively charged gene. The complex can be absorbed onto the cell surface by electrostatic action, and the gene is introduced naturally into the cell and subsequently expressed through endocytosis. Cationic polymers can be divided into polypeptides such as polylysine and poly glutamic acid, synthetic polymer material such as polyethylenimine (PEI) and polypropylene imine, and natural polymers such as chitosan, gelatin, and cyclodextrin. In some embodiments, the nucleic acid according to the invention is delivered to a target cell using a cationic polymer. In some embodiments, the cationic polymer is a polypetide polymer. In further embodiments, the polypeptide polymer is selected from the group consisting of polylysine and polyglutamic acid. In some embodiments, the cationic polymer is a synthetic polymer. In further embodiments, the synthetic polymer is selected from the group consisting of polyethylenimine (PEI) and polypropylene imine. In some embodiments, the cationic polymer is a natural polymer. In further embodiments, the natural polymer is selected from the group consisting of chitosan, gelatin, and cyclodextrin.

[0154] Also considered are calcium phosphate particles. These are biocompatible and biodegradable. Calcium plays a vital role in endocytosis and has the advantage of being readily absorbed and it poses high binding affinity. In some embodiments, the non-viral delivery system is calcium phosphate nucleotide-mediated nucleotide delivery.

[0155] Liposomes can be used for delivery of the nucleic acid of the invention into a target cell. A liposome is an artificial membrane with a thickness of 5-7 nm and a diameter of 25-500 nm. It has favourable biocompatibility and almost has no inhibition and no significant damage to normal tissues and cells such that it can exist around the target cells for a long time, enabling the target gene to be fully transfected into the target cells. Liposomes can be digested by lysosomes to release the nucleic acid in the natural mechanism, and therefore it entails a fast and convenient drug delivery, high transdermal absorption efficiency, low drug toxicity, and high stability. In some embodiments, the nucleic acid according to the invention is delivered to a target cell using liposomes. Also anticipated are nanolipsomes. Nanolipsomes are submicro bilayer lipid vesicle. Examples include but are not limited to ceramide- containing nanoliposomes and proteoliposomes.

[0156] Physical methods include but are not limited to, iontophoresis, bioballistic delivery, electrotransfection, magnetofection, sonoporation, and optoporation. Electrotransfection has been demonstrated as being particular useful for gene delivery to the eye. It is also known as electroporation or electro- permeabilization, involves applying a local and short external electric field to the cell to transiently modify the permeability of the cell membrane, facilitate the penetration of naked plasmid DNA, and promote its intracellular trafficking through electrophoresis (Bordet T et al. Drug Discovery Today, Volume 24, Number 8, August 2019). In some embodiments, the nucleic acid according to the invention is delivered to a target cell by iontophoresis, bioballistic delivery, electrotransfection, magnetofection, sonoporation, or optoporation. In some embodiments, the nucleic acid according to the invention is delivered to a target cell by electrotransfection.

[0157] In naked plasmid vector delivery, a clinical-grade plasmid DNA is prepared to transfer the gene to the tissue. Cells can be injected or electroporated with naked plasmid DNA. This method is typically considered to be safe and biocompatible. Additionally, the method is associated with a low risk of inducing immune responses. There is also no limit of the size of coding sequences. In some embodiments, the nucleic acid according to the invention is delivered to a target cell as naked DNA.

[0158] In some embodiments, the non- viral carrier is selected from the group consisting of liposomes, nanoliposomes, ccramidc-containing nanoliposomes, proteoliposomes, nanoparticlcs, calciumphosphorus silicate nanoparticles, calcium phosphate nanoparticles, silicon dioxide nanoparticles, Microparticles, poly (D-arginine), nano-dendrimers, and calcium phosphate nucleotide-mediated nucleotide delivery

Genome editing system

[0159] The agent described herein may also be a genome editing system.

[0160] In an aspect, a nucleic acid system is provided comprising one or more nucleic acids, comprising: a) a nucleic acid sequence encoding an RNA-guided endonuclease; b) a nucleic acid sequence encoding a guide RNA complementary to a target sequence associated with an insertion site in the genome of the target cell and capable of directing said RNA- guided endonuclease to said target sequence; and c) a nucleic acid sequence encoding Nfe211, for use in a method of treatment or prophylaxis of eye disorders in a subject. The nucleic acid sequence encoding Nfe211 is capable of driving expression of Nfe211 in a target cell of the subject and the nucleic acid system is suitable for directed insertion of the nucleic acid sequence encoding Nfe211 at the insertion site in the genome of the target cell.

[0161] Also provided is a system comprising: a) an RNA-guided endonuclease; b) a guide RNA complementary to a target sequence associated with an insertion site in the genome of the target cell and capable of directing said RNA-guided endonuclease to said target sequence; and c) a nucleic acid sequence encoding Nfe211, for use in a method of treatment of prophylaxis of eye disorders in a subject, where the nucleic acid sequence encoding Nfe211 is capable of driving expression of Nfe211 in a target cell of the subject and the system is suitable for directed insertion of the nucleic acid sequence encoding Nfe211 at the insertion site in the genome of the target cell.

[0162] The present invention may also use a CRISPR (“clustered regularly interspaced short palindromic repeats”) system to modulate expression of target genes.

[0163] The CRISPR or CRISPR-Cas system is derived from a prokaryotic RNA-guided defense system.

[0164] There are at least eleven different CRISPR-Cas systems, which have been grouped into three major types (1-111). Type II CRISPR-Cas systems have been adapted as a genome-engineering tool. Typically, most naturally occurring type II CRISPR-Cas systems employ three components:

• a protein endonuclease Cas (CRISPR-associated protein) having DNA nickase activity which is referred to in this specification as an RNA-guided endonuclease (or an RNA-guided DNA endonuclease),

• a “targeting” or “guide” RNA (CRISPR-RNA or crRNA) comprising a short sequence, typically of approximately 20 nucleotides, complementary to a target sequence (“protospacer”) in the genome, and

• a “scaffold” RNA (trans-acting CRISPR RNA or tracrRNA) which interacts with the crRNA and recruits the Cas endonuclease.

[0165] Typically, assembly of these components and hybridization of the crRNA with its target sequence in the chromosome results in cleavage of the chromosome by the endonuclease, at or close to the target sequence. Cleavage also requires that the target DNA contains a recognition site for the Cas enzyme (protospacer adjacent motif, or PAM) located sufficiently close to the crRNA target sequence, typically immediately adjacent the 3’ end of the target sequence. Cellular repair of the DNA break can lead to the insertion/deletion/mutation of bases and mutation at the target locus.

[0166] This three-component system has been simplified by fusing together crRNA and tracrRNA, to create a chimeric single guide RNA (sgRNA or gRNA). Hybridisation of the gRNA with the target sequence leads to cleavage of the target DNA at an adjacent/upstream PAM site. An gRNA can therefore be regarded as comprising a crRNA component (which determines the target sequence) and a tracrRNA component (which recruits the endonuclease).

[0167] The protein component of the CRISPR system is referred to as an endonuclease and may have enzymatic activity (i.e. DNA nickase activity) when associated with the appropriate RNA factors. Typically, the endonuclease will cleave chromosomal DNA. In some embodiments, the endonuclease is a Cas9 protein. Examples include Staphylococcus aureus (SaCas9), Streptococcus pyogenes (SpCas9), Neisseria meningitidis (NM Cas9), Streptococcus thermophilus (ST Cas9), Treponema denticola (TD Cas9), or variants thereof. The PAM sequences recognised by these enzymes are well known in the art. Beneficially, the new generation of SaCas9, CjCas9, and NmCas9 (2.9-3.3 kb) allows for the packaging of both Cas9 and gRNA in a single AAV vector. In some embodiments, the endonuclease is a Casl2a protein.

[0168] When using a catalytically active endonuclease, the target sequence recognised by the guide RNA may be upstream of a suitable site for insertion.

[0169] However, the endonuclease protein need not be enzymatically active. Catalytically inactive or (“dead”) endonuclease proteins may also be used in the context of the present invention, as they retain their ability to bind at the protospacer site targeted by the gRNA. A catalytically dead endonuclease may be indicated by the prefix “d”, e.g. dCas or dCasO.

[0170] The term “endonuclease” is therefore used to encompass both catalytically active and catalytically dead proteins unless the context demands otherwise.

[0171] The endonuclease may comprise a nuclear localisation sequence (NLS) effective in mammalian cells, such as the SV40 large T antigen NLS, which has the sequence PKKKRKV (SEQ ID NO: 35). Other mammalian NLS sequences are known to the skilled person. The endonuclease may comprise multiple copies of an NLS, e.g. two or three copies of an NLS. Where multiple NLS sequences are present, they are typically repeats of the same NLS.

[0172] In some embodiments, a gene encoding the endonuclease component of the nucleic acid system will be under transcriptional control of an RNA polymerase II promoter e.g. a viral or human RNA polymerase II promoter. Examples include CMV or SV40 promoter, or a mammalian “housekeeping” promoter. Genes encoding any RNA components (gRNA, crRNA or tracrRNA) will typically be under the transcriptional control of an RNA polymerase III promoter (e.g. a human RNA polymerase Uli promoter) such as the U6 or Hl promoter, or variants thereof which retain or have enhanced activity.

[0173] In some embodiments, the gene editing system described herein (e.g., a nucleic acid system or CRLSPR-based system) is used to increase expression of Nfe21L [0174] In some embodiments, it can be beneficial to employ multiple vectors and/or virions carrying different payloads. For example, for targeting integration of Nfe211 into the genome of the target cell, it may be necessary to employ one or more vectors. In one example, an AAV comprising Cas9 and the gRNA is used and a second AAV vector comprising the transgene of interest (e.g. Nfe211). In an embodiment, one or more virions may each comprise at least one of the relevant components.

[0175] The gene editing system as described herein can be used to introduce exogenous Nfe211 into the genome of a target cell. This process of introducing an exogenous gene is known as “knocking-in” or a “knock-in”. In this way exogenous Nfe211 is introduced into the target cell to increase expression.

[0176] Typically, the guide RNA directs the endonuclease (e.g. Cas9) to the target site to create a doublestrand DNA break (DSB). Cleaved ends produced by nuclease cleavage are mainly repaired by non- homologous end joining (NHEJ) or homology-directed repair (HDR). Broadly, an exogenous DNA sequence or gene can then be incorporated into the target sequencing using HDR or NHEJ. The term "homology-directed repair" or "HDR" refers to a mechanism in cells to accurately and precisely repair double-strand DNA breaks using a homologous template to guide repair. The most common form of HDR is homologous recombination (HR), a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. The term "nonhomologous end joining" or "NHEJ" refers to a pathway that repairs double-strand DNA breaks in which the break ends are directly ligated without the need for a homologous template.

[0177] In some embodiments, the nucleic acid sequence encoding Nfe211 is inserted into the genome at the insertion site through homology-directed repair. In some embodiments, the nucleic acid sequence encoding Nfc211 is flanked by 5’ homology arm and a 3’ homology arm, wherein the 5’ homology arm is homologous to a DNA sequence 5’ of the target sequence from the insertion site and the 3’ homology arm is homologous to a DNA sequence 3’ of the target sequence from the insertion site. The term “homologous nucleic acid” as used herein includes a nucleic acid sequence that is either identical or substantially similar to a known reference sequence. In one embodiment, the term “homologous nucleic acid” is used to characterize a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identical to a known reference sequence.

[0178] In some embodiments, the nucleic acid sequence encoding Nfe211 further comprises a 5’ flanking sequence comprising a target sequence and a 3’ flanking sequence comprising a target sequence. In some embodiments, the 5’ flanking sequence is 5’ of the 5’ homology arm and wherein the 3’ flanking sequence is 3’ of the 3’ homology arm. In some embodiments, the guide RNA recognizes the target sequence from the insertion site, the 5' flanking sequence, and the 3' flanking sequence. In some embodiments, the RNA- guided endonuclease cleaves the genome at the insertion site. In some embodiments, the RNA-guided endonuclease cleaves the nucleic acid comprising the nucleic acid sequence encoding Nfe211 at the 5' flanking sequence and the 3' flanking sequence.

[0179] In some embodiments, the nucleic acid comprising the nucleic acid sequence encoding Nfe211 is a plasmid. This may be called a “donor plasmid”. Typically, this produces a linear nucleic acid comprising the nucleic acid sequence encoding Nfe211. In some embodiments, the linear nucleic acid comprising the nucleic acid sequence encoding Nfe211 is inserted into the genome at the insertion site through homology- directed repair.

[0180] In alternative embodiments, the nucleic acid system further comprises at least a second a nucleic acid sequence encoding a guide RNA. In some embodiments, the second gRNA recognizes the 5' flanking sequence, and the 3’ flanking sequence only. In some embodiments, the first gRNA recognizes the target sequence from the insertion site.

[0181] In some embodiments, provided is an engineered CRLSPR-Cas vector system, comprising one or more vectors, comprising: a) a nucleic acid sequence encoding a Cas endonuclease; b) a nucleic acid sequence encoding a guide RNA complementary to a target sequence in a suitable site for insertion and capable of directing said RNA-guided endonuclease to said target sequence; and c) a nucleic acid sequence encoding Nfe211, for use in a method of treatment or prophylaxis of eye disorders in a subject, wherein the nucleic acid increases Nfe211 expression in a target cell of the subject and wherein the nucleic acid encoding Nfe211 is inserted in the genome of the target cell.

[0182] An engineered CRISPR-Cas system is also provided, where the system comprises: a) a Cas endonuclease; b) a guide RNA complementary to a target sequence in a suitable site for insertion and capable of directing said RNA-guided endonuclease to said target sequence; and c) a nucleic acid sequence encoding Nfe211, for use in a method of treatment or prophylaxis of eye disorders in a subject, wherein the nucleic acid increases Nfe211 expression in a target cell of the subject and wherein the nucleic acid encoding Nfe211 is inserted in the genome of the target cell.

