Materials and methods for treating macular degeneration

Abstract

The present invention relates to an agent that increases expression and / or activity of IRAK-M for use in a method for treating or preventing macular degeneration in a subject. The agent may be one or more of a small molecule, a nucleic acid, a vector virion, a polypeptide, a nucleic acid system, a viral vector system, or a pharmaceutical composition.

Description

[Technical field]

[0001] The present invention relates to an agent that increases expression and / or activity of IRAK-M for use in a method for treating or preventing macular degeneration in a subject. The agent may be one or more of a small molecule, a nucleic acid, a vector virion, a polypeptide, a nucleic acid system, a viral vector system, or a pharmaceutical composition. [Background technology]

[0002] Alongside other cell-autonomous responses such as metabolic regulation and autophagy, immune-mediated inflammation initiated by noxious stress (environmental factors) is at the forefront of maintaining and restoring homeostasis (1-3). Insufficient or defective immune responses not only lead to tissue damage, but also to excessive immune responses, particularly chronic inflammation or divergent or defective responses, which are harmful. Both intensify with age ("inflammatory aging") and contribute to age-related degenerative diseases (4, 5).

[0003] Age-related macular degeneration (AMD) is the leading cause of blindness in older adults, with prevalence gradually increasing with age. As life expectancy increases, AMD has become a major public health problem, as the global AMD burden is predicted to reach 288 million by 2040 (6, 7). In the United States, approximately 11 million people suffer from AMD, a prevalence similar to that of all invasive cancers combined and more than twice that of Alzheimer's disease (6). The global cost of vision impairment due to AMD alone is substantial, estimated at US$343 billion, including 74% in direct medical costs (AMD Alliance International).

[0004] Clinically, AMD is characterized by the deposition of lipoproteinaceous drusen and pigmentary abnormalities in the RPE (early AMD), the insidious lesion of insidious photoreceptor loss in the RPE (geographic atrophy, dry AMD, late type), or the frequent but not preceding choroidal neovascularization (CNV, wet AMD, late type) in 10–15% of cases. Due to the current lack of early intervention options for dry AMD, a significant proportion of AMD cases eventually progress to severe visual impairment or blindness (6, 8). In addition to the association with immune response-related genotypes and complement (9–12), unchecked inflammatory responses from immune cells (e.g., microglia / macrophages), and immunocompetent tissue-resident cells (e.g., RPE) form a crucial driving force in accelerating tissue aging toward AMD (13–17). Nevertheless, the mechanisms behind defective immune regulation with aging remain elusive.

[0005] Among the various retinal cell types, RPE is considered the most sensitive to aging, with the highest number of differentially expressed genes (DEGs) overlapping with genes related to aging and age-related retinal diseases (18, 19). Oxidative changes in aging RPE predispose to AMD by inducing altered mitochondrial metabolism, impaired and senescent intracellular RPE processing pathways (autophagy, phagolysosome, and protein trafficking), all tractable pathways that cross-regulate and also determine appropriate immune responses (20-24). Inflammatory responses can be initiated by pathogen-induced reactive oxygen species (ROS) through signaling pathways mediated by Toll-like receptors (TLRs) (25-27), which detect various pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Nonetheless, excessive or persistent TLR-mediated inflammation disrupts cell and tissue homeostasis.

[0006] The magnitude of the inflammatory response is balanced by tonic inhibitory mechanisms involving interleukin-1 receptor-associated kinase-M (IRAK-M), encoded by the IRAK3 gene, a unique IRAK family member that lacks kinase activity and acts as an anti-inflammatory molecule (28). IRAK-M suppresses inflammatory cascades mediated by TLRs or interleukin-1 receptor (IL-1R) by inhibiting the uncoupling of IRAK1 / 4 from the IRAK-MyD88 complex (Myddosome) (28-30). Dysregulated IRAK signaling contributes to metabolic insulin resistance in diabetes and obesity (30, 31). IRAK-M expression is downregulated in monocytes and adipose tissue of obese subjects and is associated with excessive oxidative stress, elevated systemic inflammation, and features of metabolic syndrome (31). IRAK-M was previously found to be expressed in mouse RPE cell lines and its expression was decreased by wortmannin (a PI3K inhibitor) or following extended periods of culture in which the cells exhibited increased mitochondrial superoxide and impaired autophagy. (13) However, the expression, role, and significance of IRAK-M regulation in retinal health and disease remain to be elucidated. Summary of the Invention

[0007] We identified that IRAK-M expression in the RPE in human and mouse retinas declines with age and oxidative stress. Data mining of RNA-seq studies revealed reduced IRAK-M expression in AMD eyes compared to age-matched controls. Immunohistochemical staining analysis of human ocular sections further confirmed the decline in ocular IRAK-M expression with aging and AMD. Reduced IRAK-M expression may impair the RPE's ability to maintain function and health. For example, IRAK-M knockout mice developed outer retinal and RPE pathology that was enhanced following oxidative insults. We showed that increased IRAK-M expression provides protection to the RPE and retina. Introducing a human IRAK-M transgene into mouse RPE preserved mitochondrial activity and promoted cell survival under oxidative stress. The inventors also demonstrated prevention of AMD-like phenotypes in two animal models, namely, prevention of light-induced retinal damage (LIRD) in wild-type mice and prevention of age-related retinal damage in IRAK-M knockout mice.

[0008] Thus, the present invention relates to an agent that increases IRAK-M expression in a target cell and / or increases IRAK-M activity in a target cell for use in a method for treating or preventing macular degeneration in a subject.

[0009] IRAK-M in a target cell can be increased by introducing exogenous IRAK-M into the target cell. Thus, the agent can be an IRAK-M polypeptide or a nucleic acid encoding IRAK-M. The following embodiments relate to approaches for increasing IRAK-M in a target cell by introducing exogenous IRAK-M into the target cell.

[0010] In an aspect of the present invention, a nucleic acid is provided for use in a method for treating or preventing macular degeneration in a subject. The nucleic acid may comprise a nucleic acid sequence encoding IRAK-M. The nucleic acid may drive expression of IRAK-M in a target cell.

[0011] In some embodiments, a promoter is operably linked to the nucleic acid sequence. The promoter can be an RPE-specific promoter. The RPE-specific promoter can be selected from the group consisting of RPE65 promoter, NA65 promoter, VMD2 promoter (also known as Best1 promoter), and Synpiii promoter. In alternative embodiments, the promoter is a ubiquitous promoter. The ubiquitous promoter can be selected from the group consisting of CMV promoter, CAG promoter, GAPDH promoter, UbiC promoter, and EF-1α promoter. In other embodiments, the promoter is the native promoter of IRAK3 or a functional fragment thereof.

[0012] In some embodiments, IRAK-M expression is increased in target cells containing nucleic acid compared to comparable cells not containing nucleic acid. Autophagy flux may be maintained or increased in target cells containing nucleic acid compared to comparable cells not containing nucleic acid. Mitochondrial activity may be maintained or increased in target cells containing nucleic acid compared to comparable cells not containing nucleic acid. Proinflammatory cytokine production may be decreased in target cells containing nucleic acid compared to comparable target cells not containing nucleic acid. Proinflammatory cytokines may be selected from the group consisting of GM-CSF and MCP-1.

[0013] The nucleic acid may be suitable for integration into the genome of a target cell by an RNA-guided endonuclease system. The RNA-guided endonuclease may be a CRISPR-Cas system.

[0014] In some embodiments, the nucleic acid is DNA. The nucleic acid can 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 can be messenger RNA or circular RNA.

[0015] In some embodiments, the nucleic acid is delivered to the target cell via a non-viral carrier, which may be selected from the group consisting of nanoparticles, liposomes, cationic polymers, and calcium phosphate particles.

[0016] In some embodiments, the nucleic acid is delivered to the target cell via a viral vector, which can be 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 poxvirus vector, anellovirus vector, and an alphavirus vector.

[0017] The nucleic acid may be a viral vector genome, which may be 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 poxvirus vector genome, anellovirus vector genome, and an alphavirus vector genome.

[0018] The macular degeneration can be age-related macular degeneration (AMD). The age-related macular degeneration (AMD) can be dry AMD. In some embodiments, the dry AMD is selected from the group consisting of early dry AMD, intermediate dry AMD, and advanced dry AMD.

[0019] The target cell may be a cell of the retina or choroid. In some embodiments, the target cell is a cell of the retina. The target cell may be a cell of the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor outer segment (POS), or retinal pigment epithelium (RPE). In some embodiments, the target cell is a cell of the RPE.

[0020] The target cell can be a myeloid cell. The myeloid cell can be a retinal myeloid cell. In some embodiments, the target cell is a CD11b + It is a myeloid cell. The nucleic acid can be administered to the subject intraocularly, intravitreally, subretinal, or periocularly. In some embodiments, the nucleic acid is administered subretinal. The nucleic acid can be administered by injection or infusion. In some embodiments, the nucleic acid is administered by subretinal injection. In some embodiments, the subject is a human. The subject can be afflicted with or at risk for developing macular degeneration.

[0021] In some embodiments, the nucleic acid sequence encodes a polypeptide comprising an amino acid sequence having at least 60% sequence identity with the amino acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid sequence encodes a functional polypeptide. The nucleic acid sequence can encode a polypeptide capable of preventing dissociation of IRAK-1 and / or IRAK-4 from MyD88 in a target cell. The nucleic acid sequence can encode a polypeptide capable of preventing formation of an IRAK-1-TRAF6 complex.

[0022] In another aspect, a vector virion for use in a method for treating or preventing macular degeneration in a subject is provided, the vector virion comprising a nucleic acid described herein.

[0023] In some embodiments, IRAK-M expression is increased in target cells containing vector virions compared to equivalent cells not containing vector virions. Autophagy flux may be maintained or increased in target cells containing vector virions compared to equivalent cells not containing vector virions. Mitochondrial activity may be maintained or increased in target cells containing nucleic acids compared to equivalent cells not containing vector virions. Proinflammatory cytokine production may be decreased in target cells containing vector virions compared to equivalent target cells not containing vector virions. The proinflammatory cytokine may be selected from the group consisting of GM-CSF and MCP-1.

[0024] 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, poxvirus, anellovirus, and alphavirus. In some embodiments, the vector virion is an adeno-associated virus (AAV). The AAV may be 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.

[0025] The vector virion may be administered to the subject intraocularly, intravitreally, subretinal, or periocularly. In some embodiments, the vector virion is administered subretinal. The vector virion may be administered by injection or infusion. In some embodiments, the vector virion is administered by subretinal injection. In some embodiments, the subject is a human. The subject may be afflicted with or at risk for developing macular degeneration.

[0026] In some embodiments, the vector virion comprises a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 1. The vector virion may comprise a nucleic acid sequence encoding a polypeptide capable of preventing dissociation of IRAK-1 and / or IRAK-4 from MyD88 in a target cell. The vector virion may comprise a nucleic acid sequence encoding a polypeptide capable of preventing formation of an IRAK-1-TRAF6 complex.

[0027] A further aspect provides an IRAK-M polypeptide for use in a method for treating or preventing macular degeneration in a subject. In some embodiments, the polypeptide comprises an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0028] The polypeptide can prevent dissociation of IRAK-1 and / or IRAK-4 from MyD88 in a target cell.The polypeptide can prevent formation of an IRAK-1-TRAF6 complex.

[0029] 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 can be conjugated to the N-terminus of the PCL. An amino acid sequence having at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 1 can be conjugated to the C-terminus of the PCL. In some embodiments, the PCL is a peptide sequence that can be cleaved by cathepsin D.

[0030] Autophagy flux may be maintained or increased in target cells comprising the polypeptide compared to comparable cells not comprising the polypeptide. Mitochondrial activity may be maintained or increased in target cells comprising the polypeptide compared to comparable cells not comprising the polypeptide. Proinflammatory cytokine production may be decreased in target cells comprising the polypeptide compared to comparable target cells not comprising the polypeptide. The proinflammatory cytokine may be selected from the group consisting of GM-CSF and MCP-1.

[0031] In an aspect of the invention, a compound according to the invention is provided for use in a method for treating or preventing macular degeneration in a subject, comprising: a) a nucleic acid sequence encoding an RNA-guided endonuclease; b) a nucleic acid sequence encoding a guide RNA that is homologous to a target sequence associated with an insertion site in the genome of the target cell and capable of directing the RNA-guided endonuclease to the target sequence; and c) a nucleic acid sequence encoding IRAK-M; The nucleic acid system is provided comprising one or more nucleic acids comprising: a nucleic acid sequence encoding IRAK-M capable of driving expression of IRAK-M in a target cell of a subject, the nucleic acid system being suitable for directing insertion of the nucleic acid sequence encoding IRAK-M into an insertion site in the genome of the target cell.

[0032] The nucleic acid sequence encoding IRAK-M may be flanked by a 5' homology arm and a 3' homology arm. In some embodiments, the 5' homology arm is homologous to a DNA sequence 5' from the insertion site of the target sequence, and the 3' homology arm is homologous to a DNA sequence 3' from the insertion site of the target sequence. The nucleic acid sequence encoding IRAK-M may further comprise a 5' flanking sequence that includes the target sequence and a 3' flanking sequence that includes the target sequence. In some embodiments, the 5' flanking sequence is 5' from the 5' homology arm, and the 3' flanking sequence is 3' from the 3' homology arm.

[0033] In an alternative embodiment, the nucleic acid sequence encoding IRAK-M is flanked by 5' and 3' target sequences, which may be identical to target sequences from the insertion site in the genome.

[0034] In some embodiments, the one or more nucleic acids are one or more viral vector genomes. The one or more viral vector genomes can be one or more adeno-associated viral vector genomes.

[0035] Further provided is a viral vector system comprising the nucleic acid system described herein. In some embodiments, the viral vector system is an adeno-associated viral vector system. Another aspect provides a pharmaceutical composition comprising a nucleic acid, vector virion, polypeptide, nucleic acid system, or viral vector system described herein, hi some embodiments, the pharmaceutical composition is formulated for ocular delivery.

[0036] IRAK-M expression can be increased by increasing endogenous IRAK-M expression in target cells. Thus, the agent can increase endogenous IRAK-M expression. The following embodiments relate to agents that can increase endogenous IRAK-M expression in target cells.

[0037] Thus, for use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease fused to one or more transcriptional activators; and b) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the promoter or regulatory sequence of the IRAK3 gene and is capable of directing the RNA-guided endonuclease to the target sequence; wherein the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0038] The transcriptional activator can be VP64. Thus, for use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease; and b) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in a promoter or regulatory sequence of the IRAK3 gene and that is capable of directing said RNA-guided endonuclease to said target sequence, said guide RNA further comprising an aptamer that is capable of specifically binding to a transcription activator; wherein the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0039] An embodiment of the invention also provides a method for treating or preventing macular degeneration in a subject, comprising: a) a nucleic acid sequence encoding an inactivated 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 that is complementary to a target sequence in a promoter or regulatory sequence of the IRAK3 gene and that is capable of directing said RNA-guided endonuclease to said target sequence, said guide RNA further comprising an RNA aptamer that is capable of specifically binding to an RNA-binding protein; wherein the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0040] The one or more transcriptional activators may be selected from the group consisting of VP64, p65, and HSF1. The RNA aptamer can bind to the RNA-binding protein dimer. The RNA-binding protein may be MS2. In some embodiments, the inactivated RNA-guided endonuclease is fused to an additional transcriptional activator. The additional transcriptional activator may be VP64.

[0041] A further aspect relates to a method for treating or preventing macular degeneration in a subject, comprising: a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease fused to an epitope repeat array comprising one or more epitopes; b) one or more nucleic acid sequences encoding epitope-binding molecules fused to one or more transcriptional activators, said epitope-binding molecules being capable of specifically binding to an epitope of the epitope repeat array; and c) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the promoter or regulatory sequence of the IRAK3 gene and is capable of directing the RNA-guided endonuclease to the target sequence; wherein the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0042] The epitope-binding molecule may comprise a nuclear localization sequence (NLS). The epitope-binding molecule may be an antibody or an antibody-like molecule. In some embodiments, the one or more transcriptional activators are selected from the group consisting of VP64, p65, and Rta.

[0043] The one or more nucleic acids may be one or more viral vector genomes. The one or more viral vector genomes may be one or more adeno-associated viral vector genomes. Another aspect relates to a compound comprising: a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease fused to one or more DNA demethylating agents; and b) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the IRAK3 gene and capable of directing the RNA-guided endonuclease to the target sequence; wherein the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0044] Further, for use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease; and b) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the IRAK3 gene and capable of directing the RNA-guided endonuclease to the target sequence, wherein the guide RNA further comprises an aptamer capable of specifically binding to a DNA demethylating agent; wherein the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0045] Embodiments include a method for treating or preventing macular degeneration in a subject, comprising: a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease; b) a nucleic acid sequence encoding an RNA-binding protein fused to one or more DNA demethylating agents; c) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the IRAK3 gene and capable of directing the RNA-guided endonuclease to the target sequence, wherein the guide RNA further comprises an RNA aptamer capable of specifically binding to an RNA-binding protein; wherein the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0046] The RNA aptamer can bind to the RNA binding protein dimer. The RNA binding protein can be MS2. In some embodiments, the inactivated RNA-guided endonuclease is fused to an additional transcription activator.

[0047] A further aspect relates to a method for treating or preventing macular degeneration in a subject, comprising: a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease fused to an epitope repeat array comprising one or more epitopes; b) one or more nucleic acid sequences encoding epitope-binding molecules fused to one or more DNA demethylating agents, the epitope-binding molecules being capable of specifically binding to an epitope of an epitope repeat array; and c) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the IRAK3 gene and capable of directing the RNA-guided endonuclease to the target sequence; wherein the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0048] The epitope-binding molecule may comprise a nuclear localization sequence (NLS). The epitope-binding molecule may be an antibody or an antibody-like molecule. In some embodiments, the DNA demethylating agent is TET1. In some embodiments, the DNA demethylating agent is LESD1.