[0183] In some embodiments, the suitable site for insertion is the AAVS1 site. The AAVS1 site or locus is also known as the “safe harbour” site or locus. The AAVS1 locus, in the intron of PPP1 R12C, provides a “safe harbour” locus because disruption of this site by the introduction of an exogenous gene does not have adverse effects on the cell. Moreover, this site is associated with robust transcription, maintaining expression of an exogenously inserted gene. Accordingly, the AAVS1 is a well-validated “safe harbour” for hosting exogenous genes, thus making it a suitable target site in the context of the present invention. [0184] Photoreceptors and RPE are postmitotic. Accordingly, these cells lack the homology-directed repair (HDR) mechanism (Ziccardi L., Int. J. Mol. Sci. 2019, 20, 5722). Site-specific transgene integration typically requires the HDR pathway. However, recent studies have identified methods for performing targeting integration using CRISPR systems in non-dividing cells. An example is described in Suzuki K., et al. 2016, Nature, Vol540, 144-149 and WO2018013932. The method described in Suzuki et al. employs a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells. The HITI is based on non-homologous end joining (NHEJ) and so can be carried out in non-dividing cells. The method as described in Suzuki et al. can be readily applied to the present invention. For example, a nucleic acid encoding Nfe211 can be knocked-in the genome of the subject's ocular- cells through CRISPR/Cas9-mediated homology- independent targeted integration (Suzuki 2016), which has been demonstrated to work in vivo in non- dividing cells such as RPE.

[0185] This method allows for directional insertion of exogenous DNA in non-dividing cells. This is achieved by employing a nucleic acid sequence comprising the gene of interest, flanked by two target sequences (e.g. a target sequence 5’ of the nucleic acid sequence encoding Nfe211 and a target sequence 3’ of the nucleic acid sequence encoding Nfe211). The target sequences in the nucleic acid sequence comprising the gene of interest are typically in the reverse direction. The target sequence in the genome is cleaved by the RNA-endonuclease forming a first half and second half of the sequence. The target sequences in the nucleic acid sequence comprising the gene of interest are also cleaved by the RNA- guided endonuclease forming a first half and second half of each target sequence. This forms a nucleic acid sequence, in the forward direction comprising a first half of a target sequence, a nucleic acid sequence comprising the gene of interest, and a second half of a target sequence. If this nucleic acid is correctly inserted in the genome it will form a sequence in the genome comprising a first half of the target sequence in the genome, a first half of the target sequence in the nucleic acid, a nucleic acid sequence comprising the gene of interest, a second half of the target sequence in the nucleic acid, and a second half of the target sequence in the genome. However, if the nucleic acid is incorrectly inserted in the genome it will form a sequence in the genome comprising a first half of target sequence in the genome, a second half of target sequence in the nucleic acid, a nucleic acid sequence comprising the gene of interest, a first half of the target sequence in the nucleic acid, and a second half of the target sequence in the genome. Thus, reforming the complete target sequence at each end of the gene of interest that has been incorrectly inserted (i.e. the gene of interest is present in the reverse orientation). HITI is expected to occur more frequently in the forward direction than the reverse direction as an intact guide RNA (gRNA) target sequence remains in the latter, which is subjected to additional endonuclease cutting until forward transgene insertion or insertions and deletions (indels) occur that prevent further gRNA binding. [0186] In some embodiments, the nucleic acid sequence encoding Nfe211 is flanked by a 5’ target sequence and a 3’ target sequence. In some embodiments, the 5’ target sequence and the 3’ target sequence are the same as the target sequence from an insertion site in the genome. In some embodiments, the nucleic acid sequence encoding a guide RNA is complementary to the 5' target sequence and the 3’ target sequence. In some embodiments, the nucleic acid sequence encoding a guide RNA is complementary to target sequence in the genome, the 5’ target sequence and the 3’ target sequence. In some embodiments, the target sequence is no longer present once the nucleic acid sequence encoding Nfe211 has been integrated into the genome in the correct orientation. In some embodiments, the target sequences present in the nucleic acid encoding Nfe211 are present in the opposite orientation to the target sequence from an insertion site in the genome. In some embodiments, the target sequences present in the nucleic acid encoding Nfe211 are present in the reverse direction. Typically, the target sequence in the genome is in the forward direction. In some embodiments, the first half and the second half of the target sequence have been cleaved by a nuclease and the first half and second half of the target sequence are inserted into the genome upstream and downstream of the exogenous DNA sequence. In this embodiment, there are no homology arms present in the nucleic acid comprising a nucleic acid sequence encoding Nfe211.

[0187] “Target sequences” herein are nucleic acid sequences recognized and cleaved by an endonuclease disclosed herein in a sequence specific manner. In some embodiments, the target sequence comprises a nuclease binding site. In some embodiments, the target sequence comprises a nick/cleavage site. In some embodiments, the target sequence comprises a protospacer adjacent motif (PAM) sequence. The target sequences include the target sequence in the genome, the 5’ target sequence and the 3’ target sequence.

[0188] In some embodiments, the suitable site for insertion is the AAVS1 site.

[0189] The viral delivery system described herein, or the non- viral delivery system described herein, may be used to introduce the nucleic acid systems described herein to a target cell.

[0190] CRISPR/Cas system components may be delivered to a target cell as a ribonucleoprotein (RNP) complex comprising a Cas9 protein and a gRNA (as described in Zhang et al., Theranostics 2021 , Vol. 11 , Issue 2). Thus, a system comprising an RNA-guided endonuclease, a guide RNA, and a nucleic acid encoding Nfe211 as described herein may be delivered to a target cell as a complex.

[0191] Zhang et al, Theranostics 2021 , Vol. 11 , Issue 2, describes various methods for delivering such complexes to target cells. The complex described herein may be delivered to a target cell by direct penetration, such as microinjection of a target cell or biolistics. A target cell membrane may be disrupted by electroporation. Electroporation may disrupt the target cell membrane, temporarily forming “nanopores” which the complex can transport across. Before electroporation, the complexes may be stabilised using an anionic polymer (e.g., polyglutamic acid). Alternatively, virus-like particles (VLPs) may be used to deliver the complex. For example, an RNA-guided endonuclease may be incorporated into lentriviral particles. Banskota et al., 2022 Cell 185, 250-265, describes the use of engineered-DNA-free- virus-like particles (eVLPs) that are able to package and deliver complexes, such as a Cas9 RNP, to target cells (for example in the retina).

[0192] Zhang et al., 2022 also describes the use of lipid nanoparticles to deliver the complex as described herein. The lipid nanoparticles may include cell-derived extracellular vesicles (EVs) and synthetic lipid nanoparticles. An example of a synthetic lipid nanoparticle includes, CRISPR^MAX (Thermo- Fisher) which has been described as successfully delivering complexes to the human retinal pigment epithelial cells (Yu et al, Biotechnol Lett (2016) 38:919-929). Alternatively, Zhang et al. 2022, describes the use of CPPs to enable the delivery of the complex. Also described are methods in which lipid moieties are added to the complex to increase the membrane permeability. Further described are polymers such as dendrimers, PBAEs, PEGylated PLL, and Chitosan nanoparticules for delivering complexes as described herein. The use of nanogels is also described in Zhang et al. Chen et al., Nat Nanotechnol (2019); 14: 974-80 describes the delivery of a complex in a nanogel to mouse retina/RPE in vivo. Nanoparticles could also be used to deliver such complexes (Zhang et al., 2022). For example, inorganic nanoparticles, such as gold nanoparticles, metal-organic frameworks (MOFs), graphene oxide, black phosphorous (BP) nanosheets, or calcium phosphate nanoparticles. In an example, Wang et al., J Controlled Release. 2020: 324: 194- 203, describes delivery of a complex (e.g., RNP) to mouse retina in vivo using a nanoparticle (a pH- responsive silica-metal- organic framework hybrid nanoparticle). Other methods described in Zhang et al., may also be used in the context of the present invention.

[0193] Any of the methods described herein may be used to deliver a complex as described herein (i.e., a system comprising an RNA-guided endonuclease, a guide RNA, and a nucleic acid encoding Nfe211 as described herein).

[0194] Delivery of the systems described herein as complexes (e.g., the protein/RNA/DNA complexes) may result in transient genome editing and thus reducing off-target effects, insertional mutagenesis, and immune responses. Delivery as a complex may also result in faster genome editing because it eliminates the need for intracellular transcription and translation.

Systems for increasing endogenous Nfe211 expression [0195] A nucleic acid system (e.g. CRISPR activation system) can be employed to increase endogenous Nfe211 expression. An example is a nucleic acid activation system (e.g., a CRISPR/Cas9 activation system).

[0196] Transcriptional activators are protein domains or whole proteins (which may be linked to deactivated endonuclease) that assist in the recruitment of co-factors, transcription factors and/or RNA polymerase for transcription of the target gene.

[0197] In an aspect, a nucleic acid system is provided comprising one or more nucleic acids, comprising: a) a nucleic acid sequence encoding a deactivated RNA-guided endonuclease fused to one or more transcriptional activators; and b) a nucleic acid sequence encoding a guide RNA complementary to a target sequence in the promoter or regulatory sequences for the Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the nucleic acid system increases Nfe211 expression in a target cell of the subject.

[0198] In some embodiments, the deactivated RNA-guided endonuclease is fused to a single transcriptional activator. For example, the deactivated RNA-guided endonuclease may be fused to VP64.

[0199] The one or more transcriptional activators may be joined to the N-terminus of the deactivated RNA- guided endonuclease. The one or more transcriptional activators may be joined to the C-terminus of the deactivated RNA-guided endonuclease. For example, VP64 may be fused to the C-terminus of the deactivated RNA-guided endonuclease. The VP64 may be fused to the deactivated RNA-guided endonuclease via a linker.

[0200] In some embodiments, the deactivated RNA-guided endonuclease is fused to more than one transcriptional activator. For example, the deactivated RNA-guided endonuclease may be fused to three transcriptional activators. In some embodiments, the transcriptional activators may be VP64, p65 and Rta. VP64 may be joined to the C-terminus of the deactivated RNA-guided endonuclease, p65 may be joined to the C-terminus of VP64, and Rta may be joined to the C-terminus of p65. An example of this system is the VP64-p65-Rta system, which is also known as VPR.

[0201] Also provided is a nucleic acid system comprising one or more nucleic acids, comprising: a) a nucleic acid sequence encoding a deactivated RNA-guided endonuclease; and b) a nucleic acid sequence encoding a guide RNA complementary to a target sequence in the promoter or regulatory sequences for Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, wherein said guide RNA further comprises an aptamer capable of specifically binding to a transcriptional activator, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the nucleic acid system increases Nfe211 expression in a target cell of the subject.

[0202] In some embodiments, the aptamer is an RNA aptamer. The transcriptional activator may be endogenous to the cell. Additionally, or alternatively, the transcriptional activator may be exogenous to the cell.

[0203] Also provided is a nucleic acid system comprising one or more nucleic acids, comprising: a) a nucleic acid sequence encoding a deactivated RNA-guided endonuclease; b) a nucleic acid sequence encoding an RNA binding protein fused to one or more transcriptional activators; and c) a nucleic acid sequence encoding a guide RNA complementary to a target sequence in the promoter or regulatory sequences for the Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, wherein said guide RNA further comprises an RNA aptamer capable of specifically binding to the RNA binding protein, for use in a method of treatment or prophylaxis of an eye disorder in a subject, where the nucleic acid system increases Nfe211 expression in a target cell of the subject.

[0204] The one or more transcriptional activators may be selected from the group consisting of VP64, p65 and HSF1 . In some embodiments, the p65 and HSF1 arc fused to an RNA binding protein.

[0205] The RNA binding protein may be MS2 (also known as a MS2 bacteriophage coat protein) and the RNA aptamer may be capable of binding to MS2. The RNA aptamer may be capable of binding to dimerised RNA binding proteins (such as dimerised MS2). Without wishing to be bound by theory, one or more RNA binding proteins are anticipated to bind to the RNA aptamer, thereby providing the one or more transcriptional activators at the target site (via the gRNA). For example, a MS2-p65- HSF1 complex guided by target-specific MS2-mediated gRNA is anticipated to enhance the binding of transcription factors to the promoter for Nfe2Ll. In some embodiments, the gRNA comprises a hairpin aptamer capable of binding MS2 (e.g., an MS2 fusion protein).

[0206] The tetraloop and stem-loop 2 of gRNA typically protrude outside of the Cas9-gRNA complex. It is also believed that these regions of the gRNA do not affect endonuclease activity. Thus, the tetraloop and/or the stem-loop 2 of gRNA may each be modified with RNA aptamers. In some embodiments, the RNA aptamer is a minimal hairpin aptamer. The minimal hairpin aptamer may be appended to the tetraloop and/or the stem loop 2 of gRNA. In some embodiments, the minimal hairpin aptamer specifically binds MS2. In some embodiments, the minimal hairpin aptamer specifically binds a MS2 dimer.

[0207] In some embodiments, the deactivated RNA-guided endonuclease is fused to an additional transcriptional activator. For example, the deactivated RNA-guided endonuclease may be fused to a single additional transcriptional activator. The deactivated RNA-guided endonuclease may be fused to VP64. An example nucleic acid system is the Synergistic Activation Mediator (SAM) system.

[0208] In another aspect, a nucleic acid system is provided comprising one or more nucleic acids, comprising: a) a nucleic acid sequence encoding a deactivated RNA-guided endonuclease fused to an epitope repeat array comprising one or more epitopes; b) one or more nucleic acid sequences encoding an epitope binding molecule fused to one or more transcriptional activators, wherein said epitope binding molecule is capable of specifically binding to an epitope of the epitope repeat array: and c) a nucleic acid sequence encoding a guide RNA complementary to a target sequence in the promoter or regulatory sequences for the Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the nucleic acid system increases Nfe211 expression in a target cell of the subject.

[0209] In some embodiments, the epitope binding molecule is an antibody or an antibody-like molecule. The one or more transcriptional activators may be fused to a single-chain variable fragment (scFv). In some embodiments, a VP64 is fused to a scFv.

[0210] A single transcriptional activator may be fused to an epitope binding molecule. For example, VP64 may be fused to an antibody or antibody-like molecule. In some embodiments, more than one transcriptional activator may be fused to an epitope binding molecule. In some embodiments, the transcriptional activators may be selected from the group consisting of VP64, p65 and Rta.

[0211] The epitope repeat array may be capable of binding multiple epitope binding molecules fused to one or more transcriptional activators. Thus, the system described herein is capable of amplifying the number of transcriptional activators at the target site. The epitope sequence may be unique (i.e., it is different to naturally occurring sequences in the target cell).

[0212] The epitope binding molecule may comprise a nuclear localization sequence (NLS) or endoplasmic reticulum localization signal (ERLS). The NLS can facilitate the transport of the epitope binding molecule to the nucleus of a target cell. In some embodiments, the NLS comprises an amino acid sequence comprising PKKKRKV (SEQ ID NO: 35). In other embodiments, the ERLS comprises an amino acid sequence KDEL (SEQ ID NO: 36).

[0213] An example of this system is known as a SunTag system, where GCN4 antibodies were fused to an NLS and VP64.

[0214] Also provided are the following systems. [0215] An aspect of the invention provides a system comprising: a) a deactivated RNA-guided endonuclease fused to one or more transcriptional activators; and b) a guide RNA complementary to a target sequence in the promoter or regulatory sequences for the Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the system increases Nfe211 expression in a target cell of the subject.

[0216] A fur ther aspect provides a system comprising: a) a deactivated RNA-guided endonuclease; b) an RNA binding protein fused to one or more transcriptional activators; c) a guide RNA complementary to a target sequence in the promoter or regulatory sequences for the Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, wherein said guide RNA comprises an RNA aptamer capable of specifically binding to the RNA binding protein, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the system increases Nfe211 expression in a target cell of the subject

[0217] Also provided is a system comprising: a) a deactivated RNA-guided endonuclease fused to an epitope repeat array comprising one or more epitopes; b) one or more epitope binding molecules fused to one or more transcriptional activators, wherein said epitope binding molecule is capable of specifically binding to an epitope of the epitope repeat array; and c) a guide RNA complementary to a target sequence in the promoter or regulatory sequences for the Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the system increases Nfe211 expression in a target cell of the subject.

[0218] As described above, the above systems may be delivered to a target cell as a complex (e.g., a protcin/RNA complex). In some embodiments, the RNA-guided endonuclease is a Cas endonuclease.

[0219] Described herein are activation systems. Example activation systems include but are not limited to, VP64-p65-Rta or VPR, deactivated endonuclease-SAM system, and deactivated endonuclease- SunTag system. Any of these activation systems can be used in the context of the present invention. An example CRISPR activation system is described in Konermann S. et al. Nature 2015: 517(7536), 583- 588.

[0220] In some embodiments, provided is an engineered CRISPR-Cas vector system, comprising one or more vectors, comprising: a) a nucleic acid sequence encoding a deactivated Cas endonuclease fused to one or more transcriptional activators; and b) a nucleic acid sequence encoding a guide RNA complementary to a target sequence in the promoter or regulatory sequences for the Nfe2Ll gene and capable of directing said Cas endonuclease to said target sequence, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the CRISPR-Cas vector system increases Nfe211 expression in a target cell of the subject.

[0221] In some embodiments, an engineered CRISPR-Cas vector system comprising one or more nucleic acids is provided, where the system comprises: a) a nucleic acid sequence encoding a deactivated Cas endonuclease; and b) a nucleic acid sequence encoding a guide RNA complementary to a target sequence in the promoter or regulatory sequences for Nfe2Ll gene and capable of directing said Cas endonuclease to said target sequence, wherein said guide RNA further comprises an aptamer capable of specifically binding to a transcriptional activator, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the CRISPR-Cas vector system increases Nfe211 expression in a target cell of the subject.

[0222] An embodiment provides an engineered CRISPR-Cas vector system, comprising one or more vectors, comprising: a) a nucleic acid sequence encoding a deactivated Cas endonuclease; b) a nucleic acid sequence encoding an RNA binding protein fused to one or more transcriptional activators; and c) a nucleic acid sequence encoding a guide RNA complementary to a target sequence in the promoter or regulatory sequences for the Nfe2Ll gene and capable of directing said Cas endonuclease to said target sequence, wherein said guide RNA further comprises an RNA aptamer capable of specifically binding to the RNA binding protein, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the CRISPR-Cas vector system increases Nfe211 expression in a target cell of the subject.

[0223] In another embodiment, provided is an engineered CRISPR-Cas vector system, comprising one or more vectors, comprising: a) a nucleic acid sequence encoding a deactivated Cas endonuclease fused to an epitope repeat array comprising one or more epitopes; b) one or more nucleic acid sequences encoding an epitope binding molecule fused to one or more transcriptional activators, wherein said epitope binding molecule is capable of specifically binding to an epitope of the epitope repeat array; and c) a nucleic acid sequence encoding a guide RNA complementary to a target sequence in the promoter or regulatory sequences of the Nfe2Ll gene and capable of directing said Cas endonuclease to said target sequence, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the CRISPR-Cas vector system increases Nfe211 expression in a target cell of the subject.

[0224] Another example of an agent capable of increasing endogenous Nfe211 expression in a target cell is a nucleic acid demethylation system (e.g., a CRISPR/Cas9 demethylation system).

[0225] DNA methylation is an epigenetic process which occurs by the addition of a methyl group to DNA, typically cytosine bases. In mammals, DNA methylation regulates gene expression by acting to repress gene transcription. Without wishing to be bound by theory, it is anticipated by that the use of a demethylating system would increase accessibility of the Nfe2Ll gene or its promoter/regulatory sequences, allowing transcription. Thus, another aspect provides a nucleic acid system comprising one or more nucleic acids, comprising: a) a nucleic acid sequence encoding a deactivated RNA-guided endonuclease fused to one or more DNA demethylating agents; and b) a nucleic acid sequence encoding a guide RNA complementary to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the nucleic acid system increases Nfe211 expression in a target cell of the subject.

[0226] The one or more DNA demethylating agents may be one or more DNA demethylating enzymes or a fragment thereof. For example, ten-eleven translocation methylcytosine dioxygenases (TET enzymes) mediate DNA demethylation by oxidizing 5 -methylcytosine (5mC) in DNA to 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5 -carboxylcytosine (5caC). In further embodiments, the DNA demethylating agent is the catalytic domain of TETI . In some embodiments, the DNA demethylating agent is TETI . Lysine- specific demethylase 1 (LESD1 , also known as KDM1A) is a lysine demethylase acting on histones H3K4mel/2 and H3K9mel/2. In some embodiments, the DNA demethylating agent is LESD1 .

[0227] In some embodiments, the deactivated RNA-guided endonuclease is fused to a single DNA demethylating agent. The one or more DNA demethylating agents may be fused to the C-terminus of the deactivated RNA-guided endonuclease. Alternatively, the one or more DNA demethylating agents may be fused to the N-terminus of the deactivated RNA-guided endonuclease.

[0228] Also provided is a nucleic acid system comprising one or more nucleic acids, comprising: a) a nucleic acid sequence encoding a deactivated RNA-guided endonuclease; and b) a nucleic acid sequence encoding a guide RNA complementary to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, wherein said guide RNA further comprises an aptamer capable of specifically binding to a demethylating agent, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the nucleic acid system increases Nfe211 expression in a target cell of the subject.

[0229] In some embodiments, the aptamer is an RNA aptamer. The demethylating agent may be endogenous to the cell. Additionally, or alternatively, the demethylating agent may be exogenous to the cell. Also provided is a nucleic acid system is provided comprising one or more nucleic acids, comprising: a) a nucleic acid sequence encoding a deactivated RNA-guided endonuclease; b) a nucleic acid sequence encoding an RNA binding protein fused to one or more DNA demethylating agents; c) a nucleic acid sequence encoding a guide RNA complementary to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, wherein said guide RNA further comprises an RNA aptamer capable of specifically binding to the RNA binding protein, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the nucleic acid system increases Nfe211 expression in a target cell of the subject.

[0230] The one or more DNA demethylating agents may be as described above. The RNA binding protein and/or the gRNA may be as described for the transcriptional activating system described above, with the exception that one or more DNA demethylating agents are fused to the RNA binding protein.

[0231] In some embodiments, the deactivated RNA-guided endonuclease is fused to an additional DNA demethylating agent. The additional DNA demethylating may be different to the one or more DNA demethylating agents.

[0232] In another aspect, a nucleic acid system is provided comprising one or more nucleic acids, comprising: a) a nucleic acid sequence encoding a deactivated RNA-guided endonuclease fused to an epitope repeat array comprising one or more epitopes; b) one or more nucleic acid sequences encoding an epitope binding molecule fused to one or more DNA demethylating agents, wherein said epitope binding molecule is capable of specifically binding to an epitope of the epitope repeat array: and c) a nucleic acid sequence encoding a guide RNA complementary to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the nucleic acid system increases Nfe211 expression in a target cell of the subject.

[0233] The epitope repeat array may be as described for the transcriptional activation system described above. The epitope binding molecule may be as described above, with the exception that one or more DNA demethylating agents are fused to the epitope binding molecule.

[0234] The invention also provides the following aspects. An aspect provides a system comprising: a) a deactivated RNA-guided endonuclease fused to one or more DNA demethylating agents; and b) a guide RNA complementary to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the system increases Nfe211 expression in a target cell of the subject. [0235] A further aspect provides a system comprising: a) a deactivated RNA-guided endonuclease; and b) a guide RNA complementary to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, wherein said guide RNA further comprises an aptamer capable of specifically binding to a DNA demethylating agent, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the system increases Nfe211 expression in a target cell of the subject.

[0236] Further provided is a system comprising: a) a deactivated RNA-guided endonuclease: b) an RNA binding protein fused to one or more DNA demethylating agents; c) a guide RNA complementary to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, wherein said guide RNA further comprises an RNA aptamer capable of specifically binding to the RNA binding protein, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the system increases Nfe211 expression in a target cell of the subject.

[0237] Another aspect, provides a system comprising: a) a deactivated RNA-guided endonuclease fused to an epitope repeat array comprising one or more epitopes; b) one or more epitope binding molecules fused to one or more DNA demethylating agents, wherein said epitope binding molecule is capable of specifically binding to an epitope of the epitope repeat array; and c) a guide RNA complementary to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene and capable of directing said RNA-guided endonuclease to said target sequence, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the system increases Nfe211 expression in a target cell of the subject.

[0238] As described above, the above systems may be delivered to a target cell as a complex (e.g., a protein/RNA complex). In some embodiments, the RNA-guided endonuclease is a Cas endonuclease.

[0239] A CRISPR-based approach for targeting DNA demethylation may allow for targeting epigenetic editing. For example, the demethylation system may comprise a deactivated endonuclease (e.g., a Cas9 nuclease) fused to a demethylation agent (e.g., TET1), and at least one Nfe211 -specific guide RNA. As with the CRISPR activation system, a CRISPR demethylation system uses modified versions of CRISPR effectors without endonuclease activity, with transcriptional activators on dCas or the gRNA. As with the system described above, the demethylation system comprises a deactivated endonuclease (e.g., dCas9), a gRNA and a DNA demethylating agent fused to the deactivated endonuclease or gRNA. Approaches for targeting DNA demethylation using CRISPR are described in Xu et a., 2016, Cell Discovery (2016) 2, 16009; doi: I0.1038/celldisc.20I6.9.

[0240] In some embodiments, provided is an engineered CRISPR-Cas vector system, comprising one or more vectors, comprising: a) a nucleic acid sequence encoding a deactivated Cas endonuclease fused to one or more DNA demethylating agents; and b) a nucleic acid sequence encoding a guide RNA complementary to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene, and capable of directing said Cas endonuclease to said target sequence, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the CRISPR-Cas vector system increases Nfe211 expression in a target cell of the subject.

[0241] In some embodiments, provided is an engineered CRISPR-Cas vector system comprising one or more nucleic acids, comprising: a) a nucleic acid sequence encoding a deactivated Cas endonuclease; and b) a nucleic acid sequence encoding a guide RNA complementary to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene and capable of directing said Cas endonuclease to said target sequence, wherein said guide RNA further comprises an aptamer capable of specifically binding to a DNA demethylating agent, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the CRISPR-Cas vector system increases Nfe211 expression in a target cell of the subject. An embodiment provides an engineered CRISPR-Cas vector system, comprising one or more vectors, comprising: a) a nucleic acid sequence encoding a deactivated Cas endonuclease; b) a nucleic acid sequence encoding an RNA binding protein fused to one or more DNA demethylating agents; and c) a nucleic acid sequence encoding a guide RNA complementary to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2L1 gene or (iii) a target sequence in the Nfe2Ll gene, and capable of directing said Cas endonuclease to said target sequence, wherein said guide RNA further comprises an RNA aptamer capable of specifically binding to the RNA binding protein, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the CRISPR-Cas vector system increases Nfe211 expression in a target cell of the subject.

[0242] In another embodiment, provided is an engineered CRISPR-Cas vector system, comprising one or more vectors, comprising: a) a nucleic acid sequence encoding a deactivated Cas endonuclease fused to an epitope repeat array comprising one or more epitopes; b) one or more nucleic acid sequences encoding an epitope binding molecule fused to one or more DNA demethylating agents, wherein said epitope binding molecule is capable of specifically binding to an epitope of the epitope repeat array; and c) a nucleic acid sequence encoding a guide RNA complementary to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene, and capable of directing said Cas endonuclease to said target sequence, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the CRISPR-Cas vector system increases Nfe211 expression in a target cell of the subject.