[0049] The one or more nucleic acids may be one or more viral vector genomes. The one or more viral vector genomes may be one or more adeno-associated viral vector genomes. Also provided is a viral vector system comprising the nucleic acid system described herein. In some embodiments, the viral vector system is an adeno-associated viral vector system.

[0050] An alternative targeting approach to increase endogenous expression of IRAK-M in target cells may use a nucleic acid binding molecule (eg, a nucleic acid binding moiety) capable of binding to a target sequence.

[0051] Accordingly, aspects provide a nucleic acid comprising a nucleic acid sequence encoding a fusion protein for use in a method for treating or preventing macular degeneration in a subject, the fusion protein comprising: a) a nucleic acid binding molecule capable of binding to a target sequence in the promoter or regulatory sequence of the IRAK3 gene; and b) comprising one or more transcriptional activators, the fusion protein inhibiting IRAK-M expression in a target cell of interest; can be increased.

[0052] An aspect of the invention is a method for treating or preventing macular degeneration in a subject, comprising: a) a nucleic acid sequence encoding a fusion protein, the fusion protein comprising (i) a nucleic acid binding molecule capable of binding to a target sequence in a promoter or regulatory sequence of the IRAK3 gene, and (ii) an epitope repeat array; and b) one or more nucleic acid sequences encoding epitope-binding molecules fused to one or more transcriptional activators, said epitope-binding molecules being capable of specifically binding to an epitope of the epitope repeat array; wherein the nucleic acid system is capable of increasing IRAK-M expression in a target cell of a subject.

[0053] A further aspect provides a nucleic acid sequence encoding a fusion protein for use in a method for treating or preventing macular degeneration in a subject, the fusion protein comprising: a) a nucleic acid binding molecule capable of binding to (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a target sequence in the IRAK3 gene; b) one or more DNA demethylating agents; wherein the fusion protein is capable of increasing IRAK-M expression in a target cell of a subject.

[0054] Also provided are methods for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding a fusion protein, the fusion protein comprising (i) a nucleic acid binding molecule capable of binding to (1) a target sequence in a promoter sequence of the IRAK3 gene, (2) a target sequence in a regulatory sequence of the IRAK3 gene, or (3) a target sequence in the IRAK3 gene, and (ii) an epitope repeat array; and b) one or more nucleic acid sequences encoding epitope-binding molecules fused to one or more DNA demethylating agents, said epitope-binding molecules being capable of specifically binding to an epitope of an epitope repeat array; wherein the nucleic acid system is capable of increasing IRAK-M expression in a target cell of a subject.

[0055] The nucleic acid binding molecule can be a TAL effector repeat array or a zinc finger array. The transcriptional activator can be the transcriptional activation domain VP64.

[0056] The DNA demethylating agent can be a DNA demethylase enzyme or a fragment thereof. In further embodiments, the DNA demethylating agent is the catalytic domain of TET1. In some embodiments, the DNA demethylating agent is TET1. In some embodiments, the DNA demethylating agent is LESD1.

[0057] Another aspect provides a small molecule for use in a method for treating or preventing macular degeneration in a subject, wherein the small molecule increases endogenous IRAK-M expression in a target cell in the subject.

[0058] The molecule can reduce DNA methylation in the promoter sequence of the IRAK3 gene, the regulatory sequence of the IRAK3 gene, and / or the IRAK3 gene sequence. In some embodiments, the small molecule is EPZ-6438. In some embodiments, the small molecule is azacytidine.

[0059] In some embodiments, the small molecule is ibudilast. The small molecule can enhance the transcription activation activity of a factor (e.g., a polypeptide) that promotes transcription from the IRAK3 promoter. In some embodiments, the small molecule is a glucocorticoid. In some embodiments, the small molecule is cortisol.

[0060] Small molecules reduce mRNA degradation and increase IRAK-M expression, downregulating IRAK-M mRNA transcripts 6 -Methyladenosine (m 6 A) It can reduce the modification. In some embodiments, the small molecule is a METTL3 inhibitor. In some embodiments, the small molecule is STM2457. In some embodiments, the small molecule is Cpd-564. In some embodiments, the small molecule is UZH2.

[0061] A further aspect provides a nucleic acid for use in a method for treating or preventing macular degeneration in a subject, wherein the nucleic acid increases endogenous IRAK-M expression in a target cell in the subject.

[0062] In some embodiments, the nucleic acid inhibits METTL3 expression. The nucleic acid can bind to and downregulate a target sequence in METTL3 mRNA, thereby increasing expression of IRAK-M.

[0063] A further aspect provides a peptide or polypeptide for use in a method for treating or preventing macular degeneration in a subject, wherein the peptide or polypeptide increases endogenous IRAK-M in target cells of the subject.

[0064] The peptide or polypeptide can activate ERK1 / 2 and / or activate PI3K and Akt1. In some embodiments, the peptide or polypeptide is adiponectin. In some embodiments, the peptide or polypeptide is globular adiponectin.

[0065] In some embodiments, IRAK-M expression is increased in target cells comprising an agent capable of increasing endogenous IRAK-M expression compared to comparable cells without the agent. Autophagy flux may be maintained or increased in target cells comprising an agent capable of increasing endogenous IRAK-M expression compared to comparable cells without the agent. Mitochondrial activity may be maintained or increased in target cells comprising a nucleic acid compared to comparable cells without the agent. Production of proinflammatory cytokines may be decreased in target cells comprising an agent compared to comparable target cells without the agent. The proinflammatory cytokine may be selected from the group consisting of GM-CSF and MCP-1.

[0066] Alternatively, or in addition, IRAK-M activity may be increased in the target cell. Thus, the agent may increase the activity of IRAK-M. The following aspects relate to increasing IRAK-M activity in the target cell.

[0067] The agent may promote binding of IRAK-M to IRAK-1 and / or IRAK-4. Alternatively, or in addition, the agent may promote binding of IRAK-M to MyD88. Provided is a small molecule for use in a method for treating or preventing macular degeneration in a subject, where the small molecule increases IRAK-M activity in target cells in the subject.

[0068] The small molecule can stimulate guanylate cyclase (GC), thereby increasing cellular cGMP. In some embodiments, the small molecule is a nitric oxide (NO) donor. In some embodiments, the small molecule is nitric oxide. In some embodiments, the small molecule is riociguat. Alternatively, the small molecule can be cGMP.

[0069] Further provided is a peptide or polypeptide for use in a method for treating or preventing macular degeneration in a subject, wherein the peptide or polypeptide increases IRAK-M activity in a target cell of the subject. In some embodiments, the agent is a polypeptide.

[0070] The peptide or polypeptide may promote binding of IRAK-M to IRAK-1, hi some embodiments, the peptide or polypeptide is α-MSH or a fragment thereof. In some embodiments, IRAK-M activity is increased in target cells comprising an agent capable of increasing IRAK-M activity compared to comparable cells without the agent. Autophagy flux may be maintained or increased in target cells comprising an agent capable of increasing IRAK-M activity compared to comparable cells without the agent. Mitochondrial activity may be maintained or increased in target cells comprising nucleic acid compared to comparable cells without the agent. Production of proinflammatory cytokines may be decreased in target cells comprising an agent capable of increasing IRAK-M activity compared to comparable target cells without the agent. The proinflammatory cytokine may be selected from GM-CSF and MCP-1.

[0071] The macular degeneration can be age-related macular degeneration (AMD). The age-related macular degeneration (AMD) can be dry AMD. In some embodiments, the dry AMD is selected from the group consisting of early dry AMD, intermediate dry AMD, and advanced dry AMD.

[0072] The target cell may be a cell of the retina or choroid. In some embodiments, the target cell is a cell of the retina. The target cell may be a cell of the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor outer segment (POS), or retinal pigment epithelium (RPE). In some embodiments, the target cell is a cell of the RPE.

[0073] The target cell can be a myeloid cell. The myeloid cell can be a retinal myeloid cell. In some embodiments, the target cell is a CD11b + It is a myeloid cell. A further aspect provides a pharmaceutical composition comprising one or more agents described herein. In some embodiments, the pharmaceutical composition is formulated for ocular delivery.

[0074] The present invention includes combinations of the described aspects and preferred features except where such combinations are expressly not permitted or are expressly avoided. Next, embodiments and experiments illustrating the principles of the present invention will be described with reference to the accompanying drawings. [Brief description of the drawings]