[0243] As described herein, the deactivated endonuclease is a mutant form of endonuclease where the endonuclease activity has been removed by point mutations in the endonuclease domain. Although deactivated endonuclease lacks endonuclease activity, it is still able to bind gRNAs and the target DNA. The deactivated endonuclease described herein may be a dCas. Typically, Cas9 is used, but other endonucleases can be used, for example, Cas 12a.

[0244] The viral delivery system described herein, or the non- viral delivery system described herein, may be used to introduce the nucleic acid systems described herein to a target cell.

[0245] In some embodiments, it can be beneficial to employ multiple vectors and/or virions carrying different payloads. For example, the nucleic acid sequences described above may be delivered via the same vector. Alternatively, the nucleic acid sequences may be delivered via multiple vectors. In an embodiment, one or more virions may each comprise at least one of the relevant components.

[0246] In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA.

Additional targeted approaches

[0247] An aspect of the invention provides a nucleic acid comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising:

(a) a nucleic acid binding molecule capable of binding to a target sequence in the promoter or regulatory sequences of the Nfe2Ll gene; and

(b) one or more transcriptional activators, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the fusion protein is capable of increasing Nfe211 expression in a target cell of the subject.

[0248] Also provided is a nucleic acid system comprising: a) a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises (i) a nucleic acid binding molecule capable of binding to a target sequence in the promoter or regulatory sequences of the Nfe2Ll gene and (ii) an epitope repeat array; and b) one or more nucleic acid sequences encoding an epitope binding molecule fused to one or more transcriptional activators, wherein said epitope binding molecule is capable of specifically binding to an epitope of the epitope repeat array, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the nucleic acid system is capable of increasing Nfe211 expression in a target cell of the subject.

[0249] Another aspect provides a nucleic acid comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising: a) a nucleic acid binding molecule capable of binding to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene; and b) one or more DNA demethylating agents, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the fusion protein is capable of increasing Nfe211 expression in a target cell of the subject.

[0250] Also provided is a nucleic acid system comprising: a) a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprising (i) nucleic acid binding molecule capable of binding to (1) a target sequence in the promoter sequence for the Nfe2L1 gene, (2) a target sequence in the regulatory sequences for the Nfe2Ll gene or (3) a target sequence in the Nfe2Ll gene and (ii) an epitope repeat array; and b) one or more nucleic acid sequences encoding an epitope binding molecule fused to one or more DNA demethylating agents, wherein said epitope binding molecule is capable of specifically binding to an epitope of the epitope repeat array, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the nucleic acid system is capable of increasing Nfe211 expression in a target cell of the subject.

[0251] The invention also provides the following fusion proteins.

[0252] An aspect of the invention provides a fusion protein comprising:

[0253] Another aspect provides a fusion protein comprising: a) a nucleic acid binding molecule capable of binding to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene; and b) one or more DNA demethylating agents, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the fusion protein is capable of increasing Nfe211 expression in a target cell of the subject.

[0254] The following systems are also provided. [0255] An aspect provides a system comprising: a) a fusion protein, wherein the fusion protein comprises (i) a nucleic acid binding molecule capable of binding to a target sequence in the promoter or regulatory sequences of the Nfe2Ll gene and (ii) an epitope repeat array; and b) one or more epitope binding molecules fused to one or more transcriptional activators, wherein said epitope binding molecule is capable of specifically binding to an epitope of the epitope repeat array, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the system is capable of increasing Nfe211 expression in a target cell of the subject.

[0256] Also provided is system comprising: a) a fusion protein, wherein the fusion protein comprising (i) nucleic acid binding molecule capable of binding to (1) a target sequence in the promoter sequence for the Nfe2Ll gene, (2) a target sequence in the regulatory sequences for the Nfe2Ll gene or (3) a target sequence in the Nfe2Ll gene and (ii) an epitope repeat array; and b) one or more epitope binding molecules fused to one or more DNA demethylating agents, wherein said epitope binding molecule is capable of specifically binding to an epitope of the epitope repeat array, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the system is capable of increasing Nfe211 expression in a target cell of the subject.

[0257] The fusion proteins and systems may be delivered to a target cell as a complex. The delivery methods described for the gene editing systems may also apply to the fusion proteins and systems described herein.

[0258] The fusion protein may further comprise a linker between the nucleic acid binding molecule and (i) the one or more transcriptional activators, (ii) the one or more demethylating agents, or (iii) the epitope repeat array.

[0259] In some embodiments, the nucleic acid binding molecule is a transcription activator-like (TAL) effector (also known as TALEs) repeat array. A fusion protein comprising a TAL effector repeat array and either (i) a transcriptional activator or (ii) a DNA demethylating agent can be used to increase endogenous expression of Nfe211 in a target cell.

[0260] TALEs are proteins secreted by some p- and y-proteobacteria. TALEs have a modular DNA- binding domains (DBD) consisting of repetitive sequences of residues. Each repeat region comprises around 34 amino acids. The residues at position 12 and 13 determine the nucleotide specificity and are known as the Repeat Variable Diresidue (RVD). The RVD is highly variable and shows a strong correlation with specific nucleotide recognition.

[0261] TAL effector repeat domains can be engineered to each bind to one nucleotide of DNA with the specificity determined by the identities of the two hypervariable residues. To construct a protein capable of recognizing a specific DNA sequence, repeats with different specificities are simply joined together into a TAL effector repeat array. Accordingly, the TAL effector repeat array can be used to bind to target sequences in Nfe2Ll gene or the promoter/regulatory sequence(s) for the Nfe2Ll gene.

[0262] The TAL effector repeat array may be fused to either (i) a transcriptional activator or (ii) a DNA demethylating agent. Alternatively, the TAL effector repeat array may be fused to an epitope repeat array as described above.

[0263] In some embodiments, provided is a nucleic acid comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising: a) a TAL effector repeat array capable of binding to a target sequence in the promoter or regulatory sequences of the Nfe2Ll gene; and b) one or more transcriptional activators, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the fusion protein is capable of increasing Nfe211 expression in a target cell of the subject.

[0264] In some embodiments, provided is a fusion protein comprising: a) a TAL effector repeat array capable of binding to a target sequence in the promoter or regulatory sequences of the Nfe2Ll gene; and b) one or more transcriptional activators, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the fusion protein is capable of increasing Nfc211 expression in a target cell of the subject.

[0265] In some embodiments, provided is a nucleic acid comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising: a) a TAL effector repeat array capable of binding to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene; and b) one or more DNA demethylating agents, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the fusion protein is capable of increasing Nfe211 expression in a target cell of the subject.

[0266] In some embodiments, provided is a fusion protein comprising: a) a TAL effector repeat array capable of binding to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene; and b) one or more DNA demethylating agents, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the fusion protein is capable of increasing Nfe211 expression in a target cell of the subject.

[0267] In some embodiments, the nucleic acid binding molecule is a zinc finger array. A fusion protein comprising a zinc finger (ZNF) array and either (i) a transcriptional activator or (ii) a DNA demethylating agent may be used to increase endogenous Nfe211 expression. [0268] Zinc-finger motifs are maintained by a zinc ion, which coordinates cysteine and histidine in different combinations allowing ZNFs to have the ability to interact with DNA and/or RNA. The ZNFs can be engineered to alter the DNA-binding specificity of the zinc-fingers. Tandem repeats of the zinc- finger domains (and/or engineered zinc-finger domains) can be used to target specific DNA (or RNA) sequence. Engineered zinc finger arrays may have between 3 and 6 individual zinc finger motifs and are capable of binding target sites ranging from 9 base pairs to 18 base pairs in length. Arrays with at least 6 zinc finger motifs may be preferred because they are capable of binding longer a target sequences, which increases specificity. The zinc finger array may be fused to either (i) a transcriptional activator or (ii) a DNA demethylating agent. Alternatively, the zinc finger array may be fused to an epitope repeat array as described above. In some embodiments, the zinc finger array comprises at least 3 zinc finger motifs. In some embodiments, the zinc finger array comprises at least 6 zinc finger motifs. The zinc finger array may be capable of binding to target sequences in Nfe2Ll gene or the promoter/regulatory sequence(s) for the Nfe2Ll gene.

[0269] In some embodiments, provided is a nucleic acid comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising: a) a zinc finger array capable of binding to a target sequence in the promoter or regulatory sequences of the Nfe2Ll gene; and b) one or more transcriptional activators, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the fusion protein is capable of increasing Nfe211 expression in a target cell of the subject.

[0270] In some embodiments, provided is a fusion protein comprising: a) a zinc finger array capable of binding to a target sequence in the promoter or regulatory sequences of the Nfe2Ll gene; and b) one or more transcriptional activators, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the fusion protein is capable of increasing Nfe211 expression in a target cell of the subject.

[0271] In some embodiments, provided is a nucleic acid comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising: a) a zinc finger array capable of binding to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene; and b) one or more DNA demethylating agents, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the fusion protein is capable of increasing Nfe211 expression in a target cell of the subject.

[0272] In some embodiments, provided is a fusion protein comprising: a) a zinc finger array capable of binding to (i) a target sequence in the promoter sequence for the Nfe2Ll gene, (ii) a target sequence in the regulatory sequences for the Nfe2Ll gene or (iii) a target sequence in the Nfe2Ll gene; and b) one or more DNA demethylating agents, for use in a method of treatment or prophylaxis of eye disorders in a subject, where the fusion protein is capable of increasing Nfe211 expression in a target cell of the subject. The transcriptional activator may be any of the transcriptional activators as described herein. Similarly, the DNA demethylating agent may any of the DNA demethylating agents as described herein.

[0273] In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA.

[0274] In some embodiments, the epitope binding molecule is an antibody or antibody-like molecule.

[0275] The viral delivery system described herein, or the non- viral delivery system described herein, may be used to introduce the nucleic acids encoding a fusion protein described herein to a target cell.

Small molecule agents

[0276] Small molecule and peptide agents may be used to increase endogenous expression of Nfe211 in target cells.

[0277] In this specification, the term small molecule refers to a low molecular weight organic compound. Small molecules are able to bind specific biological macromolecules and act as an effector, altering the activity or function of the target. Due to their small size, small molecules may have the benefit of being able to pass across cell membranes to reach targets in the cell.

[0278] An aspect of the invention provides a small molecule for use in a method of treatment or prophylaxis of eye disorders in a subject, where the small molecule increases endogenous Nfe211 expression in a target cell of the subject.

[0279] Small molecules may be used to reduce DNA methylation in the promoter sequence for the Nfe2Ll gene or the Nfe2Ll gene itself, thereby increasing accessibility of the Nfe2Ll gene or its promoter. Thus, in some embodiments, the small molecule reduces DNA methylation in the promoter sequence for the Nfe2Ll gene. In some embodiments, the small molecule reduces DNA methylation in the Nfe2Ll gene.

[0280] Example of small molecules that are capable of reducing DNA methylation in the promoter sequence for the Nfe2Ll gene or the Nfe2Ll gene include EPZ-6438 and azacytidine. Geng et al., 2020, Communications Biology, 3:306 describes the use of EPZ-6438 and azacytidine to induce Nfe2l1 expression in target cells by increasing Nfe211 transcripts. In some embodiments, the small molecule is EPZ-6438. In some embodiments, the small molecule is azacytidinc.

[0281] The small molecule may increase Nfe211 expression by recruiting one or more polypeptides that promote transcription to promoter for Nfe2Ll or stimulating release of Nfe211 from ER and its nuclear translocation.

[0282] The small molecule may increase Nfe211 expression by modifying degradation rate of Nfe2Ll RNA transcripts. Nucleic acid agents

[0283] A further aspect provides a nucleic acid for use in a method of treatment or prophylaxis of eye disorders in a subject, where the nucleic acid increases endogenous Nfe211 expression in a target cell of a subject.

[0284] The nucleic acid may inhibit expression of METTL3. The nucleic acid may be capable of binding to METTL3 mRNA. In some embodiments, the nucleic acid is capable of hybridizing to a target sequence in METTL3 mRNA. The nucleic acid may comprise a nucleic acid sequence which is at least partially complementary to a sequence in METTL3 mRNA. The nucleic acid may downregulate METTL3 expression, thereby increasing Nfe211 expression. In some embodiments, the nucleic acid may be an inhibitory nucleic acid, such as antisense or small interfering RNA, including but not limited to shRNAor siRNA. In some embodiments, the nucleic acid is selected from the group consisting of an siRNA, an shRNA, a miRNA, and an ASO.

[0285] " Short or small interfering RNAs" (siRNAs) or microRNAs" (miRNAs) depending on their origin may be used to down-regulate gene expression by binding to complementary RNAs and either triggering mRNA elimination (RNAi) or arresting mRNA translation into protein. siRNA arc derived by processing of long double stranded RNAs and when found in nature are typically of exogenous origin. Microinterfering RNAs (miRNA) are endogenously encoded small non-coding RNAs, derived by processing of short hairpins. Both siRNA and miRNA can inhibit the translation of mRNAs bearing partially complimentary target sequences without RNA cleavage and degrade mRNAs bearing fully complementary sequences.

[0286] An antisense oligonucleotide (ASO) is an oligonucleotide, preferably single stranded, that targets and binds, by complementary sequence binding, to a target oligonucleotide, e.g., mRNA. Where the target oligonucleotide is an mRNA, binding of the antisense to the mRNA blocks translation of the mRNA and expression of the gene product. Antisense oligonucleotides may be designed to bind sense genomic nucleic acid and inhibit transcription or promote degradation of a target nucleotide sequence.

[0287] Another alternative is the expression of a short hairpin RNA molecule (shRNA) in the cell. shRNAs are more stable than synthetic siRNAs. A shRNA consists of short, inverted repeats separated by a small loop sequence. One inverted repeat is complimentary to the gene target. In the cell the shRNA is processed by DICER into a siRNA which degrades the target gene mRNA and suppresses expression. In an embodiment, the shRNA is produced endogenously (within a cell) by transcription from a vector. shRNAs may be produced within a cell by transfecting the cell with a vector encoding the shRNA sequence under control of an RNA polymerase III promoter such as the human Hl or 7SK promoter or a RNA polymerase II promoter. Alternatively, the shRNA may be synthesised exogenously (in vitro) by transcription from a vector. The shRNA may then be introduced directly into the cell.

Peptide and polypeptide agents

[0288] The agent may be a peptide or polypeptide. The term "peptide" is used herein to refer to short chains of amino acids consisting of 40 or fewer amino acids linked by peptide bonds. The term "polypeptide" is used herein to refer to large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues, each being more than 40 amino acids in length. A peptide or polypeptide may be used to increase endogenous expression of Nfe211 in target cells.

[0289] Accordingly, another aspect provides a peptide or polypeptide for use in a method of treatment or prophylaxis of eye disorders in a subject, where the peptide or polypeptide increases endogenous Nfe211 expression in a target cell of the subject. The agent may be polypeptide.

[0290] The small molecules, peptides, and polypeptides as described herein can be introduced to the target cell using any of the delivery methods described herein.

Augmenting Nfe211 activity

[0291] Some agents according to the invention may augment Nfe211 activity.

[0292] An aspect provides a small molecule for use in a method of treatment or prophylaxis of eye disorders in a subject, where the small molecule increases Nfe211 activity in a target cell of the subject.

[0293] Another aspect provides a peptide or polypeptide for use in a method of treatment or prophylaxis of eye disorders in a subject, where the peptide or polypeptide increases Nfe211 activity in a target cell of a subject. The agent may be polypeptide.

[0294] The small molecules, peptides, and polypeptides as described herein can be introduced to the target cell using any of the delivery methods described herein.

Additional therapeutic agents

[0295] Additional therapeutic agents may also be used in the treatment or prophylaxis of eye disorders in a subject alongside or in combination with the agents described elsewhere in this specification. These additional therapeutic agents may target other signalling pathways or processes involved in eye disorders. Thus, the medical uses described herein may further comprise the administration of one or more additional therapeutic agents to a subject.

[0296] For example, complement activation has been strongly implicated in AMD risk and pathogenesis, particularly dry AMD risk and pathogenesis. Accordingly, the additional therapeutic agent may be an inhibitor of the complement system, such as a regulator, e.g., complement factor H (CFH) or complement factor I (CFI). The additional therapeutic agent may be a biologic that inhibits Clq, C3, C5, complement factor B (CFB), or complement factor D (CFD). Example C3 inhibitors include Pegcetacoplan (Apellis) and NGM621 (NGM Bio). An example C5 inhibitor is Avacincaptad pegol (IVERIC Bio). An example CFD inhibitor is Lampalizumab (Novartis). Gene therapy may also be used. For example, GT005 (Gyroscope), which is a CFI gene therapy. Some patients with AMD have been shown to have less CD59 present in the retina to protect cells from damage as a result of complement. Thus, in some embodiments, the additional therapeutic agent increases a soluble form of CD59 (sCD59) in target cells. An example is HMR59 (Hemera/J&J), which is a sCD59 gene therapy.

[0297] The additional therapeutic agent may be an signaling Nfe211 stimulator , which can promote Nfe211 activation and translocation to the nucleus, for example mTOR activation .

[0298] The additional therapeutic agent may be a pigment epithelium-derived factor (PEDF), one of the serpin superfamily proteins and neuroprotective factors, which has been found to be significantly reduced in expression level in Bruch’s membrane and RPE in patients with AMD (Bhutto et al. Exp Eye Res. 2006 Jan;82(l):99-110. doi: 10.1016/j.exer.2005.05.007) and diabetic retinopathy (DR) (Ogata et al., 2002, American Journal of Ophthalmology, Vol. 134(3): 348-353).

[0299] The additional therapeutic agent may be a small molecule, a peptide, a polypeptide, an antibody or antibody-like fragment, or a nucleic acid (e.g., an shRNA).

[0300] In this specification “antibody” includes a fragment or derivative of an antibody, a synthetic antibody, or a synthetic antibody fragment. In view of today's techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens. The antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]). Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications", J G R Hurrell (CRC Press, 1982). Chimeric antibodies are discussed by Neuberger et al (1988, 8th International RICMP7164916 Biotechnology Symposium Part 2, 792-799).

[0301] The additional therapeutic agent may be administered at the same time as an agent for increasing Nfe211 expression and/or increasing Nfe211 activity as described herein. For example, a composition comprising (i) the agent for increasing Nfe211 expression and/or increasing Nfe211 activity and (ii) the additional therapeutic agent may be administered to a subject. [0302] The additional therapeutic agent (e.g., the peptide, polypeptide, antibody or antibody-like fragment, or RNA molecule as described herein) may be encoded by a nucleic acid sequence.

[0303] Where the agent for increasing Nfe211 expression and/or increasing Nfe211 activity is a nucleic acid, the nucleic acid may further comprise a nucleic acid sequence encoding the additional therapeutic agent. The nucleic acid may be capable of driving expression of the additional therapeutic agent. The nucleic acid comprising at least two nucleic acid sequences (e.g., one encoding Nfe211 and the other encoding the additional therapeutic agent) may comprise a separate promoter for each nucleic acid sequence.

[0304] Alternatively, the nucleic acid sequence encoding the therapeutic agent may be delivered to a subject via a separate nucleic acid to the nucleic acid comprising a nucleic acid sequence encoding the agent for increasing Nfe211 expression and/or increasing Nfe211 activity (i.e., two different nucleic acids).

[0305] The nucleic acids may be delivered to a target cell via viral delivery systems or non-viral delivery systems.

Pharmaceutical composition and routes of administration

[0306] The agents and additional therapeutic agents described herein can be formulated in pharmaceutical compositions.

[0307] Methods for administering gene therapy vectors are well known to the skilled person. Nfe211 expression vectors may be introduced systemically (e.g., intravenously or by infusion). Nfe211 expression vectors may be introduced locally (i.e., directly to a particular tissue or organ, e.g., liver). Nfe211 expression vectors may be introduced directly into the eye (e.g., by ocular injection). For recent reviews see, e.g., Dinculescu et aL, 2005, "Adeno-associated virus-vectored gene therapy for retinal disease" Hum Gene Ther. 16:649-63; Rex et al., 2004, "Adenovirus-mediated delivery of catalase to retinal pigment epithelial cells protects neighbouring photoreceptors from photo-oxidative stress" Hum Gene Ther.

15:960-7; Bennett, 2004, "Gene therapy for Leber congenital amaurosis" Novartis Found Symp. 255: 195- 202; Hauswirth et al., "Range of retinal diseases potentially treatable by AAV-vectored gene therapy" Novartis Found Symp. 255:179-188, and references cited therein.

[0308] Administration may be peripheral, e.g. intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal. Typically, though, in the context of the invention, administration to a subject may be intraocular. In some embodiments, administration to a subject may be intravitreal, subretinal, suprachoroidal, or periocular. In some embodiments, administration is by injection or infusion. In some embodiments, administration is by subretinal injection. In some embodiments, administration is topical.

In other embodiments, administration by electroporation. [0309] Typically, the retina can be accessed via three distinct routes: intravitreal, subretinal, and suprachoroidal. The subretinal injection is typically an invasive surgical procedure in which the therapeutic composition is delivered between the photoreceptors and the RPE. This vitro-retinal technique can require an operating room and is usually performed under general anaesthesia. Intravitreal injections (Ms) on the other hand, do not need to be performed in an operating room. Suprachoroidal injections are less invasive than subretinal injection and involve accessing the retina by injecting into the space between the choroid (overlaying the RPE) and the sclera (Sahu B et al. Biomolecules 2021 , 11 , 1135).

[0310] Administration is preferably in a “prophylactically effective amount” or a “therapeutically effective amount”, this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, may depend on the individual subject and the nature and severity of their condition.

[0311] Pharmaceutical compositions may comprise, in additional to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other material well known to those skilled in the art. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient.

[0312] The nucleic acid-containing compositions of the invention can be stored and administered in a sterile physiologically acceptable carrier, where the nucleic acid is dispersed in conjunction with any agents which aid in the introduction of the DNA into cells.

[0313] Various sterile solutions may be used for administration of the composition, including water, PBS, ethanol, lipids, etc. The concentration of the DNA will be sufficient to provide a therapeutic dose, which will depend on the efficiency of transport into the cells.

[0314] Gene therapy vectors must be produced in compliance with the Good Manufacturing Practice (GMP) requirements rendering the product suitable for administration to patients. Disclosed herein are gene therapy vectors suitable for administration to patients including gene therapy vectors that are produced and tested in compliance with the GMP requirements. Gene therapy vectors subject to regulatory approval must be tested for potency and identity, be sterile, be free of extraneous material, and all ingredients in a product (i.e., preservatives, diluents, adjuvants, and the like) must meet standards of purity, quality, and not be deleterious to the patient. For example, the nucleic acid preparation is demonstrated to be mycoplasma-free. See, e.g., Islam et al., 1997, An academic centre for gene therapy research and clinical grade manufacturing capability, Ann Med 29, 579-583.

[0315] Pharmaceutical compositions may be prepared using a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective. The term “carrier” refers to diluents, binders, lubricants and disin tegrants. Those with skill in the art are familiar with such pharmaceutical carriers and methods of compounding pharmaceutical compositions using such carriers.

"Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset and the like, when administered to a human. In some embodiments, this term refers to molecular entities and compositions approved by a regulatory agency of the US federal or a state government, as the GRAS list under section 204(s) and 409 of the Federal Food, Drug and Cosmetic Act, that is subject to premarket review and approval by the FDA or similar lists, the U.S. Pharmacopeia or another generally recognised pharmacopeia for use in animals, and more particularly in humans. The pharmaceutical compositions provided herein may include one or more excipients, e.g., solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, antioxidants or antimicrobial preservatives. When used, the excipients of the compositions will not adversely affect the stability, bioavailability, safety, and/or efficacy of the active ingredients. Thus, the skilled person will appreciate that compositions are provided wherein there is no incompatibility between any of the components of the dosage form. Excipients may be selected from the group consisting of buffering agents, solubilizing agents, tonicity agents, chelating agents, antioxidants, antimicrobial agents, and preservatives.

[0316] A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0317] The terms “treatment”, “treat”, or “treating” are used herein to refer to the reduction in severity of a disease or condition, the reduction in the duration of a disease: the amelioration or elimination of one or more symptoms associated with a disease or condition, or the provision of beneficial effect to a subject with a disease or condition. The term also encompasses prophylaxis of a disease or condition or its symptoms thereof. “Prophylaxis” is known in the art to mean decreasing or reducing the occurrence or severity of a particular disease outcome. For example, delaying progression of cancer in a subject.

[0318] As used herein, the term “subject” refers to a human or any non-human animal (e g, mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” In some embodiments, the subject is human. A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder. In some embodiments, the subject is affected or is likely to be affected with a retinal disease, in particular eye disorders. [0319] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilized for realizing the invention in diverse forms thereof.

[0320] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0321] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0322] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0323] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example +/- 10%.

Sequences

Human DNA sequence:

Full-length: [2319 nucleotides, including starting methionine and stop codons]

Critical coding regulatory elements (bolded) essential for its transcriptional function:

[2016 nucleotides, including stop codon] Nfe2ll (SEQ ID NO: 1) atgctttctctgaagaaatacttaacggaaggacttctccagttcaccattctgctgagtttgattggggtacgggtggacgtggatacttacctgacctca cagcttcccccactccgggagatcatcctggggcccagttctgcctatactcagacccagttccacaacctgaggaataccttggatggctatggtatcca ccccaagagcatagacctggacaattacttcactgcccggcggctcctcagtcaggtgagggccctggacaggttccaggtgccaaccactgaggtaa atgcctggctggttcaccgagacccagaggggtctgtctctggcagtcagcccaactcaggcctcgccctcgagagttccagtggcctccaagatgtga caggcccagacaacggggtgcgagaaagcgaaacggagcagggattcggtgaagatttggaggatttgggggctgtagcccccccagtcagtgga gacttaaccaaagaggacatagatctgattgacatcctttggcgacaggatattgatctgggggctgggcgtgaggtttttgactatagtcaccgccag aaggagcaggatgtggagaaggagctgcgagatggaggcgagcaggacacctgggcaggcgagggcgcggaagctctggcacggaacctgcta gtggatggagagactggggagagcttccctgcacaggtgcctagtggggaggaccagacggccctgtccctggaagagtgccttaggctgctggaa gccacctgcccctttggggagaatgctgagtttccagcagacatttccagcataacagaagcagtgcctagtgagagtgagccccctgctcttcaaaa caacctcttgtctcctcttctgaccgggacagagtcaccatttgatttggaacagcagtggcaagatctcatgtccatcatggaaatgcaggccatgga agtgaacacatcagcaagtgaaatcctgtacagtgcccctcctggagacccactgagcaccaactacagccttgcccccaacactcccatcaatcag aatgtcagcctgcatcaggcgtccctggggggctgcagccaggacttcttactcttcagccccgaggtggaaagcctgcctgtggccagtagctccac gctgctcccgttggcccccagcaattctaccagcctcaactccaccttcggctccaccaacctgacagggctcttctttccaccccagctcaatggcaca gccaatgacacagcaggcccagagctgcctgaccctttggggggtctgttagatgaagctatgttggatgagatcagccttatggacctggccattga agaaggctttaaccctgtgcaggcctcccagctggaggaggaatttgactctgactcaggcctttccttagactcgagccatagcccttcttccctaagc agctctgaaggcagttcttcctcttcttcctcctcctcttcctcttcttcctctgcttcttcctctgcctcttcctccttttctgaggaaggtgcggttggctaca gctctgactctgagaccctggatctggaagaggccgagggtgctgtgggctaccagcctgagtattccaagttctgccgcatgagctaccaggatcca gctcagctctcatgcctgccctacctggagcacgtgggccacaaccacacatacaacatggcacccagtgccctggactcagccgacctgccaccacc cagtgccctcaagaaaggcagcaaggagaagcaggctgacttcctggacaagcagatgagccgggatgagcaccgagcccgagccatgaagatcc ctttcaccaatgacaaaatcatcaacctgcctgtggaggagttcaatgaactgctgtccaaataccagttgagtgaagcccagctgagcctcatccga gacatccggcgccggggcaagaacaagatggcggcgcagaactgccgcaagcgcaagctggacaccatcctgaatctggagcgtgatgtggagga cctgcagcgtgacaaagcccggctgctgcgggagaaagtggagttcctgcgctccctgcgacagatgaagcagaaggtccagagcctgtaccagga ggtgtttgggcggctgcgagatgagaacggacgaccctactcgcccagtcagtatgcgctccagtacgccggggacggcagtgtcctcctcatccccc gcacgatggccgaccagcaggcccggcggcaggagaggaagccaaaggaccggagaaagtga