[0075] [Figure 1A-B] IRAK-M is expressed primarily by the RPE in the retina, and its expression decreases with age and in AMD. Representative confocal images of human retinal sections from a 20-year-old donor (with no documented ocular disease) show predominant immunopositivity for IRAK-M in the pigmented RPE layer (anti-RPE65 staining). DAPI and anti-rhodopsin were used to stain nuclei and photoreceptor outer segments (POS), respectively. [Figure 1C-D] IRAK-M is expressed primarily by the RPE in the retina, and its expression decreases with age and in AMD. Representative confocal images of adult mouse retinal sections showing IRAK-M immunostaining that mostly colocalized with RPE65. [Figure 1E] IRAK-M is expressed primarily by the RPE in the retina, and its expression decreases with age and in AMD. Representative Western blotting and densitometry quantification shows an age-related decrease in IRAK-M expression levels in human RPE / choroid lysates (n=4-6). [Figure 1F]IRAK-M is expressed primarily by the RPE in the retina, and its expression decreases with age and in AMD. Representative Western blotting and densitometry quantification shows an age-related decrease in IRAK-M expression levels in mouse RPE lysates (n=4-6). [Figure 1G] IRAK-M is expressed primarily by the RPE in the retina, and its expression decreases with age and in AMD. Data mining of transcriptome data (GSE99248) shows a significant decrease in IRAK-M mRNA levels in the RPE / choroid / sclera of AMD donors compared to age-matched normal donors. Antisense RNA for IRAK-M showed a non-significant increase in AMD samples (n=7-8). [Figure 2A-B] In vitro oxidative treatment downregulates IRAK-M expression in RPE cells. (A) Human RPE cell line (ARPE-19) was treated with various concentrations of the pro-oxidant, paraquat (PQ), for up to 72 h. LDH release shows a dose-dependent cytotoxic effect of PQ after 72 h (n=3-4). (B) Stressing ARPE-19 with a subtoxic concentration of PQ (0.25 mM) led to a decrease in IRAK-M expression in ARPE19 cells after 72 h (Western blot). [Figure 2C] In vitro oxidative treatment downregulates IRAK-M expression in RPE cells, which is accompanied by (C) increased secretion of the pro-inflammatory cytokines HMGB1 (EIA, n=4-6), IL-18, and GM-CSF, and a decrease in the anti-inflammatory cytokine IL-11 (multiple cytokine array, n=4). [Fig. 2D-E] In vitro oxidative treatment downregulates IRAK-M expression in RPE cells. (D) LDH cytotoxicity assay shows subtoxic doses of PQ in human iPSC-derived RPE (n=5) after 72 h treatment. (E) Western blot shows IRAK-M expression downregulated by 72 h treatment with subtoxic PQ (0.25-0.5 mM). [Fig. 2F-G]In vitro oxidative treatment downregulates IRAK-M expression in RPE cells. LDH assay (F, n = 3–6) and representative Western blot (G) show that 72 h treatment with subtoxic PQ (0.25 mM) reduced IRAK-M expression in human primary RPE cell cultures. [Figure 3A] In vivo oxidative injury leads to a decrease in IRAK-M levels in the RPE. Retinal oxidative stress was induced in C57BL / 6J mice by fundus light induction (100 kLux, 20 min). Western blot analysis of IRAK-M expression in RPE lysates 7 days after oxidative injury (n=4 or 5). [Figure 3B-C] Oxidative injury in vivo leads to a decrease in IRAK-M levels in the RPE. Retinal oxidative stress was induced in C57BL / 6J mice by fundus light induction (100 kLux, 20 min). Representative fundus examination and OCT images obtained on day 14 show the appearance of retinal lesions (red arrows, B) and a surrogate for cell loss indicated by a decrease in outer retinal thickness (light model, C, n=8). [Figure 3D] In vivo oxidative injury leads to a decrease in IRAK-M levels in the RPE. Retinal oxidative stress was induced by intravitreal administration of PQ (2 μl, 1.5 mM) in C57BL / 6J mice. Western blot analysis of IRAK-M expression in RPE lysates 7 days after oxidative injury (n=4 or 5). [Figure 3E-F] Oxidative injury in vivo results in a decrease in IRAK-M levels in the RPE. Retinal oxidative stress was induced by intravitreal administration of PQ (2 μl, 1.5 mM) in C57BL / 6J mice. Representative fundus examination and OCT images obtained on day 14 show the appearance of retinal lesions (red arrows, E) and surrogates of cell loss indicated by both the outer and inner retina (PQ model, F, n = 9-11). [Figure 4A]IRAK-M expression is regulated by the AP-1 transcription factor in RPE. ChIP assays of ARPE-19 showed binding of AP-1 subunits c-Jun and c-Fos proteins to the IRAK-M promoter under resting conditions, which was more pronounced after 24 h of LPS stimulation. [Figure 4B] IRAK-M expression is regulated by the AP-1 transcription factor in the RPE. (B) Western blot and densitometry analysis show decreased c-Jun expression in the RPE of 13- and 19-month-old mice compared to 3 months of age (n=4). [Figure 4C-D] IRAK-M expression is regulated by AP-1 transcription factor in RPE. (C) Downregulation of c-Jun and c-Fos phosphorylation in ARPE-19 treated with PQ for 72 h was examined by Western blot. (D) c-Jun and c-Fos inhibitors downregulate IRAK-M expression. [Figure 4E-F] IRAK-M expression is regulated by AP-1 transcription factor in RPE. (E) Increasing c-Jun expression by CRISPR / Cas9 activating plasmid induces total and phosphorylated c-Jun as well as IRAK-M expression in ARPE-19. (F) LDH assay shows increased sensitivity of ARPE19 in response to PQ when c-Jun or c-Fos is inhibited (n=4). [Figure 4G] IRAK-M expression is regulated by the AP-1 transcription factor in the RPE. IRAK-M knockdown by siRNA exacerbates the effect of PQ in inducing ARPE-19 toxicity (n=4). [Figure 5A] Irak3− / − mice spontaneously develop early retinal abnormalities. In 5-month-old Irak3− / − mice, representative fundus and OCT images show an increased incidence of retinas exhibiting scattered white spots (red arrows), which are not evident at 2 mm. [Figure 5B] Irak3− / − mice spontaneously develop early retinal abnormalities. (B) Representative fundus images showing the time course of the appearance of white spots in the retina of Irak3− / − mice. [Figure 5C-D]Irak3- / - mice spontaneously develop early retinal abnormalities. (C) Time course of retinal spot incidence showed early appearance of retinal white spots in Irak3- / - mice. Each value is the ratio of the number of spotted retinas to the total number of retinas at each time point. (D) Representative fundus and OCT images show that white spots (red arrows) are associated with outer retinal abnormalities (red arrows) in 5-month-old Irak3- / - mice. [Figure 6A-B] Irak3- / - mice develop AMD-like pathology and are more vulnerable to oxidative stress. (A) Z-stack confocal images of retinal flat mounts show an abnormal CD11b+ myeloid population in the outer retina in 5-month-old Irak3- / - mice that is absent in WT counterparts. (B) Subretinal accumulation of CD11b+ cells was assessed by immunostaining on RPE / choroid flat mounts in Irak3- / - mice (n=3-10). [Figure 6C] Irak3- / - mice develop AMD-like pathology and are more vulnerable to oxidative stress.TUNEL staining on flat mounts revealed increased numbers of apoptotic cells in both retina and RPE / choroid tissues of Irak3- / - mice (5 months of age) (n=7-10). [Figure 6D] Irak3− / − mice develop AMD-like pathology and are more vulnerable to oxidative stress. Quantification of OCT images shows a surrogate for retinal cell loss, indicated by outer retinal thinning, in Irak3− / − mice at 12–13 months of age (n=6–12). [Figure 6E] Irak3- / - mice develop AMD-like pathology and are more vulnerable to oxidative stress.Multiplex cytokine arrays showed higher overall levels of serum cytokines in KO versus WT mice (12-13 months of age), with statistically significant increases in TNF-α, MCP-1, and IL-10 (n=5-6). [Figure 6F]Irak3- / - mice develop AMD-like pathology and are more vulnerable to oxidative stress. Eight-week-old WT and KO mice were subjected to retinal oxidative injury via light induction. Quantification of retinal thickness by OCT shows excessive thinning of the outer and inner retinal layers in KO mice by light induction (F) (n=8-24). [Figure 6G] Irak3- / - mice develop AMD-like pathology and are more vulnerable to oxidative stress. Eight-week-old WT and KO mice were subjected to retinal oxidative injury via intravitreal PQ injection. Quantification of retinal thickness by OCT shows reduced inner retinal thickness in PQ-induced KO mice after 14 days (n=8-24). [Figure 7A] Loss of IRAK-M in RPE cells results in impaired RPE cell homeostasis. Primary RPE cells isolated from WT or Irak3- / - mice (5 months old) were subjected to mitochondrial stress test using a Seahorse XFp analyzer, and metabolic parameters calculated from OCR (A) and ECAR (B) profiles showed reduced mitochondrial basal respiration (BR) and ATP production in KO-RPE despite no significant differences in maximal respiration (MR), proton leak (H+), non-mitochondrial respiration (NMR), and basal (BG) and maximal glycolytic capacity (MGC) between WT and KO-RPE cells (n=3). (C) Pulsed PQ or H2O2 treated (2 h treatment per day repeated for a total of 7 days) mouse primary RPE cells were analyzed for induction of senescence using a fluorescence-based SA-β-Gal assay. Mean fluorescence intensity quantified by ImageJ shows the induced SA-β-Gal signal in KO cells (n=9). Oxidative stress induction and Irak3− / − accelerated RPE senescence was also confirmed by increased p21 and decreased Lamin-B1 (D), as well as enhanced secretion of proinflammatory cytokines IL-6 (E, n=4) and HMGB1 (F, n=4). [Figure 7B]Loss of IRAK-M in RPE cells results in impaired RPE cell homeostasis. Primary RPE cells isolated from WT or Irak3- / - mice (5 months old) were subjected to mitochondrial stress test using a Seahorse XFp analyzer, and metabolic parameters calculated from OCR (A) and ECAR (B) profiles showed reduced mitochondrial basal respiration (BR) and ATP production in KO-RPE despite no significant differences in maximal respiration (MR), proton leak (H+), non-mitochondrial respiration (NMR), and basal (BG) and maximal glycolytic capacity (MGC) between WT and KO-RPE cells (n=3). (C) Pulsed PQ or H2O2 treated (2 h treatment per day repeated for a total of 7 days) mouse primary RPE cells were analyzed for induction of senescence using a fluorescence-based SA-β-Gal assay. Mean fluorescence intensity quantified by ImageJ shows the induced SA-β-Gal signal in KO cells (n=9). Oxidative stress induction and Irak3− / − accelerated RPE senescence was also confirmed by increased p21 and decreased Lamin-B1 (D), as well as enhanced secretion of proinflammatory cytokines IL-6 (E, n=4) and HMGB1 (F, n=4). [Figure 7C-D]Loss of IRAK-M in RPE cells results in impaired RPE cell homeostasis. Primary RPE cells isolated from WT or Irak3- / - mice (5 months old) were subjected to mitochondrial stress test using a Seahorse XFp analyzer, and metabolic parameters calculated from OCR (A) and ECAR (B) profiles showed reduced mitochondrial basal respiration (BR) and ATP production in KO-RPE despite no significant differences in maximal respiration (MR), proton leak (H+), non-mitochondrial respiration (NMR), and basal (BG) and maximal glycolytic capacity (MGC) between WT and KO-RPE cells (n=3). (C) Pulsed PQ or H2O2 treated (2 h treatment per day repeated for a total of 7 days) mouse primary RPE cells were analyzed for induction of senescence using a fluorescence-based SA-β-Gal assay. Mean fluorescence intensity quantified by ImageJ shows the induced SA-β-Gal signal in KO cells (n=9). Oxidative stress induction and Irak3− / − accelerated RPE senescence was also confirmed by increased p21 and decreased Lamin-B1 (D), as well as enhanced secretion of proinflammatory cytokines IL-6 (E, n=4) and HMGB1 (F, n=4). [Figure 7E-F]Loss of IRAK-M in RPE cells results in impaired RPE cell homeostasis. Primary RPE cells isolated from WT or Irak3- / - mice (5 months old) were subjected to mitochondrial stress test using a Seahorse XFp analyzer, and metabolic parameters calculated from OCR (A) and ECAR (B) profiles showed reduced mitochondrial basal respiration (BR) and ATP production in KO-RPE despite no significant differences in maximal respiration (MR), proton leak (H+), non-mitochondrial respiration (NMR), and basal (BG) and maximal glycolytic capacity (MGC) between WT and KO-RPE cells (n=3). (C) Pulsed PQ or H2O2 treated (2 h treatment per day repeated for a total of 7 days) mouse primary RPE cells were analyzed for induction of senescence using a fluorescence-based SA-β-Gal assay. Mean fluorescence intensity quantified by ImageJ shows the induced SA-β-Gal signal in KO cells (n=9). Oxidative stress induction and Irak3− / − accelerated RPE senescence was also confirmed by increased p21 and decreased Lamin-B1 (D), as well as enhanced secretion of proinflammatory cytokines IL-6 (E, n=4) and HMGB1 (F, n=4). [Figure 8A]Induction of IRAK-M expression in RPE cells preserves cell homeostasis and function against stress. OCR analysis shows that increasing endogenous IRAK-M expression by CRISPR / Cas9-activating plasmid in human iPSC-RPE cells maintains both mitochondrial respiration and glycolytic capacity upon 24-h treatment with 30 μM HO or 1 μg / ml LPS (n=3-7). (C) Stable transfectant cell lines selected from mouse B6-RPE07 that persistently express human IRAK-M were established. Measurement of LDH release from cells over 5 days from confluence shows sustained cell viability due to expression of the human IRAK-M transgene. (D) Human IRAK-M expression also reduces chronic treatment (72 h) of PQ (125 μM) or LPS (40 ng / ml)-induced cytotoxicity (n=2-4). (E&F) Primary mouse Irak3- / - RPE cells were transiently transfected for human IRAK-M expression, and after 48 hours, cells were stressed with 60 μM H2O2 for another 24 hours. OCR (E) and ECAR (F) analysis show maximal respiration sustained by human IRAK-M. No significant changes in glycolytic capacity were observed. (G) Multiplex cytokine array demonstrated that stable expression of human IRAK-M in B6-RPE07 cells inhibited proinflammatory cytokine secretion in response to stress, including LPS- or PQ-induced GM-CSF, and LPS-induced MCP-1 (n=3). [Figure 8B] Induction of IRAK-M expression in RPE cells preserves cellular homeostasis and function against stress. ECAR analysis shows that increasing endogenous IRAK-M expression by CRISPR / Cas9 activating plasmid in human iPSC-RPE cells preserves both mitochondrial respiration and glycolytic capacity upon 24-hour treatment with 30 μM HO or 1 μg / ml LPS (n=3-7). [Figure 8C-D]Induction of IRAK-M expression in RPE cells preserves cell homeostasis and function against stress. (C) A stable transfectant cell line was established from mouse B6-RPE07 that persistently expresses human IRAK-M. Measurement of LDH release from cells over a 5-day period from confluence shows sustained cell viability due to expression of the human IRAK-M transgene. (D) Human IRAK-M expression also reduces chronic treatment (72 h) of PQ (125 μM) or LPS (40 ng / ml)-induced cytotoxicity (n=2-4). [Figure 8E] Induction of IRAK-M expression in RPE cells preserves cell homeostasis and function against stress. Primary mouse Irak3- / - RPE cells were subjected to transient transfection for human IRAK-M expression, and 48 hours later, cells were stressed with 60 μM H2O2 for another 24 hours. OCR analysis shows that maximal respiration was sustained by human IRAK-M. No significant changes in glycolytic capacity were observed. [Figure 8F] Induction of IRAK-M expression in RPE cells preserves cell homeostasis and function against stress. Primary mouse Irak3- / - RPE cells were subjected to transient transfection for human IRAK-M expression, and 48 hours later, cells were stressed with 60 μM H2O2 for another 24 hours. ECAR analysis shows that maximal respiration was sustained by human IRAK-M. No significant changes in glycolytic capacity were observed. [Figure 8G] Induction of IRAK-M expression in RPE cells maintains function in cell homeostasis and stress. A multiplex cytokine array demonstrated that stable expression of human IRAK-M in B6-RPE07 cells inhibited proinflammatory cytokine secretion in response to stress, including LPS- or PQ-induced GM-CSF, and LPS-induced MCP-1 (n=3). [Figure 9] A second Western blot of human RPE / choroid lysates showing IRAK-M expression at different ages. For samples (marked in red) included in both blots (FIG. 1E and FIG. 9), the average IRAK-M expression level was used for quantitative analysis. [Figure 10A] Enrichment analysis of DEGs of RPE / choroid / sclera from AMD by Metascape. Heatmap shows the top 20 enriched clusters in downregulated mRNAs. Downregulated mRNAs or upregulated antisense RNAs suggest decreased gene expression. Enriched terms can be GO / KEGG terms, canonical pathways, reactome gene sets and WikiPathways. Enriched clusters are listed in order of greatest statistical significance. [Figure 10B] Enrichment analysis of DEGs of RPE / choroid / sclera from AMD by Metascape. Heatmap shows the top 20 enriched clusters in upregulated mRNAs. Upregulated mRNAs or downregulated antisense RNAs suggest increased gene expression. Enriched terms can be GO / KEGG terms, canonical pathways, reactome gene sets and WikiPathways. Enriched clusters are listed in order of greatest statistical significance. [Figure 10C] Enrichment analysis of DEGs of RPE / choroid / sclera from AMD by Metascape. Heatmap shows the top 20 enriched clusters in downregulated antisense RNA. Upregulated mRNA or downregulated antisense RNA suggests increased gene expression. Enriched terms can be GO / KEGG terms, canonical pathways, reactome gene sets and WikiPathways. Enriched clusters are listed in order of greatest statistical significance. [Figure 10D] Enrichment analysis of DEGs of RPE / choroid / sclera from AMD by Metascape. Heatmap shows the top 20 enriched clusters in upregulated antisense RNA. Downregulated mRNA or upregulated antisense RNA (A and D) indicate decreased gene expression. Enriched terms can be GO / KEGG terms, canonical pathways, reactome gene sets and WikiPathways. Enriched clusters are listed in order of greatest statistical significance. [Figure 11A]Data mining of an RNA-seq dataset (GSE99248) to compare normalized counts of IRAK family members (IRAK1, IRAK2, and IRAK4) mRNA in RPE / choroid / sclera tissues between AMD and controls. Genes were considered differentially expressed (DEGs) if P<0.05 and fold change >±2. [Figure 11B] Data mining of an RNA-seq dataset (GSE99248) to compare normalized counts of antisense RNA for IRAK family members (IRAK1, IRAK2, and IRAK4) in RPE / choroid / sclera tissues between AMD and controls. Genes were considered differentially expressed (DEGs) if P<0.05 and fold change>±2. [Figure 12] Data mining of an RNA-seq dataset (GSE99248) to compare normalized counts of JUN mRNA (A) and antisense RNA (B) in RPE / choroid / sclera tissues between AMD and controls. Genes were considered as differentially expressed (DEGs) if P < 0.05 and fold change > ±2. [Figure 13] (A) Overexpression of c-Jun in ARPE-19 cells does not protect cells from PQ-induced cell damage. Cells were transfected with c-Jun or IRAK-M CRISPR / Cas9 activation plasmids or control plasmids for 48 h and subsequently treated with a toxic dose of PQ (1 mM) for another 48 h. Cell culture supernatants were harvested to measure LDH release as an indicator of cytotoxicity (n=3–4). (B) Increase in endogenous IRAK-M expression in human iPSC-RPE cells by CRISPR / Cas9-based activation plasmids. Cells were transfected with plasmids for 48 h and cell lysates were prepared for Western blotting analysis of protein expression. Transfection of vehicle CRISPR / Cas9 plasmid was used as a control. [Figure 14A]Induction of IRAK-M expression in ARPE-19 cells maintains cellular homeostasis against stress. Western blot analysis of IRAK-M in cells after 48 h of transfection with CRISPR / Cas9 activating plasmid. [Figure 14B] Induction of IRAK-M expression in ARPE-19 cells maintains cellular homeostasis against stress. OCR profile and parameter analysis (n=3 or 4) showing that inhibition of LPS partially caused a decrease in maximal respiration (MR) by IRAK-M overexpression. [Figure 14C] Induction of IRAK-M expression in ARPE-19 cells maintains cellular homeostasis in response to stress. (C) ECAR profiles and parameter analysis (n=3 or 4) showing basal and maximal glycolytic activity (BG and MGC) induced by IRAK-M overexpression when cells were challenged with H2O2. [Fig. 14D-E] Induction of IRAK-M expression in ARPE-19 cells maintains cellular homeostasis against stress. (D) Autophagy tandem LC3B-GFP-RFP sensor assay and confocal images demonstrate that overexpression of IRAK-M enhances the formation of LC3B-autophagosomes (green) and LC3B-autolysosomes (red) to combat H2O2- or LPS-induced stress (n=20–25). PQ-induced cellular senescence is inhibited by IRAK-M overexpression, as evidenced by reduced SA-β-Gal activity (n=20, E) and HMGB1 release (n=6, F). [Fig. 14F-G] Induction of IRAK-M expression in ARPE-19 cells maintains cellular homeostasis in response to stress. PQ-induced cellular senescence is inhibited by IRAK-M overexpression, as evidenced by decreased SA-β-Gal activity (n=20, E) and HMGB1 release (n=6, F). (G) LDH cytotoxicity assay showing that IRAK-M overexpression inhibits PQ (1 mM)-induced cytotoxicity (n=4). [Figure 15A]Stable transfected cell colonies were selected from the mouse B6-RPE07 cell line, and two new cell lines were established to persistently express human and mouse IRAK-M, respectively. Another new cell line carried a vehicle plasmid (pUNO1) as a transfection control. qRT-PCR analysis showed strong expression of human or mouse IRAK-M genes in the cell lines, with no change in the expression of IRAK1 and IRAK4. [Figure 15B] Stable transfected cell colonies were selected from mouse B6-RPE07 cell line and two new cell lines were established to persistently express human and mouse IRAK-M, respectively. Another new cell line carried a vehicle plasmid (pUNO1) as a transfection control. To activate TLR4 signaling, the cell lines were stimulated with 1 μg / ml LPS for 30 min. NF-κB activity assay shows a decrease in nuclear NF-κB activity in cells expressing human or mouse IRAK-M (n=2). [Figure 16A] (A) Western blot analysis of the time course of total and phosphorylated c-Jun expression in ARPE-19 cells treated with PQ, H2O2, or the c-Jun inhibitor SP600125. [Figure 16B] (B) A dot plot of the BLAST sequence alignment showed close similarity in sequence between human IRAK-M (Q9Y616) and mouse IRAK-M (Q8K4B2). [Figure 17] IHC analysis of human retinal sections reveals decreased IRAK-M expression primarily in the macular RPE and choroid in aging and AMD. Mean staining intensity of IHC images of human retinal sections from two non-AMD donor eyes (a 59-year-old female and a 97-year-old female, respectively), an early AMD donor (a 95-year-old female), and a mild AMD donor demonstrate that IRAK-M expression is more severely decreased in the macular RPE in both aging and AMD, but decreased expression in the choroid is only significant in aging, whereas retinal changes are not significant (n=2 for young controls, n=5 for aged controls, and n=11 for AMD). Scale bar=100 μm. [Figure 18]AAV2 serotypes dose-dependently transduce the retina following subretinal injection. A total of 2 x 109 or 4 x 108 genome copies (gc) of AAV2 encoding EGFP driven by the CMV promoter were injected into the subretinal space per eye of 8-week-old mice. From 1 to 11 weeks post-injection, viral dose-dependent retinal transduction was examined by in vivo fundus fluorescence imaging using a Micron IV. [Figure 19A] Subretinal delivery of AAV2.CMV.hIRAK3 induces human IRAK3 expression in mouse RPE. (A) Two weeks after subretinal injection of AAV2.CMV.hIRAK3 or null AAV2.CMV (2x109 or 4x108 gc / eye), RPE / choroid and retinal tissues were analyzed for human and mouse IRAK3 mRNA expression by quantitative RT-PCR. Relative quantification of gene expression (RQ, Log10 transformation) was normalized to mouse RPS29 mRNA (n=5). [Figure 19B] Subretinal delivery of AAV2.CMV.hIRAK3 induces human IRAK3 expression in mouse RPE. (B) Virus-induced human IRAK-M protein expression was examined in mouse retinal cryosections by immunofluorescence staining with an antibody specific for only human IRAK-M or an antibody that recognizes both human and mouse IRAK-M. Representative confocal images confirm the increase in IRAK-M protein in the RPE. Scale bar = 50 μm. [Figure 20A]Subretinal delivery of AAV2.CMV.hIRAK3 inhibits light-induced outer retinal thinning. Two weeks after subretinal injection of hIRAK3 or control virus (2 × 109 gc / eye), mice were subjected to light-induced retinal degeneration in one mouse eye and then left for an additional two weeks to assess retinal damage and response to treatment. (A) Representative fundus examination and OCT images obtained 14 days after light induction show light-induced focal outer retinal lesions. (B) Each value of outer retinal thickness was the average thickness of 200-800 µm from the ONH overlying the light-affected area, measured from OCT images using an ImageJ macro. Quantification of the data shows the protective effect of AAV2.CMV.hIRAK3 treatment against light-induced photoreceptor loss, as shown by inhibiting outer retinal thinning (n = 10-11). [Figure 20B] Subretinal delivery of AAV2.CMV.hIRAK3 inhibits light-induced outer retinal thinning. Two weeks after subretinal injection of hIRAK3 or control virus (2 × 109 gc / eye), mice were subjected to light-induced retinal degeneration in one mouse eye and then left for an additional two weeks to assess retinal damage and response to treatment. (A) Representative fundus examination and OCT images obtained 14 days after light induction show light-induced focal outer retinal lesions. (B) Each value of outer retinal thickness was the average thickness of 200-800 µm from the ONH overlying the light-affected area, measured from OCT images using an ImageJ macro. Quantification of the data shows the protective effect of AAV2.CMV.hIRAK3 treatment against light-induced photoreceptor loss, as shown by inhibiting outer retinal thinning (n = 10-11). [Figure 21]AAV2.CMV.hIRAK3 gene therapy prevents light-induced retinal cell death. Two weeks after subretinal injection of hIRAK3 or null AAV2.CMV vectors (2x109gc / eye), mice were subjected to light-induced retinal degeneration in one eye and maintained for an additional two weeks. Eyes were harvested and sections were processed for TUNEL staining and analysis. DAPI was used to stain cell nuclei. The number of TUNEL+ cells (identified using confocal images showing apoptotic cells (TUNEL positive) in the retinas of different groups) was quantified and averaged from three sections of each eye of three or six mice per group. Scale bar = 100 μm. [Figure 22] AAV2.CMV.hIRAK3 gene therapy protects photoreceptor mitochondria from light damage. Two weeks after light induction, mouse retinal cryosections were fixed for MitoView Green staining to assess mitochondrial content. Slides were counterstained with DAPI. MFI analysis of MitoView Green stained confocal images shows a decrease in mitochondrial content of PR inner segments due to light damage in control AAV2-injected eyes, which is significantly inhibited by AAV2.CMV.hIRAK3 treatment. Graph shows the average of MFI measured in three different fields from two sections of 3–6 mice. Scale bar = 100 µm. [Figure 23A-B] AAV2.CMV.hIRAK3 gene therapy reduces retinal plaques in Irak3- / - mice. 2 x 109 gc of AAV2.CMV.hIRAK3 were injected into the subretinal space of one eye of Irak3- / - mice (2-4 months old) and control AAV2 was injected into the contralateral eye. Mice were then housed under normal conditions for 6 months and the retina was evaluated using fundus examination and OCT at the indicated time points. (A) In Irak3- / - mice (8 months old) administered AAV at 2 months of age, representative fundus images show retinal white spots. A line divides the retina into two sides based on the injection site. (B) The time course of the incidence of spotted retina (number of spots >3) shows that IRAK3 gene delivery significantly slowed the appearance of retinal spots in IRAK3-KO mice during aging. Each value is the ratio of the number of spotted retina to the total number of retinas (n = 15 or 16) at each time point. [Fig. 23C-D]AAV2.CMV.hIRAK3 gene therapy reduces retinal plaques in Irak3− / − mice. 2×109 gc of AAV2.CMV.hIRAK3 were injected into the subretinal space of one eye of Irak3− / − mice (2–4 months old) and control AAV2 was injected into the contralateral eye. Mice were then housed under normal conditions for 6 months and the retina was evaluated using fundus examination and OCT at the indicated time points. (C) Retinal plaque counts were performed blindly on the whole retina or (D) the injected side for comparison of AAV2.CMV.hIRAK3- and null vector-treated eyes in aged KO mice (8–10 months old). [Figure 24] AAV2.CMV.hIRAK3 gene therapy inhibits retinal thinning in aged IRAK3- / - mice. Irak3- / - mice (2-4 months old) were administered 2x109gc of AAV2.CMV.hIRAK3 or null AAV2.CMV subretinally. Outer retinal thickness was averaged from the temporal and nasal sides of OCT images for all animals in each group. Quantification of the data showed a reduction in outer retinal thickness in the central region 200 μm away from the optic nerve head in aged Irak3- / - mice (8-10 months old) compared to age-matched WT mice, which was significantly restored by IRAK3 gene therapy. [Fig. 25A-B] Putative endogenous promoter sequence of human IRAK3 gene. Three fragments before exon 1 of human IRAK-M gene (ENSG00000090376) were selected. (A) The 0.88 kb fragment is the predicted "core promoter" containing CpG islands and H3K methylation marks. (B) The 1.36 kb fragment is the predicted maximum promoter size that the AAV backbone CMV.GFP.WPRE.io2 can accommodate given IRAK3 gene size plus WPRE and io2 elements. [Figure 25C] The predicted endogenous promoter sequence of the human IRAK3 gene. Three fragments before exon 1 of the human IRAK-M gene (ENSG00000090376) were selected. The 1.6 kb fragment is the largest promoter size that an AAV vector can generally accommodate for a given IRAK3 gene size. [Figure 26A-B]Comparison of AAV2-mediated IRAK3 gene delivery and protective effects of different promoters in RPE cells and LIRD. (A-C) Murine B6-RPE07 cells were transduced with null AAV2.CMV, AAV2.CMV.hIRAK3, AAV2.Best1.hIRAK3, AAV2.Endo1.hIRAK3, AAV2.Endo2.hIRAK3 or AAV2.Endo3.hIRAK3 (MOI 50,000gc). Three days after gene transduction, (A) exogenous human IRAK3 and (B) endogenous murine IRAK3 mRNA expression was analyzed by qPCR (n=4). [Fig. 26C-D] Comparison of different promoters for AAV2-mediated IRAK3 gene delivery and protective effect in RPE cells and LIRD. (A-C) Murine B6-RPE07 cells were transduced with null AAV2.CMV, AAV2.CMV.hIRAK3, AAV2.Best1.hIRAK3, AAV2.Endo1.hIRAK3, AAV2.Endo2.hIRAK3 or AAV2.Endo3.hIRAK3 (MOI 50,000gc). Three days after gene transduction, (C) cytotoxicity was measured by LDH assay (n=4). (D) Considering the greater transgene expression by Endo3 promoter compared to Endo1 and Endo2 promoters, human ARPE-19 cells were transduced with AAV2.Endo3.hIRAK3 compared to AAV2.CMV.hIRAK3 and AAV2.Best1.hIRAK3, followed by paraquat treatment for 4 days. The LDH assay was used to assess cytotoxicity in response to oxidative stressors (n=4). [Figure 26E] Comparison of different promoters for AAV2-mediated IRAK3 gene delivery and protective effects in RPE cells and LIRD. (E) Subretinal delivery of AAV2.hIRAK3 with different promoters suppresses light-induced outer retinal thinning. Three to five weeks after subretinal injection of the indicated AAV2 (2 × 109 gc / eye), mice were subjected to LIRD and then left for up to two weeks for OCT evaluation. Data show the percentage of ORL thinning relative to control retinas without light induction (average from 8–9 eyes / group). [Figure 27]AAV5 induces human IRAK-M gene expression in RPE cells in a dose-dependent manner. Murine B6-RPE07 cells were transduced with null AAV5.CMV or AAV5.CMV.hIRAK3 at an MOI of 50,000 or 100,000 gc. Three days after transduction, (A) exogenous human IRAK3 and (B) endogenous murine IRAK3 mRNA expression was analyzed by qPCR (n=3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0077] Age-related macular degeneration (AMD) is a progressive degenerative disease. Impairment of nutritional, immune, and metabolic functions of the retinal pigment epithelium (RPE) and the surrounding microenvironment typically results in macular damage, RPE and photoreceptor (PR) loss, and gradual loss of central vision (94, 95). Accumulation of lipoproteinaceous drusen deposits in early nonexudative (dry) AMD can develop into a geographic atrophy (late dry) stage or a neovascular exudative (wet) stage (96). Despite the increasing burden associated with the rapidly increasing prevalence of AMD due to the aging population, treatment options to prevent RPE and PR loss in AMD are lacking. The unmet need for treatment of dry AMD is urgent.

[0078] Alongside chronological aging, the interplay between oxidative stress and chronic inflammation resulting from genotype-predisposing susceptibility and environmental stressors are major contributing factors in AMD. Inflammation-induced drusen formation is not only a hallmark of early AMD but also a key source of proinflammatory factors that trigger the breakdown of macular function (97). Central inflammatory players involved in AMD progression include dysregulated complement cascade components, inflammasome activation, cytokines and immune-responsive cells including dendritic cells, microglia, macrophages and RPE (97, 98).

[0079] The RPE is highly susceptible to disruptions in aging (19) and inflammatory stressors. Impairments in RPE intracellular process pathways, including autophagy, phagolysosome, mitochondrial metabolism, protein trafficking, and aging, coupled with oxidative stress, result in further inflammation (23). The magnitude of the inflammatory response is balanced by inhibitory mechanisms, such as regulation by interleukin-1 receptor-associated kinase-M (IRAK-M), encoded by the IRAK3 gene (28). Dysregulation and impairment of IRAK-M signaling are associated with oxidative stress and systemic inflammation, which are involved in metabolic disorders such as insulin resistance and obesity, all of which are associated with age-related eye diseases, including AMD (31, 99, 100).

[0080] The present invention is based on the finding that IRAK-M expression in RPE is decreased by aging and subsequent oxidative stress. Reduced IRAK-M expression has also been identified in the eyes of AMD patients. The inventors have found that reduced IRAK-M expression impairs the ability of cells to maintain function and health. Increasing IRAK-M expression provides protection to RPE and retina. Thus, the present invention relates to increasing IRAK-M expression for the prevention and treatment of macular degeneration.

[0081] Several strategies can be used to achieve increased IRAK-M expression in a subject, which are described in more detail below. Medical Use Thus, the present invention relates to an agent as described herein for increasing IRAK-M expression in a target cell and / or increasing IRAK-M activity in a target cell for use in a method for treating or preventing macular degeneration in a subject.

[0082] An option for increasing IRAK-M expression in a target cell may involve introducing exogenous IRAK-M into the target cell. Thus, the agent may be an IRAK-M polypeptide or a nucleic acid encoding IRAK-M. In some embodiments, the agent is heterologous. Another option for increasing IRAK-M expression involves increasing endogenous IRAK-M expression in the target cell. Thus, the agent may increase endogenous IRAK-M expression.

[0083] Alternatively, the agent may increase the activity of IRAK-M in the target cell. Further provided is a method for treating or preventing macular degeneration in a subject, the method comprising administering to the subject an agent described herein. Further provided is the use of an agent described herein for the manufacture of a medicament for treating or preventing macular degeneration in a subject.

[0084] The invention also relates to a nucleic acid, vector virion, polypeptide, nucleic acid system, viral vector system, or pharmaceutical composition for use in a method for treating or preventing macular degeneration in a subject, wherein the nucleic acid increases IRAK-M expression in a target cell of the subject.

[0085] Also provided is a method for treating or preventing macular degeneration in a subject, comprising administering to the subject a nucleic acid, vector virion, polypeptide, nucleic acid system, viral vector system, or pharmaceutical composition described herein. In another aspect, there is provided a use of a nucleic acid, vector virion, polypeptide, nucleic acid system, viral vector system, or pharmaceutical composition described herein for the manufacture of a medicament for treating or preventing macular degeneration in a subject.

[0086] Macular degeneration Macular degeneration is a medical condition that can cause vision loss, with the central field of vision becoming blurred or completely lost. The term "macular degeneration" refers to any of several conditions in which the retina macula degenerates or becomes dysfunctional, for example, as a result of reduced proliferation of macular cells, increased death or rearrangement of macular cells (e.g., RPE cells), loss of normal biological function, or a combination of these events.

[0087] In particular, the present invention relates to age-related macular degeneration. As used herein, "age-related macular degeneration" or "AMD" includes early, intermediate, and advanced / late AMD, including 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), the supportive monolayer of cells underlying the photoreceptors, is commonly seen in AMD patients. The retinal pigment epithelium (RPE) is a multifunctional monolayer of neuroepithelial-derived cells adjacent to the photoreceptor (PR) cells and choroid complex. The RPE is typically composed of a monolayer of hexagonal cells densely packed with pigment granules.

[0088] In some embodiments, the macular degeneration is age-related macular degeneration. Typically, AMD involves the progressive accumulation of characteristic yellow deposits called drusen in the macula (part of the retina) between the RPE and the underlying choroid. Drusen are formed from extracellular proteins and lipids. The accumulation of drusen damages the retina over time. AMD can be divided into three stages: early, intermediate, and late, based in part on the extent (size and number) of drusen. In some embodiments, application of the treatment of the present invention results in a reduction of drusen in target cells compared to cells not including the treatment of the present invention. In some embodiments, application of the treatment of the present invention to target cells prevents the formation of drusen in target cells compared to cells not including the treatment of the present invention.

[0089] In some embodiments, the age-related macular degeneration (AMD) is early AMD. Early AMD is usually diagnosed based on the presence of medium-sized drusen. Early AMD tends to be asymptomatic. In some embodiments, the age-related macular degeneration (AMD) is intermediate AMD. Intermediate AMD is typically diagnosed by large drusen and / or retinal pigmentary abnormalities. Intermediate AMD may result in some vision loss, but is generally asymptomatic. In some embodiments, the age-related macular degeneration (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 may be caused by atrophy or by the development 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.

[0090] In some embodiments, the age-related macular degeneration (AMD) is dry AMD. Dry AMD encompasses all forms of AMD that are not wet AMD, including early and intermediate AMD, and an advanced form of dry AMD called geographic atrophy. In some embodiments, the age-related macular degeneration (AMD) is geographic atrophy. Geographic atrophy, also known as atrophic AMD, is an advanced form of dry AMD. It is characterized by progressive and irreversible loss of retinal cells, resulting in loss of visual function. Typically, three areas of the retina atrophy in geographic atrophy. These are the choriocapillaris, the retinal pigment epithelium, and the overlying photoreceptors.

[0091] In contrast, wet AMD (also called neovascular or exudative AMD) is a wet form of advanced AMD. It is characterized as vision loss due to abnormal blood vessel growth in the choriocapillaris through Bruch's membrane (choroidal neovascularization). It is usually, but not always, preceded by dry AMD. The growth 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, leading to blood and protein leakage below the macula. These blood vessels bleed, leak, and scar, eventually causing irreversible damage to photoreceptors, and if left untreated, rapid vision loss occurs. In some embodiments, the age-related macular degeneration (AMD) is not wet AMD. In some embodiments, the age-related macular degeneration (AMD) is dry AMD, excluding wet AMD.

[0092] Typically, in patients with macular degeneration, the retina and choroid are affected. Thus, in some embodiments, the target cell is a cell of the retina or choroid. In some embodiments, the target cell is a cell of the retina. The retina is the innermost light-sensitive tissue in the eye. The retina comprises several layers, including a layer that contains photoreceptors. The main functional layers of the retina are the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor outer segment (POS), the retina that supports the retina, and the retinal pigment epithelium (RPE). In some embodiments, the target cell is a cell of the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor outer segment (POS), or retinal pigment epithelium (RPE). In some embodiments, the target cell is a cell of the retinal pigment epithelium (RPE).

[0093] In some embodiments, the target cells are myeloid cells. In some embodiments, the myeloid cells are retinal myeloid cells. In some embodiments, the target cells are CD11b + Myeloid cells. CD11b + The cells include yolk sac-derived tissue-resident microglia, which are specialized in maintaining retinal tissue and neuronal homeostasis.

[0094] In some embodiments, a method for treating or preventing macular degeneration in a subject comprises contacting a target cell or tissue with an agent described herein. In some embodiments, a method for treating or preventing macular degeneration in a subject comprises contacting a target cell or tissue with a nucleic acid, vector virion, polypeptide, nucleic acid system, viral vector system, or pharmaceutical composition described herein.

[0095] IRAK-M IRAK-M is an inactive kinase encoded by the IRAK3 gene. Specifically, IRAK-M (also known as IRAK3) is a cytoplasmic pseudokinase that belongs to the IRAK family. The IRAK family consists of two active kinases (IRAK-1 and IRAK-4) and two inactive kinases (IRAK-2 and IRAK-M).

[0096] IRAK-M is a negative regulator of the TLR / IL-1R-induced proinflammatory cascade. IRAK-M prevents the dissociation of IRAK-1 and IRAK-4 from MyD88 and the formation of the IRAK-1-TRAF6 complex, thus preventing downstream TLR / IL-1R signaling.

[0097] Exogenous IRAK-M polypeptides and / or peptides can be delivered directly to the cytoplasm of ocular cells. Thus, aspects of the invention provide IRAK-M polypeptides and / or peptides for use in a method for treating or preventing macular degeneration in a subject.

[0098] The human sequence of IRAK-M is provided below (UniProt Q9Y616). SEQ ID NO:1: MAGNCGARGALSAHTLLFDLPPALLGELCAVLDSCDGALGWRGLAERLSSSWLDVRHIEKYVDQGKSGTRELLWSWAQKNKTIGDLLQVLQEMGHRRAIHLITNYGAVLSPSEKSYQEGGFPNILFKETANVTVDNVLIPEHNEKGILL KSSISFQNIIEGTRNFHKDFLIGEGEIFEVYRVEIQNLTYAVKLFKQEKKMQCKKHWKRFLSELEVLLLFHHPNILELAAYFTETEKFCLIYPYMRNGTLFDRLQCVGDTAPLPWHIRIGILIGISKAIHYLHNVQPCSVICGSISSAN ILLDDQFQPKLTDFAMAHFRSHLEHQSCTINMTSSSSKHLWYMPEEYIRQGKLSIKTDVYSFGIVIMEVLTGCRVVLDDPKHIQLRDLLRELMEKRGLDSCLSFLDKKVPPCPRNFSAKLFCLAGRCAATRAKLRPSMDEVLNTLESTQ ASLYFAEDPPTSLKSFRCPSPLFLENVPSIPVEDDESQNNNLLPSDEGLRIDRMTQKTPFECSQSEVMFLSLLDKKPESKRNEEACNMPSSSCEESWFPKYIVPSQDLRPYKVNIDPSSEAPGHSCRSRPVESSCSSKFSWDEYEQYKKE An embodiment provides a polypeptide for use in a method for treating or preventing macular degeneration in a subject, the polypeptide comprising an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0099] In some embodiments, the polypeptide has an amino acid sequence that has 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: 1. In some embodiments, the amino acid sequence of the polypeptide consists of SEQ ID NO:1.

[0100] In some embodiments, the nucleic acid or vector virion comprising the nucleic acid comprises a nucleic acid sequence encoding a polypeptide having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid or vector virion comprising the 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: 1. In some embodiments, the nucleic acid or vector virion comprising the nucleic acid comprises a nucleic acid sequence consisting of SEQ ID NO:1.

[0101] Percent (%) identity of an amino acid sequence with respect to a reference sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum % sequence identity, without 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).