SEQ ID NO: 2 Human protein sequence (whole sequence, 772 amino acids, including starting methionine):

MLSLKKYLTEGLLQFTILLSLIGVRVDVDTYLTSQLPPLREIILGPSSAYTQTQFHNLRNTLDGYGIHPKSIDLDNYFTARRLLS

QVRALDRFQVPTTEVNAWLVHRDPEGSVSGSQPNSGLALESSSGLQDVTGPDNGVRESETEQGFGEDLEDLGAVAPPV SGDLTKEDIDUDILWRQDIDLGAGREVFDYSHRQKEQDVEKELRDGGEQDTWAGEGAEALARNLLVDGETGESFPAQV PSGEDQTALSLEECLRLLEATCPFGENAEFPADISSITEAVPSESEPPALQNNLLSPLLTGTESPFDLEQQWQDLMSIMEM

QAMEVNTSASEILYSAPPGDPLSTNYSLAPNTPINQNVSLHQASLGGCSQDFLLFSPEVESLPVASSSTLLPLAPSNSTSLN

STFGSTNLTGLFFPPQLNGTANDTAGPELPDPLGGLLDEAMLDEISLMDLAIEEGFNPVQASQLEEEFDSDSGLSLDSSHS PSSLSSSEGSSSSSSSSSSSSSSASSSASSSFSEEGAVGYSSDSETLDLEEAEGAVGYQPEYSKFCRMSYQDPAQLSCLPYLEH

VGHNHTYNMAPSALDSADLPPPSALKKGSKEKQADFLDKQMSRDEHRARAMKIPFTNDKIINLPVEEFNELLSKYQLSE AQLSLIRDIRRRGKNKMAAQNCRKRKLDTILNLERDVEDLQRDKARLLREKVEFLRSLRQMKQKVQSLYQEVFGRLRDE

NGRPYSPSQYALQYAGDGSVLLIPRTMADQQARRQERKPKDRRK

Mouse DNA sequence (Nfe2ll):

SEQ ID NO: 3: Full-length: [2226 nucleotides, including starting methionine and stop codons] Critical coding regulatory elements (bolded essential for its transcriptional funchon: [1923 nucleotides, including stop codon] atgctttctctgaagaaatatttaacggaaggacttctccagttcaccatcctgctgagtctgattggggttcgggtggacgtggatacttacctgacctca cagctcccccctctccgggagatcatcctggggcccagctctgcctatacccagacccagttccacaacctgaggaataccttggatggctatgggatcc accccaagagcatagacctggacaattacttcactgcccggcggctccttagtcaggtgagggccctggataggttccaggtgcctaccactgaggtca atgcttggctggtccaccgagacccggaggggtctgtctctggcagccagcccaactcaggcctcgccctcgagagttccagtggcctccaagatgtg acaggcccagacaacggggtgagagaaagcgaaacggagcagggattcggtgaagatttggaggacctgggggctgtagcccctcctgtcagtgg agacttaaccaaagaggatatagatctgattgacatcctttggcgacaggatattgatctgggggctgggcgtgaggtttttgactacagtcatcgcca gaaggagcaggatgtggataaggaactgcaagatggacgagaacgagaggacacctggtcaggcgagggtgcggaagctctggcccgagacctg ctagtagatggagagactggggagagcttccctgcacagtcccagctgacgtttccagcatcccagaagcagtgcctagtgagagtgagtcccccgc ccttcagaacagccttctatctcctcttctgacggggacagaatcaccatttgatttggaacagcagtggcaagatctcatgtccatcatggaaatgcag gctatggaagtaaatacatcagcaagtgagattctgtacaatgcccctcctggagaccctcttagcaccaactacagccttgcacccaacactcccatc aatcagaatgtcagcctgcatcaggcgtccctggggggctgcagtcaggacttctccctcttcagccccgaggtggagagcctgcctgtggctagcag ctccacactgcttccactcgtccccagcaactccaccagtctcaactccaccttggctctaccaacctagcagggctttctttccatcccagctcaatgg cacagccaatgacacatcaggccctgagctacctgacccccttgggggcctgttagacgaagctatgctggatgagatcagcctgatggacctggcc attgaggagggcttcaacccggtgcaggcttcccagctcgaagaggagtttgactctgactcaggcctctccttggactccagccatagcccttcctctc tgagcagctctgaagggagctcttcttcttcctcctcctcctcttcctcttctgcttcctcctctgcctcttcttccttctctgaggagggtgctgttggttacag ctctgactctgagaccctagacctagaagaggctgagggtgcagtgggctaccagccggaatactccaagtctgccgcatgagctatcaggatcctt ctcagctctcttgcctccctacttagagcatgtgggccacaatcatacatacaatatggcacccagtgcccttgactctgctgatctaccaccacccag caccctcaagaaaggtagcaaggaaaagcaggctgacttcctggacaagcagatgagccgagatgagcacagagcccgagccatgaagatcccat tcaccaatgacaagatcatcaacctgcctgtagaagaattcaatgagctgctgtccaaataccagctgagcgaggcccagctcagcctcatccgggat atccggcgccggggcaaaaacaagatggctgcacagaactgccgcaagcgcaagttggacaccatcctaaacctagaacgtgatgtggaggactt gcagcgagataaggcccgattgcttcgagaaaaggtagagttccttcggtctctgcgacagatgaagcagaaggtccaaagcttataccaggaggtg ttgggcggctgcgggatgagcatgggaggccctactcacccagtcagtatgcccttcagtatgctggggatggcagtgtcctcctcattcctcgcacg atggctgaccagcaggctcggcgacaggagagaaagccaaaggaccggaggaagtga

SEQ ID NO: 4 Mouse protein sequence (whole sequence, 741 amino acids, including starting methionine): M LSLKKYLTEGLLQFTILLSLIGVRVDVDTYLTSQLPPLREIILGPSSAYTQTQFHNLRNTLDGYGIHPKSIDLDNYFTARRLLS QVRALDRFQVPTTEVNAWLVHRDPEGSVSGSQPNSGLALESSSGLQDVTGPDNGVRESETEQGFGEDLEDLGAVAPPV SGDLTKEDIDLIDILWRQDIDLGAGREVFDYSHRQKEQDVDKELQDGREREDTWSGEGAEALARDLLVDGETGESFPAQ FPADVSSIPEAVPSESESPALQNSLLSPLLTGTESPFDLEQQWQDLMSIMEMQAMEVNTSASEILYNAPPGDPLSTNYSLA PNTPINQNVSLHQASLGGCSQDFSLFSPEVESLPVASSSTLLPLVPSNSTSLNSTFGSTNLAGLFFPSQLNGTANDTSGPEL PDPLGGLLDEAMLDEISLMDLAIEEGFNPVQASQLEEEFDSDSGLSLDSSHSPSSLSSSEGSSSSSSSSSSSSASSSASSSFSE EGAVGYSSDSETLDLEEAEGAVGYQPEYSKFCRMSYQDPSQLSCLPYLEHVGHNHTYNMAPSALDSADLPPPSTLKKGS KEKQADFLDKQMSRDEHRARAM KIPFTNDKIINLPVEEFNELLSKYQLSEAQLSLIRDIRRRGKNKMAAQNCRKRKLDTIL NLERDVEDLQRDKARLLREKVEFLRSLRQMKQKVQSLYQEVFGRLRDEHGRPYSPSQYALQYAGDGSVLLIPRTMADQQ ARRQERKPKDRRK

EXAMPLES

[0324] Materials and Methods related to Examples below

[0325] Animals and Animal Procedures. Transgenic mice overexpressing Nfc211 (Nfc211^0E, MGI:5804124) were recovered frozen sperm (RBRC10149) purchased from RIKEN BioResource Research Center (Kyoto, Japan) and were previously described in (30). Transgenic mice expressing Ub^G76v-GFP and Gyr^/_ mice were previously described in (58, 59). The Bho^{F>2 ill/F}'^{2 i}" mice were purchased from Jackson Labs (stock # 005105). Mice with floxed (Nfe211^fl/fl) fifth exon of Nfe211 gene (NM_008686.3) were generated at Cyagen (Santa Clara, CA, USA) using previously published genetic strategy (45). ChxlO-Crc mice expressing Crc in retina were previously described in (31). Tsc2^{Rod K0} mice were derived by crossing Tsc2^fl/fl (Jackson Labs, stock # 027458) and iCre75 (Jackson Labs, stock # 015850) mouse lines and were previously described in (24, 60). Rhodopsin knockout mice were previously described in (67). Breeding schemes for all mouse lines and littermates used in experiments are indicated in Table S2. Animals were reared under a normal day/night cycle and handled according to the protocols approved by the Institutional Animal Care and Use Committee of the University of Florida (#202009934). Littermates of both sexes were used and processed as a group. Mouse genotypes were determined using real-time PCR with specific probes designed for each gene (Transnetyx, Memphis, TN, USA). All lines were tested negative for rdl and rd8 mutations. All experiments were performed using littermate controls. Noninvasive experiments with animals (ERG and OCT) were performed as previously described (24).

[0326] Western blotting, proteasome activity assays, polyubiquitin- and rhodopsin- enrichment assays. Western blotting was performed using previously described protocols with antibodies listed in Table S3 (24). Retina or liver tissue was sonicated in RIPA lysis buffer (20-188, EMD Millipore, Burlington, MA, USA), supplemented with a Halt™ Protease or Halt™ Protease and Phosphatase (78429 and 78440, Thermo Fisher Scientific, Waltham, MA, USA) inhibitor cocktails. The samples used for western blotting of polyubiquitinated chains were prepared in the presence of 5pM PR-619 (Life Sensors, Malvern, PA, USA) inhibitor of deubiquitinases/deubiquitylases/ubiquitin-like isopeptidases. The total protein concentration was measured using the Pierce 660 nm Protein Assay Reagent (22660, Thermo Fisher Scientific). Samples were brought to the same concentration in Laemmli Buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 20mM DTT, 10% Glycerol, 0.01% Bromophenol blue), heated at 95 °C for 5 min, and immediately used for experiments. Samples containing 35 to 50 pg of total protein were resolved on a precast 4-20% Tris-Glycine Gel (5678094, Bio-Rad Laboratories, Hercules, CA, USA), transferred to a 0.45-pm PVDF (polyvinylidene fluoride) membrane (IPFL00010, EMD Millipore, Burlington, MA, USA) using wet transfer. Detailed information for blocking methods, antibody dilutions, and detection methods are listed in Table S3. Protein bands were visualized with the Odyssey Infrared Imaging System (LLCOR Biosciences, Lincoln, NE, USA) or ChemiDoc Imager system (Bio-Rad Laboratories, Hercules, CA, USA). Western blots were quantified using ImageJ using rectangle tool: on each quantified blot, the individual bands/lanes of the same size were selected. The measurements were normalized on values obtained for housekeeping proteins ( -actin or HSC70). The linear range of detection was established in pilot experiments with serial dilutions of samples, adjustments of antibody concentrations, and imaging settings.

[0327] Chymotrypsin peptidase assay was performed as previously described (24). Subcellular fractionation was performed on fresh liver tissue (-100 mg from each mouse) using NE-PER Nuclear and Cytoplasmic Extraction Reagents (78833, Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer's instructions. Lamin A/C and Vimentin were used as markers for nuclei and cytosolic/membrane fractions. Polyubiquitinated proteins were enriched using TUBE2 (Tandem Ubiquitin Binding Entities 2) polyubiquitin binding protein domains bound to magnetic beads (UM-402M, Life Sensors, Malvern, PA, USA). Lysates prepared from Rho ⁷ retinas were used to control for anti-rhodopsin antibody specificity and from Rho^{H2 il l7H2 i}" mice as samples lacking wild-type rhodopsin. Retinas were disrupted in Dounce tissue homogenizer and cleared by centrifugation at 14K g for 10 min under refrigeration. Lysates were brought to the same protein concentration and rotated with TUBE2 beads for two hours in the cold room (-200 pg of total protcin/30pl of beads). Following incubation, the beads were washed three times, and captured proteins were eluted by heating beads in 100 pl of Laemmli Buffer at 95 °C for 5 min. Aliquots containing input and flowthrough fractions (15 pg of protein) and 20pl of eluates were used for western blot analysis with anti-polyubiquitin (FK2) and anti-rhodopsin (1D4) antibodies. For rhodopsin immunoprecipitation, retinal lysates (~200pg of total protein) were incubated for two hour in the cold room with 5 pg of anti-rhodopsin 1D4 antibody preincubated with 25pl of Dynabeads (10003D, Invitrogen, Waltham, MA, USA) following the manufacturer's instructions. Rhodopsin was eluted by heating beads in 100 pl of Laemmli Buffer (95 °C for 10 minutes), and 20 pl of eluates were used for western blot analysis with anti-polyubiquitin (FK2) or anti-rhodopsin (B630) antibodies. The lysates for rhodopsin or polyubiquitin enrichment were prepared in Lysis Buffer (50mM Tris-HCl, pH 7.5, 0.15M NaCl, ImM EDTA, 1% NP-40, 10% glycerol) supplemented with protease/phosphatase/deubiquitinases inhibitors described above; TBST (20mM Tris-HCl, pH 8.0, 0.15M NaCl, 0.1% Tween- 20) was used to wash beads.