[0102] In some embodiments, the polypeptide of the present invention is formulated for ocular delivery. In some embodiments, the polypeptide of the present invention is functional. In some embodiments, the polypeptide can prevent dissociation of IRAK-1 and / or IRAK-4 from MyD88 in target cells. In some embodiments, the polypeptide can prevent formation of IRAK-1-TRAF6 complex.

[0103] We found that increased IRAK-M expression provided protection to the RPE and retina more generally. Introduction of a human IRAK-M transgene into mouse RPE preserved mitochondrial activity and promoted cell survival under oxidative stress. We also identified an increase in autophagic flux associated with increased IRAK-M expression.

[0104] In some embodiments, the autophagy flux is increased in the target cells compared to cells that are not modified with an agent described herein (e.g., a small molecule, a nucleic acid, a vector virion, a polypeptide, a nucleic acid system, a viral vector system, or a pharmaceutical composition described herein). Autophagy flux can be used to determine autophagy activity in cells. By "autophagy flux" is meant the amount of autophagic degradation that occurs in the target cells. Autophagy flux can be measured using a number of different techniques, as described in Yoshii SR and Mizushima N. Int J Mol Sci. 2017 Sep;18(9):1865. For example, autophagy flux can be measured by immunoblotting for LC3-II. LC3-II is commonly used as an autophagosomal marker, as the amount of LC3-II reflects the number of autophagosomes and autophagy-related structures. Similarly, the amount of p62 in tissues can be measured. Since p62 directly binds to LC3-II and is selectively degraded by autophagy, degradation of p62 is another widely used marker to monitor autophagy activity. Also, the dynamics of the autophagy pathway can be measured by fluorescent live-cell imaging using LC3-RFP-GFP tandem sensors combined with lysosomal probes such as Lysotracker or LAMP1 / 2 labels, which allow detection of LC3+neutral pH autophagosomes, LC3+acidic pH autolysosomes, and lysosomal activity.

[0105] In some embodiments, mitochondrial activity is maintained or increased in target cells compared to cells not modified with the agents described herein. Mitochondria are eukaryotic organelles that have their own mitochondrial DNA. Oxygen respiration (aerobic respiration) and ATP production occur in mitochondria. Synthesis of ATP via oxidative phosphorylation is the most common function attributed to mitochondria. This process is typically determined indirectly by measuring mitochondrial oxygen (O2) consumption, or respiration. Mitochondria play a central role in establishing and regulating cellular redox homeostasis. In general, maintaining or increasing mitochondrial activity means maintaining or increasing the expression or activity of electron transport components, ATP synthesis proteins, TCA cycle components, or coproporphyrinogen oxidase (CPOX). It also includes maintaining or increasing ATP synthesis.

[0106] Methods for measuring mitochondrial activity include, but are not limited to, measuring the rate of mitochondrial respiratory O2 flux (including O2-dependent quenching of porphyrin-based fluorophores and amperometric O2 sensors), oxidant release (e.g., fluorescent, chemiluminescent, and electrochemical / nanoparticle-based methods for detecting oxidants), measurement of mitochondrial membrane potential, ATP production by bioluminescence, calcium retention capacity, measurement of mitochondrial NAD(P)H, and the like.

[0107] Inflammation may be reduced in target tissues compared to target tissues that are not modified with the agents described herein. Proinflammatory cytokine production may be reduced in target cells that contain the agent compared to comparable target cells that do not contain the agent. In some embodiments, IRAK-M expressed in target cells inhibits the production of proinflammatory cytokines (e.g., under stress). The proinflammatory cytokines may be selected from the group consisting of GM-CSF and MCP-1. The levels of these proinflammatory cytokines may be measured using techniques such as flow cytometry or Western blot. The concentration of proinflammatory cytokines in target cells may be reduced compared to target cells that are not modified with the agents described herein.

[0108] In some embodiments, the polypeptide is a recombinant polypeptide modified for delivery to target cells. In one example, IRAK-M can be conjugated to a peptide described in Bhattacharya et al. to mediate delivery to RPE cells. Bhattacharya 2017, Journal of Controlled Release, vol. 251, pp. 37-48, describes a peptide-based delivery system that allows controlled cargo release in RPE cells. The system described is typically used for intravitreal administration. Other possible delivery routes are described herein. Peptide-based delivery systems include peptide-based cleavable linkers (PCLs) with cell-penetrating peptides (CPPs) conjugated to the N-terminus, and cargo (e.g., IRAK-M) is conjugated to the C-terminus. Examples of 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 that allow intracellular delivery of molecular cargo.

[0109] Cell penetrating peptides (CPPs) are typically short peptides that facilitate cellular uptake and incorporation of molecules (e.g., polypeptides). CPPs typically deliver cargo into cells via endocytosis. CPPs generally have an amino acid composition that includes a sequence that contains a large amount of positively charged amino acids (e.g., lysine or arginine) or an alternating pattern of polar charged and non-polar hydrophobic amino acids. Examples of 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, and the like. Examples of CPPs for RPE cells are provided in Bhattacharya 2017, Journal of Controlled Release 251, 37-48. Non-limiting examples include GRKKRRQRRPPQ (SEQ ID NO:2), rrrrrrrrr (SEQ ID NO:3), RLVSYNGIIFFLK (SEQ ID NO:4), FNLPLPSRPLLR (SEQ ID NO:5) (wherein "r" is D-Arg). In some embodiments, the CPP further comprises a short flexible linker between the CPP and the PCL. In some embodiments, the short flexible linker has the amino acid sequence GGS. The PCL is a peptide-based cleavable linker. Other cleavable linkers can be used in the context of the present invention. Examples of 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:7), and KGSALISWIKR (SEQ ID NO:8), where "r" is D-Arg.

[0110] Thus, examples of CPPs conjugated to PCL include, but are not limited to, GRKKRRQRRPPQGGSKGKPILFFRLKr (SEQ ID NO: 9), GRKKRRQRRPPQGGSKPILFFRLGK (SEQ ID NO: 10), GRKKRRQRRPPQGGSKGSALISWIKR (SEQ ID NO: 11), rrrrrrrrGGSKGKPILFFRLKr (SEQ ID NO: 12), rrrrrrrrrGGSKPILFFRLGK (SEQ ID NO: 13), rrrr rrrrrGGSKGSALISWIKR (SEQ ID NO: 14), RLVSYNGIIFFLKGGSKGKPILFFRLKr (SEQ ID NO: 15), RLVSYNGIIFFLKGGSKPILFFRLGK (SEQ ID NO: 16), RLVSYNGIIFFLKGGSKGSALISWIKR (SEQ ID NO: 17), FNLPLPSRPLLRGGSKGKPILFFRLKr (SEQ ID NO: 18), FNLPLPSRPLLRGGSKPILFFRLGK (SEQ ID NO: 19), FNL PLPSRPLLRGGSKGSALISWIKR (SEQ ID NO: 20), GRKKRRQRRPPQKGKPILFFRLKr (SEQ ID NO: 21), GRKKRRQRRPPQKPILFFRLGK (SEQ ID NO: 22), GRKKRRQRRPPQKGSALISWIKR (SEQ ID NO: 23), rrrrrrrrrKGKPILFFRLKr (SEQ ID NO: 24), rrrrrrrrrKPILFFRLGK (SEQ ID NO: 25), rrrrrrrrrKGSALISWIKR (SEQ ID NO: SEQ ID NO:26), RLVSYNGIIFFLKKGKPILFFRLKr (SEQ ID NO:27), RLVSYNGIIFFLKKPILFFRLGK (SEQ ID NO:28), RLVSYNGIIFFLKKGSALISWIKR (SEQ ID NO:29), FNLPLPSRPLLRKGKPILFFRLKr (SEQ ID NO:30), FNLPLPSRPLLRKPILFFRLGK (SEQ ID NO:31), and FNLPLPSRPLLRKGSALISWIKR (SEQ ID NO:32).

[0111] Any one of the above peptides can be directly or indirectly conjugated to an IRAK-M polypeptide. In some embodiments, a molecule is provided that includes a PCL having a CPP conjugated to its N-terminus and an IRAK-M polypeptide or peptide (e.g., IRAK-M) conjugated to the C-terminus of the PCL (e.g., CPP-PCL-IRAK-M). In some embodiments, a molecule is provided that includes a PCL having a CPP conjugated to its N-terminus and an IRAK-M polypeptide having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 conjugated to the C-terminus of the PCL (e.g., CPP-PCL-IRAK-M).

[0112] Gene Therapy Gene therapy involves the introduction of genetic material into target cells with the goal of modulating the expression of an altered particular protein, thereby reversing the biological disorder causing that alteration. The present invention contemplates a nucleic acid sequence encoding an IRAK-M protein for use in a method for treating or preventing macular degeneration in a subject.

[0113] In human cells, IRAK-M is encoded by the IRAK3 gene. In the context of the present invention, "IRAK3 gene" refers to a DNA sequence (e.g., found in the genome) that encodes IRAK-M. The IRAK3 gene can be operably linked to any suitable transcriptional and / or translational regulatory sequence in the nucleic acid and vector systems described herein.

[0114] The term "nucleic acid" as used herein refers to DNA or RNA, or molecules containing both ribo- and deoxyribonucleotides. Nucleic acids include genomic DNA, cDNA, and oligonucleotides, including sense and antisense nucleic acids. Nucleic acids can 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.

[0115] In some embodiments, the nucleic acid sequence encoding IRAK-M is exogenous. In some embodiments, the nucleic acid sequence encoding IRAK-M is heterologous. The term "exogenous" as used herein refers to a nucleic acid encoding a protein that is not normally made in an appreciable or therapeutic amount in an ocular cell. Exogenous nucleic acid also includes a nucleic acid that is normally found in the genome of an ocular cell, but that is no longer expressed or is expressed in a reduced amount compared to non-disease tissue. Thus, a genetically engineered ocular cell may contain extra copies of a gene that is normally found in its genome. The term "heterologous" with respect to a nucleic acid refers to a nucleic acid that does not naturally occur in the target cell.

[0116] In some embodiments, the nucleic acid is an episome. An episome is a genetic element that can replicate independently of a target cell and in association with the chromosome into which it becomes integrated. The nucleic acid can 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 (approximately 4 kb) circular replicon. In some embodiments, the nucleic acid is a messenger RNA or a circular RNA.

[0117] In some embodiments, the nucleic acid can be integrated into the genome of the host. In alternative embodiments, the nucleic acid is not inserted into the genome of the host. Nucleic acid that is randomly integrated into the genome of the host may cause adverse events after insertional mutagenesis. Nucleic acid that is not randomly inserted into the genome of the host advantageously avoids any insertional mutagenesis.

[0118] As will be understood by those skilled in the art, nucleic acids for gene therapy contain elements necessary for the transcription and translation of the inserted coding sequence, which may include, for example, promoters, enhancers, and other regulatory elements. Promoters may be constitutive or inducible. Promoters can be selected to target preferential gene expression in target tissues 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). The promoter may also be active in any cell or tissue type.

[0119] The nucleic acid encoding IRAK-M is typically operably linked to regulatory elements such as promoters and enhancers that drive transcription of DNA in target cells of an individual. The promoter can drive the expression of IRAK-M in all cell types. Alternatively, the promoter can drive the expression of IRAK-M only in certain cell types, such as cells of the retina, such as RPE. In some embodiments, the promoter is a universal promoter. The term "universal promoter" refers to a promoter that is active in any cell, tissue, and / or cell cycle stage. Typically, a universal 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), miniCAG (SV40 intron) promoter, SV40 promoter, CBA / CB7 promoter, smCBA promoter, CBh promoter, MeCP2 promoter, shCMV promoter, CMVd2 promoter, core CMV promoter, SV40 mini promoter, SCP3 promoter, EF1-α promoter, PGK promoter, GAPDH promoter, and UbC promoter.

[0120] In some embodiments, the promoter is an RPE-specific promoter. In further embodiments, the RPE-specific promoter is selected from the group consisting of RPE65 promoter, NA65 promoter, VMD2 promoter (also known as Best1 promoter) and Synpiii promoter. Suitable promoters, particularly retina-specific promoters, are described in Buck et al., Int. J. Mol. Sci. 2020, vol. 21, p. 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.

[0121] In yet another embodiment, the promoter is the native promoter of IRAK3 or a functional fragment thereof. Preferably, the native promoter is the promoter region spanning from -1 to -1698 from the transcription start site of IRAK3 (identified in Pino-Yanes et al. (2011) Am J Respir Cell Mol Biol vol. 45, pp. 740-745). The present inventors identified and tested three fragments upstream of the first exon of the human IRAK3 gene (Ensembl ID: ENSG00000090376). These fragments were selected as putative endogenous IRAK3 promoters and shown to drive robust expression in cells. These three fragments are shown below as SEQ ID NOs: 46-48.

[0122] 0.88 kb fragment (herein "Endo1") - SEQ ID NO:46: TTAGAGTGTGATGGGCTGAGTGGGGTTGTGAGTGATTATCTTCTTTTTTCAGTTTTTTTCTGGGTTTTCCAAGTGTTCCTCGATGAACATGGATAGTTTTTCTGACAGGATAAAAAAGAAGTAGTCCGGGACAGTGGCTAACACCCCGAATCCCAGCACTTTGGGAAGCCGGAGGTGGGAGGATCGCTTGAGGCCAGGAGTTTGAAACCAGCCTGGGCAGCATAACGACACTCCCTCTCTACGAAAAACGAAAAAAAATAATTAGCCGGACGTGGTGGCGTGCGACTGTGGTCCCAGCTACTCGGGAGGCTGAGGTGGGAGGATCGCTTGAGCCCAATAGGTGGAGGCTCCGTGAGCTGAGATAGCGCCACTGCGCTCCTGCCTGGGCGACAGAGTGAGAACCTGACTCAAAACAAAGAAAAAAGGAAGAAAAGAAAGGAAGGGAAGAAGGAAGGAAGGGAGAAGCTTTCAAAAATAAACTTTTGTAAGAAGTAATGACACCGCTAGCCGTCCACACCAGGAGACCGCCTAGCCGTGGGGCACGGTGGGCTCCTGGGAGCTCTGAGCTCTGGGCTTTCTCCAGTTCGCACTCTGCTTGTCTCGGCAGCTCCGTCCCCACCGCAGAGGTGTGAAGGGGCGCAAAGCCAGCGAAGGGAGAACCCGGGTCGGGTAACCCCCAGGCCTGGCCAGGCGGACGCAGGGGCATCTCGGGCGAGGCGCGCCTTGCGTCACGTGGGCACCGCCCCTGCAGTGACCGGAGAACGGCGTGTTCCTAGGGCTCTGCTGCCGTCGTGGAAGCAGGATTTCCGCGGTTGTGTAACGGCCTGTCGCAGGCGTGCAGGGACCTGGACTCCGCCTCGTCCCCGGGGCTCGGGCAGCCGAGCC 1.36 kb fragment (referred to as "Endo2" in this specification) - SEQ ID NO: 47: 1.6 kb fragment (herein "Endo3") - SEQ ID NO:48: It is also expected that the fragments of SEQ ID NOs: 46 to 48 can function as a promoter of IRAK3. An example of a functional fragment of SEQ ID NOs: 46 to 48 is shown in SEQ ID NO: 49 below. SEQ ID NO: 49 contains an H3K methylation mark and a CpG island, and also contains a predicted TATA box (GATAAA), all of which are typical features of a promoter.

[0123] SEQ ID NO:49: GATAAAAAAGAAGTAGTCCGGGACAGTGGCTAACACCCCGAATCCCAGCACTTTGGGAAGCCGGAGGTGGGAGGATCGCTTGAGGCCAGGAGTTTGAAACCAGCCTGGGCAGCATAACGACACTCCCTCTCTACGAAAAACGAAAAAAAATAATTAGCCGGACGTGGTGGCGTGCGACTGTGGTCCCAGCTACTCGGGAGGCTGAGGTGGGAGGATCGCTTGAGCCCAATAGGTGGAGGCTCCGTGAGCTGAGATAGCGCCACTGCGCTCCTGCCTGGGCGACAGAGTGAGAACCTGACTCAAAACAAAGAAAAAAGGAAGAAAAGAAAGGAAGGGAAGAAGGAAGGAAGGGAGAAGCTTTCAAAAATAAACTTTTGTAAGAAGTAATGACACCGCTAGCCGTCCACACCAGGAGACCGCCTAGCCGTGGGGCACGGTGGGCTCCTGGGAGCTCTGAGCTCTGGGCTTTCTCCAGTTCGCACTCTGCTTGTCTCGGCAGCTCCGTCCCCACCGCAGAGGTGTGAAGGGGCGCAAAGCCAGCGAAGGGAGAACCCGGGTCGGGTAACCCCCAGGCCTGGCCAGGCGGACGCAGGGGCATCTCGGGCGAGGCGCGCCTTGCGTCACGTGGGCACCGCCCCTGCAGTGACCGGAGAACGGCGTGTTCCTAGGGCTCTGCTGCCGTCGTGGAAGCAGGATTTCCGCGGTTGTGTAACGGCCTGTCGCAGGCGTGCAGGGACCTGGACTCCGCCTCGTCCCCGGGGCTCGGGCAGCCGAGCC In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 49, or a functional fragment thereof. In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 49. In some embodiments, the promoter consists of the nucleic acid sequence of SEQ ID NO: 49. In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 46, or a functional fragment thereof. In some embodiments, the promoter consists of the nucleic acid sequence of SEQ ID NO: 46. In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 47, or a functional fragment thereof. In some embodiments, the promoter consists of the nucleic acid sequence of SEQ ID NO: 47. In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 48, or a functional fragment thereof. In some embodiments, the promoter consists of the nucleic acid sequence of SEQ ID NO: 48.