[0328] Transcriptomics analysis and RNA ISH. Bulk R Aseq of retinas and differentially expressed gene analysis was performed as previously described in (62). For bulk RNAseq transcriptomics studies, the total RNA was prepared from the retinas and livers of five Nfe21^0E (4 males and 1 female) and WT (4 males and 1 female) littermate mice of 6-7 weeks of age. Differentially expressed genes are shown in Microsoft Excel files Datasets SI (retina) and S2 (liver). RT-qPCR was performed using primers listed in Table S4 as previously described (24). RNA ISH was performed on 5-pm-thick paraffin sections prepared from formalin-fixed eyes with RNAscope probes (Advanced Cell Diagnostics, Hayward, CA, USA) listed in Table S5 on the automated Leica Bond platform (Leica Microsystems GmbH, Wetzlar, Germany) following manufacturer’s instructions. The single-cell libraries were prepared with pooled retinas from one male and one female littermate mice (Nfe211^0E and WT) using lOx Chromium Platform and following protocols described in (63). Sequencing was performed at UF Interdisciplinary Center for Biotechnology Research and single-cell data analysis was performed as described in our previous studies (62, 64).

[0329] Histology and Microscopy. Histology analysis was performed on 5-pm-thick paraffin sections cut through the superior-inferior line of the eye and containing optic nerve, stained with hematoxylin and eosin stain (H&E), and quantified as described in (24). An accumulation of the ub^{G76v GFP} reporter was assessed in 20-pm-thick frozen retinal sections prepared from the eyes fixed in 4% paraformaldehyde PBS solution (65). Rod outer segments were stained with WGA (wheat germ agglutinin, Alexa Fluor 555 conjugate, W32464, Thermo Fisher). Samples for littermate mice were processed together and imaged on a Leica TCS SP8 confocal microscope using the same settings.

[0330] All proteasomal assays, western blotting, RT-qPCR, RNA ISH, and microscopy experiments describe biological replicates and are representative of three and more independently performed experiments on separate groups of mice. The number of independent biological samples analyzed with OCT, ERG and H&E are shown in the corresponding figure legends.

[0331] Statistical analysis. Statistical differences were considered significant when the p-value <0.05, as determined by the two-tailed Student's test using GraphPad Software. Differential gene expression analysis of rod scRNAseq datasets was performed using function FindMarkers from Seurat package, p- Values in the figures are indicated as follows: *p< 0.05, **p < 0.01, ***p< 0.001, **** < 0.0001, and ns, for p > 0.05.

[0332] Example 1: Nfe211 sets proteasomal levels and activity in the retina

[0333] To identify the role of Nfe211 in control of proteasomal levels in the retina, we took advantage of a previously developed transgenic mouse (hereafter Nfe211^0E mice) driving expression of Nfe211 under control of the broadly active MafGD (Maf gene regulatory domain) promoter (30). These mice, which were developed to study glucose metabolism in the liver, were viable, fertile, and rescued otherwise lethal whole-body knockout of the Nfe211 gene (30). In the following experiments, we examined changes in proteasomal levels in the retinas and livers of Nfe211^OE mice. We used retinal lysates prepared from retina-specific Nfe211 -knockout mice to control for the specificity of anti-Nfe211 antibodies and to determine the contribution of basal Nfe211 activity to defining proteasome levels. Retina-specific Nfe211- knockout mice (hereafter, Nfe211^{Retina K0} mice) were generated by crossing mice bearing the floxed Nfe2ll allele and ChxlO-Cre mice expressing Cre recombinase in all retinal neurons and Muller cells early in development (31).

[0334] A Anorogenic chymotrypsin-peptidase assay, a commonly used method to evaluate proteasome activity, showed an 85% higher rate of substrate proteolysis in the livers and a 34% higher rate of substrate proteolysis in the retinas of Nfe211^OE mice compared to those of wild-type littermate mice (Fig. 1A). The rate of proteolysis was reduced by 31% in the retinas of the Nfe211^{Retina K0} mice (Fig. 1A). Changes in proteasome activity were paralleled by changes in proteasome levels, which were increased in the retinas and livers of the Nfc211^0E mice and reduced in the retinas of the Nfc211^{Retina K0} mice (Fig. 1BC). A targeted RT-qPCR transcriptional analysis indicated a 50-100% increase in components representative of the 20S core particle (al, a5, ]35), 19S regulator (PSMD1, PSMD11, PSMC4, and PSMC6), and components of ubiquitin-independent regulators (PSME1 and PSME4) in the livers of the Nfe211^0E mice (Fig. IB). In the retinas, the levels of proteasomal components were 5 to 50% higher in the Nfc211^0E mice and 25 to 50% lower in the Nfc211^{Retina K0} mice (Fig. IB). Transcriptional changes translated into changes in protein levels (Fig. 1C-F): increased amounts of representative proteasome subunits in the Nfe211^0E mice (49 to 141% in the livers and 14 to 27% in the retinas) and reduced amounts (a 16 to 34% decrease) in the retinas of the Nfe211^{Retina K0} mice.

[0335] The detection of basal Nfe211 levels in normal unstressed cells and tissues is challenging (32). Therefore, the staining of endogenous Nfe211 protein from wild-type mice in the western blots was weak, but a prominent band was observed for lysates prepared from Nfe211^0E mice (Fig. 1D-F). This band was absent in the control retinas of the Nfe211^{Retina K0} mice run on the same SDS-PAGE gel (Fig. 1FG). In Nfe211 -overexpressing mice, the western blot bands representing Nfe211 in the retinas were less intense than those representing Nfe211 in the livers, suggesting a less robust increase, which may explain the smaller effect on proteasomal expression observed in the retinas (Fig. 1G). According to the currently accepted model of Nfe211 regulation, Nfe211 is an endoplasmic reticulum (ER)-resident protein that undergoes a continuous cycle of synthesis, insertion into the ER membrane, glycosylation/deglycosylation, extrusion through the ER-associated degradation (ERAD) pathway and proteasomal degradation with a half-life of approximately a few minutes (33). The proteolytic products of the Nfe211 protein lacking the N-terminal transmembrane domain, when not destroyed by proteasomes, translocate to the nucleus, where they drive the expression of proteasome subunits. The details of the Nfe211 protein life cycle, a full list of its regulators, and the number and size of proteolytic products are still under investigation and continuously revised (33, 34). Nevertheless, our data are in agreement with the general picture of Nfe211 regulation. On the one hand, overexpression of Nfe211 increased the fraction of Nfe211 escaping proteasomal degradation and reaching the nucleus, as evident from its enrichment in the nuclear fraction (Fig. 1H). On the other hand, the retinas of Nfe211^{Retina K0} mice showed reduced levels of proteasomes, indicating the contribution of basal Nfe211 levels to the amounts of proteasomes in retinas (Fig. 1BF).

[0336] Example 2: AAV-driven NFE2L1 overexpression stimulates proteasomal biogenesis.

[0337] We used A AV-based gene delivery system to stimulate proteasomal genes through NFE2L1 overexpression (Fig 2). A mixture of IO¹⁰ particles of AAV5 containing full-length sequence coding for human NFE2L1 protein tagged with HA epitope on C-terminus and 10⁸ particles AAV2 coding for mCherry both driven by small chicken beta actin promoters (CBA) were delivered subretinally in 4 weeks-old wild-type mice. Control mice were injected with a mix of IO¹⁰ particles of AAV5-eGFP and 10⁸ particles AAV2- mCherry. Three weeks post-injection, the harvested retinas were analyzed for expression with WB and proteolytic assay. This analysis demonstrated 25 to 70% upregulation of key proteasomal components 19S and 20S, and less abundant regulators 11 S and PA200. It also demonstr ated about 40% increase in proteasomal activity. Therefore, these data support an AAV-based gene delivery system of NFE2L1 as an approach to stimulate proteasomal biogenesis.

. An RNA in situ hybridization (RNA ISH) analysis of Nfe211^0Emice showed a panretinal increase in Nfe211 transcript levels in all retinal layers, including increased staining in the outer nuclear layer (ONL) containing photoreceptor nuclei (Fig. 2A). The analysis of control retinas from Nfe211^{Retina KO} mice showed nearly complete but mosaic loss of Nfe211 transcripts (Fig. 2A). Next, we used single-cell RNAseq (scRNAseq) and a cell-selective strategy to confirm an increase of proteasome transcripts in rods. As shown in Fig. 2B, we readily distinguish and isolate rod photoreceptors in UMAP plots in our single-cell datasets. An increase in Nfe211 and proteasome transcripts was evident from the elevated average values and a higher fraction of sequenced rods containing transcripts of interest in Nfe211^0E mice compared to wild-type littermates (Fig. 2C). Bulk RNA-seq of Nfe211^0E retinas did not show a coordinated increase in proteasome levels (Fig. 2D, see also Dataset SI), indicating that the measure was likely not sensitive enough to identify an -20-30% change, which we detected with targeted RT-qPCR, and confirmed with western blotting and peptidase measurements as described above. At the same time, an analysis of Nfe211^0E livers with bulk RNA-seq showed a robust transcriptional response (Fig. 2E), with top changes impacting genes belonging to "Protein Ubiquitination Pathway" (Fig. 2F). These transcriptional changes included a coordinated upregulation of transcripts for major proteasomal subunits (Fig. 2G). In the next set of experiments, we showed that -30% increase in proteasomal activity and Nfe211 overexpression was sufficient to stimulate degradation of ubiquitinated proteins in rods of mice stressed by misfolded transmembrane proteins.

[0338] Example 3: Nfe211 overexpression counteracts ubiquitin-proteasome insufficiency in a heterozygote Rho^P23H/WT knock-in mouse model of human blindness

[0339] Previously, we and other researchers reported that photoreceptor neurons in genetically diverse mouse models of retinal degeneration accumulated the ubiquitin-proteasome reporter Ub^G76V-GFP, which served as a readout for the degradation of short-lived ubiquitinated proteins (76, 35-38). The specific changes in the photoreceptors of these models that interfere with the processing of the Ub^G76V-GFP reporter, impairing its clearance, are unknown. Similarly, it is unclear whether the rate-limiting steps in processing ubiquitinated proteins in these diverse models are the same. Nevertheless, an accumulation of this reporter can be interpreted to be a manifestation of limited UPS capacity (UPS insufficiency) to degrade proteins, which becomes evident in photoreceptors showing an increased burden of misfolded and mistargeted proteins (35). These mice allow us to assess the impact of proteasome increase on protein degradation in vivo.

[0340] A heterozygote Rho^p23H/WT knock-in mouse is an established model of human blindness called retinitis pigmentosa (39. 40), expressing both mutant and wild-type rhodopsin in rod photoreceptors. A proline-to-histidine (P23H) amino acid substitution destabilized the structure of the transmembrane protein rhodopsin, driving its constant ERAD-associated polyubiquitination and proteasomal degradation, which stresses rod photoreceptors, eventually leading to their death (39, 40). As shown in Fig. 3A and described in previous studies, an accumulation of Ub^G76V-GFP reporter was observed in the photoreceptors of these mice. Therefore, Rho^p23H/WT mouse line expressing the Ub^G76V-GFP reporter might serve as an efficient model to examine new methodologies to modulate the efficiency of UPS functioning in vivo (16, 24). [0341] As shown in Fig. 4 A, overexpression of Nfe211 improved clearance of Ub^G76v-GFP reporter in photoreceptors of Rho^p23H/WT mice detected with confocal microscopy. In agreement with the imaging findings, western blot (Fig. 4BC) quantification showed an approximately two-fold reduction in steadystate reporter level. Control experiments confirmed the increase in proteasome activity and higher levels of proteasomes in Rho^p23H/WT7Nfe211^OE retinas (Fig. 4DEF), similar to the changes described for the Nfe211^OE mice above. Quantitative western blotting did not detect changes in autophagy markers, LC3-I and LC3-II (Microtubule-associated proteins 1 A/1B light chain 3B-I/II) or their ratios but showed a slight increase in the levels of SQSTM1 (Sequestosome-1) protein (Fig. 4G). The western blot of ubiquitination patterns in retinal lysates probed with FK2 antibody generated against polyubiquitinated chains (Fig. 4HI) and P4D1 antibody raised against monoubiquitin (Fig. 4JK) did not reveal a difference between Rho^p23H/WT/Nfe211^OE and Rho^p23H/WT littermate mice. Similarly, the ubiquitination patterns in the extracts prepared from the retinas of Nfe211^0E mice and wild-type littermates were undistinguishable. Therefore, Nfe211 overexpression does not impact the steady-state levels of polyubiquitinated proteins as detected with western blotting. Yet, better clearance of reporter in combination with elevated proteasomal activity suggests some extent of improvement in UPS functioning in stressed rods, at least with the processing of some protein substrates. According to the current concept in the field, it is thought that mutant P23H rhodopsin efficiently and almost entirely degraded, with a small fraction of rhodopsin immunoprecipitated from retinas of Rho^p23H/WT mice found to be modified with ubiquitin (39, 40). In our analysis, we did not detect differences in the total levels of rhodopsin and ubiquitinated rhodopsin in fractions enriched for polyubiquitin (using polyubiquitin binding domains) or rhodopsin (using antirhodopsin antibody) in Rho^p23H/WT/Nfe211^OE mice. Thus, Nfe211 overexpression does not appear- to complement already efficient rhodopsin degradation, at least as could be assessed by levels of ubiquitinated rhodopsin with available tools. Yet, better clearance of reporter points to benefits of Nfe211 overexpression and an improvement in UPS functioning, e.g., aiding with degradation of an everyday basal load of damaged, misfolded or mistranslated proteins in already stressed Rho^{p23H/W T} rods. In the experiments described in the next section, we applied in vivo imaging and physiological methods to show quantitatively that Nfe211 overexpression and higher proteasomal activity slowed down vision loss in Rho^p23H/WT mice.