[0124] In this specification, the term "operably linked" may include the situation where a selected nucleotide sequence and a regulatory nucleotide sequence, such as a promoter sequence, are covalently linked in such a manner as to place expression of the 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 it is capable of effecting transcription of the nucleotide coding sequence that forms part or all of the selected nucleotide sequence. The resulting transcription product may then, if appropriate, be translated into a desired protein or polypeptide.

[0125] In some embodiments, the introduction of a nucleic acid encoding IRAK-M results in a genetically engineered target cell or tissue. The term "genetically engineered" as used herein refers to a cell or tissue that has been subjected to recombinant DNA manipulation, such as the introduction of an exogenous nucleic acid. For example, the cell contains an exogenous nucleic acid. Generally, the exogenous nucleic acid is produced using recombinant DNA technology.

[0126] The therapeutic nucleic acid can be delivered in vivo. Alternatively, the therapeutic nucleic acid can be delivered ex vivo, whereby the patient's cells are extracted and cultured outside the body. The cells are then genetically modified by the introduction of the therapeutic nucleic acid, and then reintroduced into the patient. In a preferred embodiment, the nucleic acid is delivered in vivo. In the context of the present invention, it is preferred that the expression of the nucleic acid encoding IRAK-M continues as long as possible. It is also preferred that there is low immunogenicity, since the immune response of the host can determine transgenic expression.

[0127] Gene delivery to target tissue / cell is an important step in gene therapy. This step can be performed by a gene delivery vehicle called vector. A vector for gene therapy is a vehicle that carries a gene of interest to a target cell. There are two types of vectors: viral and non-viral. In some embodiments, the vector is a viral vector. In alternative embodiments, the vector is a non-viral vector.

[0128] Viral Vector Gene Delivery System Recombinant viral vectors, which are preferably replication-deficient, have been used as vehicles to deliver transgenes to target cells.

[0129] 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 derived from the following viruses: retrovirus, adenovirus, adeno-associated virus, SV40 type virus, polyomavirus, Epstein-Barr virus, papillomavirus, herpes virus, vaccinia virus, poliovirus, orthomyxovirus, paramyxovirus, papovavirus, picornavirus, lentivirus, poxvirus, anellovirus, and alphavirus. In some embodiments, 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 poxvirus vector, anellovirus vector, and an alphavirus vector.

[0130] In some embodiments, the viral vector is an adeno-associated viral vector. In some embodiments, the nucleic acid is a viral vector genome. Aspects of the invention provide a vector virion for use in a method for treating or preventing macular degeneration in a subject. The vector virion comprises a nucleic acid that encodes IRAK-M and comprises a nucleic acid sequence capable of driving expression of IRAK-M in a target cell. In some embodiments, the vector virion is a recombinant vector virion.

[0131] Virion particles comprising the vector genome of the invention are typically generated in a packaging cell that can replicate the viral genome, express viral proteins (e.g., structural virion proteins and associated enzymes), and assemble virion particles. Also provided are packaging cells that comprise a nucleic acid construct encoding the vector genome described herein. Packaging cells may also require helper virus functions, e.g., from adenovirus, E1-deleted adenovirus, or herpesvirus. Techniques for producing virion particles are well known in the art. Packaging cells are typically eukaryotic cells, such as mammalian cells, e.g., primate cells, e.g., human cells. In some embodiments, cell lines are used. In some embodiments, 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 methods for producing vector virions.

[0132] The size of the transgene that can be incorporated into a viral vector depends on various factors, including the particular virus on which the vector is based, the packaging capabilities of the virion, and which (if any) native viral genes have been deleted from the vector.

[0133] Non-limiting examples of viral vectors are provided below. Adenovirus: Adenoviruses are commonly used in gene therapy due to their ability to successfully transduce many cell types. They generally have a packaging capacity of approximately 30-40 kb of nucleic acid.

[0134] To improve safety, different generations of adenoviral vectors have been created. First generation adenoviral vectors were engineered by removing the E1 region, making them replication defective, and the E3 region. Newer second generation adenoviruses have been engineered with additional deletions or mutations in the viral E2 and E4 regions, preventing the transcriptional control of viral gene expression and viral genome replication, respectively. Further improvements in the safety and efficacy of adenoviral vectors came with the development of "gutless" or "helper-dependent" adenoviral vectors, in which all viral sequences have been deleted except for the inverted terminal repeats (ITRs) and a packing signal that allows space for approximately 36 kb of cargo genes. This third generation virus requires an additional adenoviral helper virus, similar in composition to the first common virus, to aid in replication and packaging (although these viruses contain loxP sites inserted adjacent to the packing signal). These third generation vectors retain the advantages of first generation adenoviral vectors in terms of high efficiency in vivo transduction and transgene expression and can mediate high levels and long-term transgene expression in the absence of toxicity.

[0135] 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.

[0136] Lentivirus: Lentiviruses are RNA viruses of the Retroviridae family. The packaging capacity of this viral vector ranges from 8 to 9 kb of nucleic acid. They possess reverse transcriptase, through which they can integrate retrotranscribed proviral DNA into the host cell chromosome. Lentiviruses can integrate their genome into the host cell, resulting in stable expression. However, genome integration can result in insertional mutagenesis. Therefore, non-integrating lentiviral vectors are typically developed by rendering them integrase-deficient. These persist as episomal dsDNA circles that can transduce non-dividing cells. These non-integrating vectors allow efficient and sustained transgenic expression in postmitotic tissues.

[0137] In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a non-integrating lentiviral vector. Adeno-associated virus: Adeno-associated viruses are replication-deficient parvoviruses with a single-stranded DNA genome that is approximately 4.7 kb in length, including an ITR of 145 nucleotides. Several features make them suitable for retinal gene therapy, such as lack of pathogenicity, minimal immunogenicity, ability to transduce non-dividing cells, and ability to mediate sustained levels of therapeutic gene expression. Adeno-associated viruses are among the smallest viruses, with an uncoiled icosahedral capsid of approximately 22 nm. Because they require the presence of a helper virus for replication to occur, adeno-associated viruses are classified as dependoviruses, which are naturally replication-deficient and non-pathogenic. Importantly, AAV recombinant genomes persist as episomes in transduced cells, resulting in long-lasting expression of the transgene in non-dividing retinal cells (Bordet T et al., Drug Discovery Today, Vol. 24, No. 8, August 2019). AAVs are also routinely used in ocular gene therapy (Buck TMInt. J. Mol. Sci. 2020, 21, 4197).

[0138] A preferred viral gene delivery vector is an AAV vector. "AAV" is an abbreviation for adeno-associated virus, and can be used to refer to the virus itself or its derivatives. This term encompasses all serotypes and variants, both naturally occurring and engineered. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also called recombinant AAV vector (or "rAAV vector"). rAAV can contain a polynucleotide of interest (e.g., a nucleic acid sequence encoding IRAK-M). In general, rAAV vectors contain 5' and 3' adeno-associated virus inverted terminal repeats (ITRs) and a polynucleotide of interest operably linked to a sequence that regulates its expression in target cells.

[0139] 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 the various serotypes of AAV, as well as the sequences of the natural terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases, such as GenBank. See, e.g., GenBank Accession Nos. 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).

[0140] AAV can infect both dividing and non-dividing cells and has a broad tropism that allows it to infect many cell types depending on the specific serotype. Recombinant vectors of AAV (rAAV) used in gene therapy are mainly based on serotype 2 (AAV2); this was the first human serotype described and was the best characterized AAV serotype. Because the AAV capsid protein is responsible for its tropism and therefore its effectiveness, pseudotyping strategies were previously developed, where pseudotyped or hybrid AAV vectors encode the serotype rep, usually AAV2, and the cap gene of a different serotype.

[0141] The vector may be a pseudotyped AAV vector. The term "pseudotyped AAV vector" as used herein refers to a vector particle that includes a rAAV vector genome and a native AAV capsid that includes AAV Rep proteins, where the Cap, Rep and ITRs of the vector genome are derived from at least two different AAV serotypes. Examples of AAV chimeric vectors include, but are not limited to, AAV2 / 5, AAV2 / 6, and AAV2 / 8.

[0142] Signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, and some or all of the internal sequences of the genome (encoding replication and structural capsid proteins, rep-cap) are replaced with foreign DNA, such as an expression cassette, with the rep and cap proteins provided in trans. The sequences located between the ITRs of the AAV vector are sometimes referred to as "payloads". In some embodiments, the payload is a nucleic acid that includes a nucleic acid sequence encoding IRAK-M. The actual capacity of any particular AAV particle may vary depending on the viral proteins employed.

[0143] The vector can be an engineered AAV vector. For example, the engineered AAV vector is the SH10 vector described in Klimczak RR et al., 2009, PLoS One vol. 4(10):e7467. The AAV engineered vector can have a mutated capsid, particularly a tyrosine mutated capsid. Other known suitable engineered capsids include AAV2tYF, AAV2.7m8, R100, AAV2.GL, AAV2.NN, AAV44.9, and AAV44.9(E531D).

[0144] The technology of producing AAV vector particles in packaging cells is standard in the art.For example, the production of pseudotyped AAV is disclosed in International Publication No. WO01 / 83692.In various embodiments, AAV capsid protein can be modified to enhance the delivery of recombinant vector.Modifications to capsid protein are generally known in the art.See, for example, US Patent Application Publication No. 2005 / 0053922 and US Patent Application Publication No. 2009 / 0202490.

[0145] A non-limiting, exemplary method of making packaging cells is to create a cell line that stably expresses all the components required for AAV particle production. For example, a plasmid (or a plurality of plasmids) containing the AAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the AAV genome, and a selection marker, such as 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 advantage of this method is that the cells are selected and suitable for large-scale production of AAV. This can also be achieved by using adenovirus or baculovirus instead of plasmid to introduce the AAV genome and / or the rep and cap genes into the packaging cell.

[0146] In some embodiments, the viral vector is an adeno-associated viral 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(E531D).

[0147] Non-viral gene transfer: Non-viral systems typically include all 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 guns, electroporation, particle bombardment, ultrasound application, and magnetofection.

[0148] Non-viral gene transfer typically has the advantages of being more cost-effective, reducing immune system induction, and no size limit for transgenic DNA. Non-viral DNA vectors can include plasmids or minicircles. Non-viral RNA vectors can include messenger RNA or circular RNA.

[0149] In some embodiments, the non-viral carrier is selected from the group consisting of nanoparticles, liposomes, cationic polymers, and calcium phosphate particles. In some embodiments, the nucleic acid is delivered to the target cell via a non-viral delivery system, hi some embodiments, the non-viral delivery system is selected from the group consisting of nanoparticles, liposomes, cationic polymers, calcium phosphate particles, gene guns, electroporation, particle bombardment, ultrasound application, and magnetofection.

[0150] Nanoparticles (NPs) can be used to deliver plasmid DNA containing functional copies of genes to target tissues, such as the retina. NPs are usually engulfed by target cells via phagocytosis or endocytosis. Typically, nanoparticle compositions can cross the plasma membrane, escape endosomes, and transport plasmid DNA to the nucleus (Sahu B et al., Biomolecules 2021, vol. 11, p. 1135).

[0151] Generally, nanoparticles encapsulate or adsorb DNA or RNA onto their surface. The uptake of nanoparticles by target cells depends on their composition and net charge. There are many different types of nanoparticles, including but not limited to lipid-based NPs, peptide-based NPs, polymer-based NPs, and metal NPs.

[0152] Lipid nanoparticles are stable, biocompatible, and do not induce immune responses after administration (e.g., to the eye). Typically, lipid-based NPs are composed of cationic lipids (with a positively charged, hydrophilic head, and a hydrophobic tail, such as DOTAP) and helper lipids (such as cholesterol). The positively charged heads bind to the negatively charged phosphate groups of DNA, forming the compact structure of lipoplexes. When DNA is wrapped in lipoplexes, it is protected from degradation. The lipid-DNA complex enters the cell by endocytosis.

[0153] Peptide-based NPs generally contain cationic peptides that are lysine / arginine rich and form tight compact structures with DNA. Polymer-based NPs generally contain cationic polymers mixed with DNA to form nano-sized polyplexes. Some examples of polymer-based vectors are polyethylene (PEI), dendrimers, and polyphosphate esters. Examples of synthetic polymers include, but are not limited to, poly(L-ornithine), polyethyleneimine, and poly(amidoamine) dendrimers. Some examples of natural polymers include, but are not limited to, chitosan, dextran, and gelatin. An example of a metal NP is gold NP (AuNP). DNA-gold nanoparticles are easy to generate and have high tolerance and low toxicity. Other nanoparticles that can be considered are calcium silicate-phosphorus nanoparticles, calcium phosphate nanoparticles, and silicon dioxide nanoparticles.

[0154] In some embodiments, the nucleic acids of the present invention are delivered to target cells using nanoparticles. In some embodiments, the nanoparticles are lipid-based nanoparticles. In some embodiments, the nanoparticles are peptide-based nanoparticles. In some embodiments, the nanoparticles are polymer-based nanoparticles. In some embodiments, the nanoparticles are metal nanoparticles, optionally gold nanoparticles.

[0155] The positive charge on the surface of the cationic polymer can form a positive complex with the negatively charged gene. The complex is absorbed to the cell surface by electrostatic action, and the gene is naturally introduced into the cell and then expressed by endocytosis. Cationic polymers can be divided into polypeptides such as polylysine and polyglutamic acid, synthetic polymer materials such as polyethyleneimine (PEI) and polypropyleneimine, and natural polymers such as chitosan, gelatin, and cyclodextrin.

[0156] In some embodiments, the nucleic acids of the present invention are delivered to target cells using a cationic polymer. In some embodiments, the cationic polymer is a polypeptide 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 polyethyleneimine (PEI) and polypropyleneimine. 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.

[0157] Calcium phosphate particles are also considered. They are biocompatible and biodegradable. Calcium plays an important role in endocytosis and has the advantage of being easily absorbed, resulting in high binding affinity. In some embodiments, the non-viral delivery system is calcium phosphate nucleotide-mediated nucleotide delivery.

[0158] Liposomes can be used to deliver the nucleic acid of the present invention to target cells. Liposomes are artificial membranes with a thickness of 5-7 nm and a diameter of 25-500 nm. It has favorable biocompatibility, is mostly uninhibited and without significant damage to normal tissues and cells, and can exist around target cells for a long time, allowing the target gene to be completely transfected into the target cells. Liposomes can be digested by lysosomes and release nucleic acids by natural mechanisms, thus resulting in rapid and convenient drug delivery, high transdermal absorption efficiency, low drug toxicity, and high stability. In some embodiments, the nucleic acid of the present invention is delivered to target cells using liposomes. Nanoliposomes are also contemplated. Nanoliposomes are submicron bilayer lipid vesicles. Examples include, but are not limited to, ceramide-containing nanoliposomes and proteoliposomes.

[0159] Physical methods include, but are not limited to, iontophoresis, bioballistic delivery, electrotransfection, magnetofection, sonoporation, and optoporation. Electrotransfection has been shown to be particularly useful for gene delivery to the eye. It is also known as electroporation or electropermeabilization, and involves applying a localized and short external electric field to cells to temporarily alter the permeability of the cell membrane, facilitating the penetration of naked plasmid DNA and promoting its intracellular transport by electrophoresis (Bordet T et al., Drug Discovery Today, Vol. 24, No. 8, August 2019). In some embodiments, the nucleic acid of the present invention is delivered to the target cell by iontophoresis, bioballistic delivery, electrotransfection, magnetofection, sonoporation, or optoporation. In some embodiments, the nucleic acid of the present invention is delivered to the target cell by electrotransfection.

[0160] In naked plasmid vector delivery, clinical grade plasmid DNA is prepared to transfer genes into tissues. Cells can be injected or electroporated with naked plasmid DNA. This method is typically considered safe and biocompatible. In addition, this method is associated with a low risk of inducing an immune response. Also, the size of the coding sequence is not limited. In some embodiments, the nucleic acid of the present invention is delivered to target cells as naked DNA.

[0161] In some embodiments, the non-viral carrier is selected from the group consisting of liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, nanoparticles, calcium silicate-phosphorus nanoparticles, calcium phosphate nanoparticles, silicon dioxide nanoparticles, microparticles, poly(D-arginine), nanodendrimers, and calcium phosphate nucleotide-mediated nucleotide delivery. Genome editing system The agents described herein may also be genome editing systems.

[0162] In an embodiment, the nucleic acid system comprises a nucleic acid sequence for use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an RNA-guided endonuclease; b) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence associated with an insertion site in the genome of the target cell and capable of directing the RNA-guided endonuclease to the target sequence; and c) a nucleic acid sequence encoding IRAK-M; The nucleic acid system is provided comprising one or more nucleic acids comprising: a nucleic acid sequence encoding IRAK-M capable of driving expression of IRAK-M in a target cell of a subject, the nucleic acid system being suitable for directed insertion of the nucleic acid sequence encoding IRAK-M into an insertion site in the genome of the target cell.

[0163] Also provided are methods for treating or preventing macular degeneration in a subject, a) RNA-guided endonucleases; b) a guide RNA that is complementary to a target sequence associated with the insertion site in the genome of the target cell and capable of directing the RNA-guided endonuclease to the target sequence; and c) a nucleic acid sequence encoding IRAK-M; A system is provided comprising a nucleic acid sequence encoding IRAK-M capable of driving expression of IRAK-M in a target cell of a subject, the system being suitable for directed insertion of the nucleic acid sequence encoding IRAK-M at an insertion site in the genome of the target cell.