[0342] Example 4: Nfe211 overexpression delays vision loss in a heterozygote Rho^P23H/WT knock-in model of human blindness

[0343] Optical coherence tomography (OCT) allows efficient quantitative in vivo studies of retinal structures (41). As shown in Fig. 5 AB, the ONL, containing photoreceptor nuclei, in the Rho^p23H/WT mice became progressively thinner with age (marked in blue), and was barely discernable after the mice reached six months of age. Overexpression of Nfe211 delayed ONL thinning in the Rho^p23H/WT mice (Fig. 5ABC). The ONL thickness around the optic nerve head (ONH) in Rho^p23H/WT/Nfe211^OE mice, presented in the form of spider diagrams, was consistently thicker compared to that in Rho^{p25ll/W l} littermates older than 45 days (Fig. 5D). A morphometric analysis confirmed an increase in photoreceptor survival in Nfe211^OE-overexpressing animals (Fig. 6). We analyzed retinal cross sections containing optic nerve head and cut along the superior-inferior line of eye. Samples prepared from 6-month-old Rho^p23H/WT/Nfe211^OE mice showed from 1 to 3 (Fig. 6B) additional rows of surviving photoreceptor nuclei and 10 to 90% longer outer segments (Fig. 6C) throughout the entire retina compared to those in the Rho^p23H/WT littermate mice. OCT (Fig. 5CD) studies and morphometric analysis did not identify long term adverse effects of Nfe211 overexpression: the retinal structure and the number of nuclei the Nfe211^0E mice were indistinguishable from that in the wild-type littermates. In the next set of experiments, we showed that better photoreceptor preservation in the Rho^p23HAVT/Nfe211 ^OE mice translated into improved retinal function.

[0344] Electroretinography (ERG) is a physiological method for quantitatively assessing retinal function in vivo (42). In typical ERG studies, dark-adapted mice are exposed to bright flashes with increasingly intense light to assess scotopic (rod), mesopic (mixed rod and cone), and photopic (cone) vision. In ERG traces, the a-wave responses originate from rod and cone photoreceptors, and the b-wave represents amplified responses from retinal neurons downstream of photoreceptors. The amplitudes of both waves are effective quantitative measures of the number and health of surviving photoreceptors. The ERG recordings of Rho^p23H/WT/Nfe211^OEmice consistently yielded higher responses, with the maximum a- and b-wave amplitudes recorded to be 50 and 100% higher in 3- and 6-month-old animals compared to those in Rho^p23H/WT littermates (Fig. 7ABC and 7EFG). ERG responses of Nfe211^0E mice and their wild type littermates were indistinguishable from each other (Fig. 7ABD and 7EFH).

[0345] An OCT analysis (Fig. 5A) showed that Nfe211 overexpression delayed vision loss in Rho^p23H/WT mice for approximately two months. Notably, this form of retinal degeneration progresses slower in humans than in mice (years instead of months) (39). Therefore, months of delayed photoreceptor loss in mice might translate into years of preserved vision in humans. Furthermore, in this study, we focused only on target validation, and the extent of the observed treatment might be limited by the characteristics of the available Nfe211^0E transgenic mouse line. A more efficient approach to augment the Nfe211 pathway may potentially lead to greater improvement.

[0346] Example 5: Nfe211 overexpression and genetic activation of mTORCl counteract UPS insufficiency and drive proteasome biogenesis in rods stressed with misfolded cytosolic proteins [0347] Ongoing pathological changes in photoreceptors and the nature of proteotoxic stress can reduce the efficiency of Nfe211 activation, thus limiting the extent of the proteasome increase. Therefore, in a final set of experiments, we sought to generalize our findings and examine the modulation of proteasome biogenesis in an alternative model of photoreceptor degeneration caused by misfolded cytosolic protein- induced stress. In Gyi ⁷ mice, the misfolded G|3i subunit could not be stabilized without its functional partner, the Gyi subunit. Continuous degradation of misfolded G]31 subunit overloads UPS in rod photoreceptors, until they die (35). Consistent with this model, we previously found that the Ub^G76V-GFP reporter accumulated in the rods of Gyi ' mice (35). This form of photoreceptor degeneration is different from that in Rho^p23II/WT mice, which is caused by stress induced by misfolded transmembrane protein rhodopsin continuously degraded via ERAD pathway (40). As discussed below, we probed the effectiveness of Nfe211 overexpression to stimulate proteasome biogenesis and counteract UPS insufficiency in Gyi ^/_ mice, but also compared effects with those caused by genetic activation of mTORCl. We crossed Gy, ⁷ and Nfe211 -overexpressing mice to generate the Gyi ⁷7Nfe211^OE mouse line. Genetic activation of the mTORCl pathway was achieved by deleting its negative regulator Tsc2 specifically in the rods of the Gyi ⁷ mice (hereafter, the Gyf⁷7Tsc2^{Rod K0} mouse line).

[0348] As shown in Fig. 8ABC, both the overexpression of Nfe211 and chronic mTORCl activation improved clearance of Ub^G76V-GFP reporter in GD i ⁷ photoreceptors. Western blot quantification revealed that the level of the Ub^G76v-GFP reporter in lysates prepared with retinas of Gyi ^/7Nfe211^OE and Gyi ⁷ /Tsc2^{Rod K0} mice was reduced by 44% and 85%, respectively (Fig. 8DE). Chymotrypsin-like peptidase assay showed higher rates of substrate proteolysis in the retinas of both mouse lines, a 22% increase in Gyi ⁷7Nfe211^OE and a 28% increase in Gyf⁷7Tsc2^{Rod K0} mice compared to Gyr⁷ littermates (Fig. 8F). We previously reported that mTORCl -mediated proteasomal activity in the retinas of rod-specific Tsc2 knockout mice, to some extent, was phosphorylation-dependent (24). Therefore, we studied the impact of lambda protein phosphatase treatment (X PP) on proteasome activity. Phosphatase treatment slightly reduced proteasome activity (3-4%) in the retinal lysates prepared from Gy, ⁷ and Nfe211 -overexpressing Gyi ⁷ littermate mice (Fig. 8F), but this trend did not reach a level of statistical significance. In Gyi ⁷ /Tsc2^{Rod K0} mice, phosphatase treatment decreased proteolytic activity by 15%. Nevertheless, even after treatment, the proteolytic activity remained -16% higher than that in phosphatase-treated Gyi ⁷ littermates (Fig. 8F). Quantitative western blotting demonstrated higher levels of proteasomes in the retinas of both Gyi ⁷7Nfe211^OE and Gyp ⁷7Tsc2^{Rod K0} mice, with a slightly higher effect after mTORCl activation, particularly for the PSMD11 and [15 proteasome subunits (Fig. 8GH). Notably, changes in proteasome expression and activity in Gyp⁷TTsc2^{Rod KO} were most likely low-end estimates since they were measured in whole-retina lysates, but Tsc2 was removed only from rod photoreceptors. [0349] Western blotting for the Nfe211 protein in the retinas of Gyi ' mice produced a weak signal, with a prominent band shown for the Nfe211^0E mice, and a slight elevation in Gyr^/7Tsc2^{Rod K0} animals (Fig. 8H). Due to complex posttranslational modifications and weak signal, detecting subtle changes in Nfe211 levels in the retina by western blotting was challenging (Fig. 8H). Therefore, we performed a targeted RT-qPCR analysis to confirm the elevation in Nfe211 in the Gyi ^/7Tsc2^{Rod K0} mice (Fig. 81). We used RNA ISH to confirm the mTORCl -mediated increase in the number of Nfe211 transcripts in the ONL (which contains mostly rod photoreceptor nuclei) of Gyi ^/7Tsc2^{Rod K0} mice as an additional control (Fig. 8J, see also Fig. S4). Interestingly, the mTORCl -mediated increase of proteasome transcripts in the G71 ^/7Tsc2^Rod ^KO and Tsc2^{Rod KO} mice were comparable (Fig. 81). This observation contrasts our previous findings for degenerating retinas of Rho^p23H/WT/Tsc2^{Rod K0} mice stressed by misfolded transmembrane P23H mutant protein, in which this transcriptional response was suppressed (24). Therefore, the type of proteotoxic stress and form of retinal degeneration may indeed impact the efficiency of Nfe211 activation, particularly mediated via chronic stimulation of mTORCl pathway. Thus, our analysis showed that in retina of Gyi ⁷ mice stressed by misfolded cytosolic protein, both Nfe211 overexpression and chronic mTORCl activation were efficient in driving proteasome biogenesis and counteracting UPS insufficiency. However, whereas in Nfe211 overexpressing mice higher proteasomal activity could be attributed to an increase in proteasomal amounts, chronic stimulation of mTORCl pathway could augment proteasomal capacity through a combination of increasing proteasomal pool and phosphorylation-mediated stimulation of proteasomes. Notably, this in vivo comparison might point to natural limits (-30%) to which proteasomal activity could be enhanced through either mechanism in the retina. Future studies might have to consider more potent combinatorial approaches to stimulate proteasomal degradation.

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Claims

CLAIMS What is claimed is:

1 . A method of treatment or prophylaxis of an eye disorder in a subject, the method comprising administering a therapeutically effective amount of a composition comprising a nucleic acid encoding a polypeptide wherein the nucleic acid comprises a nucleic acid sequence encoding a polypeptide comprising at least 50% , at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NOs 2 or 4, and wherein expression of the nucleic acid increases proteosome activity in a target cell.

2. The method according to claim 1 , wherein a promoter is operably linked to the nucleic acid sequence.

3. The method of claim 2, wherein the promoter is selected from the group consisting of a CMV promoter, a Bcstl promoter, and the native promoter for Nfc2Ll or a functional fragment thereof.

4. The method according to any one of the preceding claims, wherein the nucleic acid is delivered to a target cell via a viral vector.

5. The method of claim 4, wherein the viral vector is selected from the group consisting of an adeno- associated virus vector, an adenovirus vector, a retrovirus vector, an orthomyxovirus vector, a paramyxovirus vector, a papovavirus vector, a picornavirus vector, a lentivirus vector, a herpes simplex virus vector, a vaccinia virus vector, a pox virus vector, an anellovirus vector, and an alphavirus vector.

6. The method according to any one of the preceding claims, wherein the nucleic acid is a viral vector genome.

7. The nucleic acid for use according to claim 6, wherein the viral vector genome is selected from the group consisting of an adeno-associated virus vector genome, an adenovirus vector genome, a retrovirus vector genome, an orthomyxovirus vector genome, a paramyxovirus vector genome, a papovavirus vector genome, a picornavirus vector genome, a lentivirus vector genome, a herpes simplex virus vector genome, a vaccinia virus vector genome, a pox virus vector genome, an anellovirus vector genome, and an alphavirus vector genome.

8. The method according any one of the preceding claims, wherein the eye disorder is selected from an inherited eye condition, macular degeneration or glaucoma.

9 . The method according any one of the preceding claims, wherein the target cell is a cell of the retina or the choroid.

10. The method according to claim 9 , wherein the target cell is a cell of the RPE, rod photoreceptor, cone photoreceptor or a ganglion cell.

11. The method according any one of the preceding claims, wherein the nucleic acid is administered intraocularly, intravitreally, subretinally, or periocularly to a subject.

12. The method according to claim 11, wherein the nucleic acid is administered by subretinal injection.

13. The method according to any one of the preceding claims, wherein the nucleic acid sequence encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2 and 4.

14. A vector virion for use in a method of treatment or prophylaxis of an eye disorder in a subject, wherein the vector virion comprises a nucleic acid comprising a nucleic acid sequence encoding a polypeptide comprising at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NOs 2 or 4 and wherein the nucleic acid is capable of increasing proteosome activity in a target cell.

15. The vector virion for use according to claim 14, wherein a promoter is operably linked to the nucleic acid sequence.

16. The vector virion for use according to claim 15, wherein the promoter is selected from the group consisting of a CMV promoter, a Bestl promoter, photoreceptor-specific - promoter (Rhodopsin or Rhodopsin Kinase 1 or similar), RPE-specific (a Bestl promoter or similar), neuron-specific promoter (synapsin (SYN)), and the native promoter for Nfe2Ll or a functional fragment thereof.

17 . The vector virion for use according to any one of claims 14-16, wherein the nucleic acid is suitable for integration into the genome of the target cell by an RNA-guided endonuclease system.

18. The vector virion for use according to any one of claims 14-17 , wherein the vector virion is selected from the group consisting of adeno-associated virus, adenovirus, retrovirus, orthomyxovirus, paramyxovirus, papovavirus, picornavirus, lentivirus, herpes simplex virus, vaccinia virus, pox virus, anello virus, and alphavirus.

19. The vector virion for use according to claim 18, wherein the vector virion is an adeno- associated virus (AAV).

20. The vector virion for use according to claim 19, wherein the AAV is selected from the group consisting of AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), and AAV type 9 (AAV-9).

21. An method fortreatment or prophylaxis of an eye disorder in a subject, the method comprising administering a therapeutically effective amount of a composition comprising a polypeptide comprising at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NOs 2 or 4.

22. The method of claim 21, wherein the polypeptide further comprises a cell penetrating peptide (CPP).