[0164] The present invention may also use the CRISPR ("clustered regularly interspaced short palindromic repeats") system to modulate expression of target genes. CRISPR or CRISPR-Cas systems originate from prokaryotic RNA-guided defense systems. There are at least 11 different CRISPR-Cas systems, which are classified into three main types (I-III). Type II CRISPR-Cas systems have been adapted as genome engineering tools.

[0165] Typically, most naturally occurring type II CRISPR-Cas systems employ three components: the protein endonuclease Cas (a CRISPR-associated protein) with DNA nickase activity, referred to herein as RNA-guided endonuclease (or RNA-guided DNA endonuclease); a "targeting" or "guide" RNA (CRISPR-RNA or crRNA) that contains a short sequence, typically around 20 nucleotides, that is complementary to a target sequence in the genome (the "protospacer"); and A "scaffold" RNA (trans-acting CRISPR RNA or tracrRNA) that interacts with the crRNA and recruits the Cas endonuclease.

[0166] Typically, assembly of these components and hybridization of the crRNA with its target sequence in the chromosome results in endonucleolytic cleavage of the chromosome at or near the target sequence. Cleavage also requires that the target DNA contains a recognition site for the cas enzyme (protospacer adjacent motif, or PAM) sufficiently close to the crRNA target sequence, typically immediately adjacent to the 3' end of the target sequence. Cellular repair of the DNA break can result in base insertions / deletions / mutations and mutations at the target locus.

[0167] This three-component system has been simplified by fusing the crRNA and tracrRNA together to create a chimeric single guide RNA (sgRNA or gRNA). Hybridization of the gRNA with the target sequence results in cleavage of the target DNA at the adjacent / upstream PAM site. Thus, the gRNA can be considered as including a crRNA component (which determines the target sequence) and a tracrRNA component (which recruits the endonuclease).

[0168] The protein components of the CRISPR system are called endonucleases, and when bound to the appropriate RNA factor, they can have enzymatic activity (i.e., DNA nickase activity). Typically, endonucleases cleave chromosomal DNA. In some embodiments, the endonucleases are Cas9 proteins. 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 recognized by these enzymes are well known in the art. Beneficially, the new generation SaCas9, CjCas9, and NmCas9 (2.9-3.3 kb) allow both Cas9 and gRNA to be packaged in a single AAV vector. In some embodiments, the endonuclease is a Cas12a protein.

[0169] When using a catalytic endonuclease, the target sequence recognized by the guide RNA may be upstream of the suitable site for insertion. However, the endonuclease protein does not have to be enzymatically active. Catalytically inactive or ("dead") endonuclease proteins can also be used in conjunction with the present invention since they retain the ability to bind to the protospacer site targeted by the gRNA. Catalytically dead endonucleases can be indicated by the prefix "d" (e.g., dCas or dCas9).

[0170] Thus, the term "endonuclease" is used to encompass both catalytically active and catalytically dead proteins, unless the context requires otherwise.

[0171] The endonuclease may contain a nuclear localization sequence (NLS) that is effective in mammalian cells, such as the SV40 large T antigen NLS having the sequence PKKKRKV (SEQ ID NO: 33). Other mammalian NLS sequences are known to those skilled in the art. The endonuclease may contain multiple copies of the NLS, for example, two or three copies of the NLS. When multiple NLS sequences are present, they are typically repeats of the same NLS.

[0172] In some embodiments, the gene encoding the endonuclease component of the nucleic acid system is under the transcriptional control of an RNA polymerase II promoter, such as a viral or human RNA polymerase II promoter. Examples include the CMV or SV40 promoter, or a mammalian "housekeeping" promoter. The gene encoding any RNA component (gRNA, crRNA or tracrRNA) is typically under the transcriptional control of an RNA polymerase III promoter (e.g., a human RNA polymerase UII promoter), such as the U6 or H1 promoter, or a variant thereof that retains or enhances activity.

[0173] In some embodiments, a gene editing system described herein (e.g., a nucleic acid-based or CRISPR-based system) is used to increase expression of IRAK-M. In some embodiments, it may be beneficial to use multiple vectors and / or virions carrying different payloads. For example, it may be necessary to employ one or more vectors to target the integration of IRAK-M into the genome of a target cell. In one example, an AAV containing Cas9 and gRNA and a second AAV vector containing a transgene of interest (e.g., IRAK-M) are used. In one embodiment, one or more virions may each contain at least one associated component. Using the gene editing system described herein, exogenous IRAK-M can be introduced into the genome of a target cell. This process of introducing an exogenous gene is known as "knock-in" or "knock-in". In this way, exogenous IRAK-M is introduced into a target cell to increase expression.

[0174] Typically, guide RNA directs an endonuclease (e.g., Cas9) to the target site to create a double-stranded DNA break (DSB). The broken ends generated by nuclease cleavage are repaired primarily by non-homologous end joining (NHEJ) or homology-directed repair (HDR). In a broad sense, exogenous DNA sequences or genes can then be incorporated into the target sequence using HDR or NHEJ. The term "homology-directed repair" or "HDR" refers to a mechanism in cells to accurately and precisely repair double-stranded DNA breaks using a homologous template to guide the repair. The most common form of HDR is homologous recombination (HR), which is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules. The term "non-homologous end joining" or "NHEJ" refers to a pathway to repair double-stranded DNA breaks that directly ligates the broken ends without the need for a homologous template.

[0175] In some embodiments, the nucleic acid sequence encoding IRAK-M is inserted into the genome at the insertion site via homology directed repair. In some embodiments, the nucleic acid sequence encoding IRAK-M is flanked by a 5' homology arm and a 3' homology arm, where 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.

[0176] In some embodiments, the nucleic acid sequence encoding IRAK-M further comprises a 5' flanking sequence comprising the target sequence and a 3' flanking sequence comprising the target sequence. In some embodiments, the 5' flanking sequence is 5' to the 5' homology arm and the 3' flanking sequence is 3' to the 3' homology arm. In some embodiments, the guide RNA recognizes the target sequence, the 5' flanking sequence and the 3' flanking sequence from the insertion site. 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 IRAK-M in the 5' flanking sequence and the 3' flanking sequence.

[0177] In some embodiments, the nucleic acid comprising the nucleic acid sequence encoding IRAK-M is a plasmid. This may be referred to as a "donor plasmid". Typically, this produces a linear nucleic acid comprising the nucleic acid sequence encoding IRAK-M. In some embodiments, the linear nucleic acid comprising the nucleic acid sequence encoding IRAK-M is inserted into the genome at the insertion site through homology-directed repair.

[0178] In alternative embodiments, the nucleic acid system further comprises at least a second nucleic acid sequence encoding a guide RNA. In some embodiments, the second gRNA recognizes only the 5' and 3' flanking sequences. In some embodiments, the first gRNA recognizes the target sequence from the insertion site.

[0179] In some embodiments, for use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding a Cas endonuclease; b) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence at a suitable site for insertion and capable of directing the RNA-guided endonuclease to the target sequence; and c) a nucleic acid sequence encoding IRAK-M; An engineered CRISPR-Cas vector system is provided that includes one or more vectors, comprising: a nucleic acid that increases expression of IRAK-M in a target cell of a subject, and a nucleic acid encoding IRAK-M is inserted into the genome of the target cell.

[0180] Also provided is an engineered CRISPR-Cas system for use in a method for treating or preventing macular degeneration in a subject, the system comprising: a) Cas endonucleases; b) a guide RNA that is complementary to the target sequence at an appropriate site for insertion and capable of directing the RNA-guided endonuclease to the target sequence; and c) a nucleic acid sequence encoding IRAK-M; wherein the nucleic acid increases expression of IRAK-M in a target cell of the subject, and the nucleic acid encoding IRAK-M is inserted into the genome of the target cell.

[0181] In some embodiments, the suitable site for insertion is the AAVS1 site. The AAVS1 site or locus is also known as a "safe harbor" site or locus. The AAVS1 locus, located in the intron of PPP1R12C, provides a "safe harbor" locus because disruption of this site by introduction of an exogenous gene does not adversely affect the cell. Moreover, this site is associated with robust transcription, maintaining the expression of exogenously inserted genes. Thus, AAVS1 is a well-validated "safe harbor" for harboring exogenous genes, and therefore a suitable target site in the context of the present invention.

[0182] Photoreceptors and RPE are postmitotic. Thus, these cells lack homology directed repair (HDR) mechanisms (Ziccardi L., Int. J. Mol. Sci. 2019, vol. 20, pp. 5722). Site-specific transgene integration typically requires the HDR pathway. However, recent studies have identified methods to perform targeted integration using the CRISPR system in non-dividing cells. An example is described in Suzuki K. et al., 2016, Nature, vol. 540, pp. 144-149 and International Publication No. WO2018 / 013932. The method described in Suzuki et al. employs a homology-independent targeted integration (HITI) strategy that allows for robust DNA knock-in in both dividing and non-dividing cells. HITI is based on non-homologous end joining (NHEJ) and therefore can be performed in non-dividing cells. The method described in Suzuki et al. can be easily applied to the present invention. For example, nucleic acids encoding IRAK-M can be knocked into the genome of target ocular cells via CRISPR / Cas9-mediated homology-independent targeted integration (Suzuki 2016) and have been demonstrated to act in vivo in non-dividing cells such as the RPE.

[0183] This method allows for the directional insertion of exogenous DNA into non-dividing cells. This is accomplished by employing a nucleic acid sequence containing a gene of interest flanked by two target sequences (e.g., a target sequence 5' to the nucleic acid sequence encoding IRAK-M and a target sequence 3' to the nucleic acid sequence encoding IRAK-M). The target sequences in the nucleic acid sequence containing the gene of interest are typically in inverted orientation. The target sequences in the genome are cleaved by an RNA-endonuclease to form a first half and a second half of the sequence. The target sequences in the nucleic acid sequence containing the gene of interest are also cleaved by an RNA-guided endonuclease to form a first half and a second half of each target sequence. This forms a nucleic acid sequence that includes, in front, the first half of the target sequence, the nucleic acid sequence containing the gene of interest, and the second half of the target sequence. If this nucleic acid is correctly inserted into the genome, it forms a sequence in the genome that includes the first half of the target sequence in the genome, the first half of the target sequence in the nucleic acid, the nucleic acid sequence containing the gene of interest, the second half of the target sequence in the nucleic acid, and the second half of the target sequence in the genome. However, if the nucleic acid is incorrectly inserted into the genome, it will form a sequence in the genome that includes the first half of the target sequence in the genome, the second half of the target sequence in the nucleic acid, the nucleic acid sequence containing the gene of interest, the first half of the target sequence in the nucleic acid, and the second half of the target sequence in the genome. Thus, it reconstructs the complete target sequence at each end of the incorrectly inserted gene of interest (i.e., the gene of interest is present in the reverse orientation). HITI is expected to occur more frequently in the forward direction than in the reverse direction, since the latter will be subject to additional endonuclease cleavage until an intact guide RNA (gRNA) target sequence remains at the latter, resulting in a forward transgene insertion or multiple insertions and deletions (indels) that prevent further gRNA binding.

[0184] In some embodiments, the nucleic acid sequence encoding IRAK-M is flanked by 5' and 3' target sequences. In some embodiments, the 5' and 3' target sequences are the same as the target sequence from the insertion site in the genome. In some embodiments, the nucleic acid sequence encoding the guide RNA is complementary to the 5' and 3' target sequences. In some embodiments, the nucleic acid sequence encoding the guide RNA is complementary to the target sequence, 5' and 3' target sequences in the genome. In some embodiments, the target sequence is no longer present when the nucleic acid sequence encoding IRAK-M is integrated into the genome in the correct orientation. In some embodiments, the target sequence present in the nucleic acid encoding IRAK-M is present in the opposite orientation from the insertion site in the genome to the target sequence. In some embodiments, the target sequence present in the nucleic acid encoding IRAK-M is present in the reverse orientation. Typically, the target sequence in the genome is in the forward orientation. In some embodiments, the first and second halves of the target sequence are cleaved by a nuclease, and the first and second halves 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 IRAK-M.

[0185] A "target sequence" herein is a nucleic acid sequence that is 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. Target sequences include target sequences in a genome, 5' target sequences and 3' target sequences.

[0186] In some embodiments, a suitable site for insertion is the AAVS1 site. The viral delivery systems described herein, or the non-viral delivery systems described herein, can be used to introduce the nucleic acid systems described herein into target cells.

[0187] CRISPR / Cas system components can be delivered to target cells as a ribonucleoprotein (RNP) complex containing the Cas9 protein and gRNA (Zhang et al., Theranostics 2021, Vol. 11, No. 2). Thus, a system containing an RNA-guided endonuclease, a guide RNA, and a nucleic acid encoding IRAK-M as described herein can be delivered to target cells as a complex.

[0188] Zhang et al., Theranostics 2021, vol. 11, no. 2, describes various methods for delivering such complexes to target cells. The complexes described herein can be delivered to target cells by direct penetration, such as microinjection or biolistic bombardment, of the target cells. The target cell membrane can be disrupted by electroporation. Electroporation can disrupt the target cell membrane and temporarily form a "nanopore" through which the complexes can be transported. Prior to electroporation, the complexes can be stabilized using an anionic polymer (e.g., polyglutamic acid). Alternatively, virus-like particles (VLPs) can be used to deliver the complexes. For example, RNA-guided endonucleases can be incorporated into Rentori virus particles. Banskota et al., 2022, Cell vol. 185, pp. 250-265, describe the use of engineered DNA-free virus-like particles (eVLPs) that can package and deliver complexes such as Cas9 RNP to target cells (e.g., the retina).

[0189] Zhang et al., 2022, also describe the use of lipid nanoparticles to deliver the complexes described herein. Lipid nanoparticles can include cell-derived extracellular vesicles (EVs) and synthetic lipid nanoparticles. Synthetic lipid nanoparticles have been described to successfully deliver CRISPR complexes to human retinal pigment epithelial cells. MAX(Thermo Fisher) (Yu et al., Biotechnol Lett (2016) 38:919-929). Alternatively, Zhang et al., 2022, describes the use of CPPs to enable delivery of the complexes. Also described is a method of adding lipid moieties to the complexes to increase membrane permeability. Additionally, polymers are described for delivering the complexes described herein, such as dendrimers, PBAE, PEGylated PLL, and chitosan nanoparticles. The use of nanogels is also described in Zhang et al. Chen et al., Nat Nanotechnol (2019); 14:974-80, describes the in vivo delivery of the complexes in nanogels to mouse retina / RPE. Nanoparticles can 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 phosphorus (BP) nanosheets, or calcium phosphate nanoparticles. In one example, Wang et al., J Controlled Release. 2020;324:194-203, describes the delivery of a complex (e.g., RNP) to the mouse retina in vivo using nanoparticles (pH-responsive silica-metal-organic framework hybrid nanoparticles). Other methods described in Zhang et al. may also be used in the context of the present invention.

[0190] Any of the methods described herein can be used to deliver the complex described herein (i.e., a system comprising an RNA-guided endonuclease described herein, a guide RNA, and a nucleic acid encoding IRAK-M).

[0191] Delivery of the systems described herein as complexes (e.g., protein / RNA / DNA complexes) can result in transient genome editing, thus reducing off-target effects, insertional mutagenesis, and immune responses. Delivery as a complex can also result in faster genome editing, since the need for intracellular transcription and translation is eliminated.

[0192] Increased endogenous gene expression The following agents can increase endogenous IRAK-M expression: A system to increase endogenous IRAK-M expression A nucleic acid system (e.g., a CRISPR activation system) can be employed to increase endogenous IRAK-M expression. An example is a nucleic acid activation system (e.g., a CRISPR / Cas9 activation system).

[0193] Transcriptional activators are protein domains or entire proteins (which may be linked to an inactivated endonuclease) that assist in the recruitment of cofactors, transcription factors and / or RNA polymerase to transcribe a target gene.

[0194] In an embodiment, a compound according to the present invention is provided for use in a method for treating or preventing macular degeneration in a subject, comprising: a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease fused to one or more transcriptional activators; and b) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the promoter or regulatory sequence of the IRAK3 gene and is capable of directing the RNA-guided endonuclease to the target sequence; wherein the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0195] In some embodiments, the inactivated RNA-guided endonuclease is fused to a single transcriptional activator. For example, the inactivated RNA-guided endonuclease can be fused to VP64.

[0196] One or more transcriptional activators can be connected to the N-terminus of the inactivated RNA-guided endonuclease. One or more transcriptional activators can be connected to the C-terminus of the inactivated RNA-guided endonuclease. For example, VP64 can be fused to the C-terminus of the inactivated RNA-guided endonuclease. VP64 can be fused to the inactivated RNA-guided endonuclease via a linker.

[0197] In some embodiments, the inactivated RNA-guided endonuclease is fused to more than one transcriptional activator. For example, the inactivated RNA-guided endonuclease can be fused to three transcriptional activators. In some embodiments, the transcriptional activators can be VP64, p65, and Rta. VP64 can be connected to the C-terminus of the inactivated RNA-guided endonuclease, p65 can be connected to the C-terminus of VP64, and Rta can be connected to the C-terminus of p65. An example of this system is the VP64-p65-Rta system, also known as VPR.

[0198] Also provided are methods for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease; and b) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in a promoter or regulatory sequence of the IRAK3 gene and that is capable of directing said RNA-guided endonuclease to said target sequence, said guide RNA further comprising an aptamer that is capable of specifically binding to a transcription activator; wherein the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0199] In some embodiments, the aptamer is an RNA aptamer. The transcriptional activator can be endogenous to the cell. Additionally, or alternatively, the transcriptional activator can be exogenous to the cell.

[0200] Also provided are methods for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated 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 that is complementary to a target sequence in a promoter or regulatory sequence of the IRAK3 gene and that is capable of directing said RNA-guided endonuclease to said target sequence, said guide RNA further comprising an RNA aptamer that is capable of specifically binding to an RNA-binding protein; A nucleic acid system is provided that includes one or more nucleic acids comprising: the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0201] The one or more transcriptional activators may be selected from the group consisting of VP64, p65, and HSF1. In some embodiments, p65 and HSF1 are fused to an RNA binding protein.

[0202] The RNA binding protein can be MS2 (also known as MS2 bacteriophage coat protein), and the RNA aptamer can bind to MS2. The RNA aptamer can bind to a dimerized RNA binding protein (such as dimerized MS2). Without wishing to be bound by theory, it is expected that one or more RNA binding proteins will bind to the RNA aptamer, thereby providing one or more transcriptional activators (via gRNA) to the target site. For example, it is expected that the MS2-p65-HSF1 complex guided by the target-specific MS2-mediated gRNA will enhance the binding of the transcription factor to the promoter of IRAK3. In some embodiments, the gRNA comprises a hairpin aptamer that can bind to MS2 (e.g., MS2 fusion protein).

[0203] The tetraloop and stem loop 2 of the gRNA typically protrude outside the Cas9-gRNA complex. Also, these regions of the gRNA are believed not to affect endonuclease activity. Thus, the tetraloop and / or stem loop 2 of the gRNA can each be modified with an RNA aptamer. In some embodiments, the RNA aptamer is a minimal hairpin aptamer. The minimal hairpin aptamer can be added to the tetraloop and / or stem loop 2 of the gRNA. In some embodiments, the minimal hairpin aptamer specifically binds to MS2. In some embodiments, the minimal hairpin aptamer specifically binds to MS2 dimer.

[0204] In some embodiments, the inactivated RNA-guided endonuclease is fused to an additional transcription activator. For example, the inactivated RNA-guided endonuclease can be fused to a single additional transcription activator. The inactivated RNA-guided endonuclease can be fused to VP64. An example of a nucleic acid system is the synergistic activation mediator (SAM) system.

[0205] In another aspect, a compound according to the present invention for use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease fused to an epitope repeat array comprising one or more epitopes; b) one or more nucleic acid sequences encoding epitope-binding molecules fused to one or more transcriptional activators, said epitope-binding molecules being capable of specifically binding to an epitope of the epitope repeat array; and c) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the promoter or regulatory sequence of the IRAK3 gene and is capable of directing the RNA-guided endonuclease to the target sequence; wherein the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0206] In some embodiments, the epitope-binding molecule is an antibody or antibody-like molecule. One or more transcriptional activators may be fused to a single chain variable fragment (scFv). In some embodiments, VP64 is fused to an scFv.

[0207] 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 activator may be selected from the group consisting of VP64, p65, and Rta.

[0208] Epitope repeat arrays can bind multiple epitope-binding molecules fused to one or more transcriptional activators.Thus, the system described herein can amplify the number of transcriptional activators at a target site.Epitope sequences can be unique (i.e., different from the sequences naturally occurring in target cells).

[0209] The epitope-binding molecule may comprise a nuclear localization sequence (NLS). The NLS may facilitate the transport of the epitope-binding molecule to the nucleus of the target cell. In some embodiments, the NLS comprises an amino acid sequence comprising SEQ ID NO: 33.

[0210] An example of this system is known as the SunTag system, in which the GCN4 antibody was fused to the NLS and VP64. The following system is also provided:

[0211] An aspect of the present invention is a) an inactivated RNA-guided endonuclease fused to one or more transcriptional activators; b) a guide RNA that is complementary to a target sequence in the promoter or regulatory sequence of the IRAK3 gene and is capable of directing said RNA-guided endonuclease to said target sequence; wherein the system increases IRAK-M expression in target cells of a subject.

[0212] Another aspect of the invention provides a method for treating or preventing macular degeneration in a subject, comprising: a) an inactivated RNA-guided endonuclease; and b) a guide RNA that is complementary to a target sequence in the promoter or regulatory sequence of the IRAK3 gene and is capable of directing said RNA-guided endonuclease to said target sequence, said guide RNA further comprising an aptamer that is capable of specifically binding to a transcription activator; wherein the system increases IRAK-M expression in target cells of a subject.

[0213] A further aspect relates to a method for treating or preventing macular degeneration in a subject, comprising: a) an inactivated RNA-guided endonuclease; b) an RNA-binding protein fused to one or more transcriptional activators; and c) a guide RNA that is complementary to a target sequence in a promoter or regulatory sequence of the IRAK3 gene and is capable of directing said RNA-guided endonuclease to said target sequence, said guide RNA comprising an RNA aptamer that is capable of specifically binding to an RNA-binding protein; wherein the system increases IRAK-M expression in target cells of a subject.

[0214] Also provided are methods for treating or preventing macular degeneration in a subject, a) an inactivated RNA-guided endonuclease fused to an epitope repeat array containing one or more epitopes; b) one or more epitope-binding molecules fused to one or more transcriptional activators, said epitope-binding molecules capable of specifically binding to an epitope of said epitope repeat array; and c) a guide RNA that is complementary to a target sequence in the promoter or regulatory sequence of the IRAK3 gene and is capable of directing the RNA-guided endonuclease to the target sequence; wherein the system increases IRAK-M expression in target cells of a subject.

[0215] As described above, the above systems can be delivered to the target cell as a complex (e.g., a protein / RNA complex). In some embodiments, the RNA-guided endonuclease is a Cas endonuclease.

[0216] The present specification describes an activation system.Examples of activation systems include, but are not limited to, VP64-p65-Rta or VPR, inactivated endonuclease-SAM system, and inactivated endonuclease-SunTag system.Any of these activation systems can be used in the context of the present invention.An example of CRISPR activation system is described in Konermann S. et al., Nature 2015:517 (7536), pp. 583-588.

[0217] In some embodiments, for use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated Cas endonuclease fused to one or more transcriptional activators; and b) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the promoter or regulatory sequence of the IRAK3 gene and is capable of directing the Cas endonuclease to the target sequence; An engineered CRISPR-Cas vector system is provided, comprising one or more vectors comprising:

[0218] In some embodiments, there is provided an engineered CRISPR-Cas vector system comprising one or more nucleic acids for use in a method for treating or preventing macular degeneration in a subject, the system comprising: a) a nucleic acid sequence encoding an inactivated Cas endonuclease; and b) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in a promoter or regulatory sequence of the IRAK3 gene and that is capable of directing the Cas endonuclease to the target sequence, the guide RNA further comprising an aptamer that is capable of specifically binding to a transcription activator; wherein the CRISPR-Cas vector system increases IRAK-M expression in target cells of a subject.

[0219] Embodiments include a method for treating or preventing macular degeneration in a subject, comprising: a) a nucleic acid sequence encoding an inactivated 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 that is complementary to a target sequence in the promoter or regulatory sequence of the IRAK3 gene and is capable of directing the Cas endonuclease to the target sequence; wherein the guide RNA further comprises an RNA aptamer capable of specifically binding to an RNA binding protein, and wherein the CRISPR-Cas vector system increases IRAK-M expression in a target cell of the subject.

[0220] In another embodiment, for use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated Cas endonuclease fused to an epitope repeat array comprising one or more epitopes; b) one or more nucleic acid sequences encoding epitope-binding molecules fused to one or more transcriptional activators, said epitope-binding molecules being capable of specifically binding to an epitope of the epitope repeat array; and c) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the promoter or regulatory sequence of the IRAK3 gene and is capable of directing the Cas endonuclease to the target sequence; An engineered CRISPR-Cas vector system is provided, comprising one or more vectors comprising:

[0221] Another example of an agent that can increase endogenous IRAK-M expression in a target cell is a nucleic acid demethylation system (e.g., the CRISPR / Cas9 demethylation system). DNA methylation is an epigenetic process that occurs by the addition of methyl groups, typically cytosine bases, to DNA. In mammals, DNA methylation regulates gene expression by acting to repress gene transcription. Without wishing to be bound by theory, it is expected that the use of a demethylation system increases the accessibility of the IRAK3 gene or its promoter / regulatory sequences, allowing transcription.

[0222] Accordingly, another aspect provides a method for treating or preventing macular degeneration in a subject, comprising: a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease fused to one or more DNA demethylating agents; and b) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the IRAK3 gene and capable of directing the RNA-guided endonuclease to the target sequence; wherein the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0223] The one or more DNA demethylating agents can be one or more DNA demethylases or fragments thereof. For example, 10-11 translocation enzyme methylcytosine dioxygenase (TET enzyme) mediates 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 TET1. In some embodiments, the DNA demethylating agent is TET1. Lysine-specific demethylase 1 (LESD1, also known as KDM1A) is a lysine demethylase that acts on histones H3K4me1 / 2 and H3K9me1 / 2. In some embodiments, the DNA demethylating agent is LESD1.

[0224] In some embodiments, the inactivated RNA-guided endonuclease is fused to a single DNA demethylation agent. One or more DNA demethylation agents can be fused to the C-terminus of the inactivated RNA-guided endonuclease. Alternatively, one or more DNA demethylation agents can be fused to the N-terminus of the inactivated RNA-guided endonuclease.

[0225] Also provided are methods for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease; b) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the IRAK3 gene and capable of directing the RNA-guided endonuclease to the target sequence; wherein the guide RNA further comprises an aptamer capable of specifically binding to a demethylating agent, and the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0226] In some embodiments, the aptamer is an RNA aptamer. The demethylating agent can be endogenous to the cell. Additionally, or alternatively, the demethylating agent can be exogenous to the cell.

[0227] Also provided are methods for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease; b) a nucleic acid sequence encoding an RNA-binding protein fused to one or more DNA demethylating agents; c) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the IRAK3 gene and capable of directing the RNA-guided endonuclease to the target sequence; wherein the guide RNA further comprises an RNA aptamer capable of specifically binding to an RNA binding protein, and the nucleic acid system increases IRAK-M expression in a target cell of the subject.

[0228] The one or more DNA demethylating agents can be as described above. The RNA binding protein and / or gRNA can be as described for the transcription activation system above, except that the one or more DNA demethylating agents are fused to an RNA binding protein.

[0229] In some embodiments, the inactivated RNA-guided endonuclease is fused to an additional DNA demethylating agent, which may be different from the one or more DNA demethylating agents.

[0230] In another aspect, a compound according to the present invention for use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease fused to an epitope repeat array comprising one or more epitopes; b) one or more nucleic acid sequences encoding epitope-binding molecules fused to one or more DNA demethylating agents, the epitope-binding molecules being capable of specifically binding to an epitope of an epitope repeat array; and c) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the IRAK3 gene and capable of directing the RNA-guided endonuclease to the target sequence; wherein the nucleic acid system increases IRAK-M expression in a target cell of a subject.

[0231] The epitope repeat array can be as described for the transcription activation system above. The epitope binding molecule can be as described above, except that one or more DNA demethylating agents are fused to the epitope binding molecule.

[0232] The present invention also provides the following aspects. Embodiments include a method for treating or preventing macular degeneration in a subject, comprising: a) an inactivated RNA-guided endonuclease fused to one or more DNA demethylating agents; and b) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a guide RNA that is complementary to a target sequence in the IRAK3 gene and is capable of directing the RNA-guided endonuclease to the target sequence; wherein the system increases IRAK-M expression in target cells of the subject.

[0233] A further aspect relates to a method for treating or preventing macular degeneration in a subject, comprising: a) an inactivated RNA-guided endonuclease; and b) a guide RNA that is complementary to (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a target sequence in the IRAK3 gene and is capable of directing the RNA-guided endonuclease to the target sequence, wherein the guide RNA further comprises an aptamer that is capable of specifically binding to a DNA demethylating agent; wherein the system increases IRAK-M expression in target cells of the subject.

[0234] Further, for use in a method for treating or preventing macular degeneration in a subject, a) an inactivated RNA-guided endonuclease; b) an RNA-binding protein fused to one or more DNA demethylating agents; and c) a guide RNA that is complementary to (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a target sequence in the IRAK3 gene and is capable of directing the RNA-guided endonuclease to the target sequence, wherein the guide RNA further comprises an RNA aptamer that is capable of specifically binding to an RNA-binding protein; wherein the system increases IRAK-M expression in target cells of a subject.

[0235] Another aspect relates to a compound comprising: a) an inactivated RNA-guided endonuclease fused to an epitope repeat array containing one or more epitopes; b) one or more epitope-binding molecules fused to one or more DNA demethylating agents, said epitope-binding molecules being capable of specifically binding to an epitope of an epitope repeat array; and c) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a guide RNA that is complementary to a target sequence in the IRAK3 gene and is capable of directing the RNA-guided endonuclease to the target sequence; wherein the system increases IRAK-M expression in target cells of a subject.

[0236] As described above, the above system can be delivered to the target cell as a complex (e.g., a protein / RNA complex). In some embodiments, the RNA-guided endonuclease is a Cas endonuclease.

[0237] CRISPR-based approaches targeting DNA demethylation can allow for targeting epigenetic editing. For example, the demethylation system can include an inactivated endonuclease (e.g., Cas9 nuclease) fused to a demethylation agent (e.g., TET1) and at least one IRAK-M specific guide RNA. Similar to the CRISPR activation system, the CRISPR demethylation system uses a modified CRISPR effector without endonuclease activity and has a transcription activator on the dCas or gRNA. Similar to the above system, the demethylation system includes an inactivated endonuclease (e.g., dCas9), gRNA, and a DNA demethylation agent fused to the inactivated endonuclease or gRNA. An approach for targeting DNA demethylation using CRISPR is described in Xu et al., 2016, Cell Discovery (2016), vol. 2, pp. 16009; doi:10.1038 / celldisc.2016.9.

[0238] In some embodiments, for use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated Cas endonuclease fused to one or more DNA demethylating agents; and b) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the IRAK3 gene and capable of directing the Cas endonuclease to the target sequence; An engineered CRISPR-Cas vector system is provided that includes one or more vectors comprising:

[0239] In some embodiments, for use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated Cas endonuclease; and b) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the IRAK3 gene and capable of directing the Cas endonuclease to the target sequence, wherein the guide RNA further comprises an aptamer capable of specifically binding to a DNA demethylating agent; An engineered CRISPR-Cas vector system is provided that includes one or more nucleic acids, comprising:

[0240] Embodiments include a method for treating or preventing macular degeneration in a subject, comprising: a) a nucleic acid sequence encoding an inactivated Cas endonuclease; b) a nucleic acid sequence encoding an RNA-binding protein fused to one or more DNA demethylating agents; and c) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the IRAK3 gene and capable of directing the Cas endonuclease to the target sequence, wherein the guide RNA further comprises an RNA aptamer capable of specifically binding to an RNA-binding protein; wherein the CRISPR-Cas vector system increases IRAK-M expression in a target cell of a subject.

[0241] In another embodiment, for use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated Cas endonuclease fused to an epitope repeat array comprising one or more epitopes; b) one or more nucleic acid sequences encoding epitope-binding molecules fused to one or more DNA demethylating agents, the epitope-binding molecules being capable of specifically binding to an epitope of an epitope repeat array; and c) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the IRAK3 gene and capable of directing the Cas endonuclease to the target sequence; An engineered CRISPR-Cas vector system is provided that includes one or more vectors comprising:

[0242] As described herein, inactivated endonucleases are mutant forms of endonucleases whose endonuclease activity is eliminated by point mutations in the endonuclease domain. Inactivated endonucleases lack endonuclease activity but can still bind to gRNA and target DNA. Inactivated endonucleases described herein can be dCas. Typically, Cas9 is used, but other endonucleases (e.g., Cas12a) can be used.

[0243] The viral delivery systems described herein, or the non-viral delivery systems described herein, can be used to introduce the nucleic acid systems described herein into target cells. In some embodiments, it may 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 embodiments, one or more virions may each contain at least one of the associated components.

[0244] In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. Additional Targeting Approaches Aspects of the invention provide a nucleic acid comprising a nucleic acid sequence encoding a fusion protein for use in a method for treating or preventing macular degeneration in a subject, the fusion protein comprising: (a) a nucleic acid binding molecule capable of binding to a target sequence in the promoter or regulatory sequence of the IRAK3 gene; and (b) one or more transcriptional activators; wherein the fusion protein is capable of increasing IRAK-M expression in a target cell of a subject.

[0245] Also provided are methods for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding a fusion protein, the fusion protein comprising (i) a nucleic acid binding molecule capable of binding to a target sequence in a promoter or regulatory sequence of the IRAK3 gene, and (ii) a nucleic acid sequence comprising an epitope repeat array; and b) one or more nucleic acid sequences encoding epitope-binding molecules fused to one or more transcriptional activators, said epitope-binding molecules being capable of specifically binding to an epitope of the epitope repeat array; wherein the nucleic acid system is capable of increasing IRAK-M expression in a target cell of a subject.

[0246]

[0013] In another aspect, a nucleic acid is provided comprising a nucleic acid sequence encoding a fusion protein for use in a method for treating or preventing macular degeneration in a subject, the fusion protein comprising: a) a nucleic acid binding molecule capable of binding to (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a target sequence in the IRAK3 gene; and b) one or more DNA demethylating agents; wherein the fusion protein is capable of increasing IRAK-M expression in a target cell of a subject.

[0247] Also provided are methods for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding a fusion protein, the fusion protein comprising: (i) a nucleic acid sequence comprising: (1) a target sequence in a promoter sequence of the IRAK3 gene, (2) a target sequence in a regulatory sequence of the IRAK3 gene, or (3) a nucleic acid binding molecule capable of binding to a target sequence in the IRAK3 gene; and (ii) an epitope repeat array; and b) one or more nucleic acid sequences encoding epitope-binding molecules fused to one or more DNA demethylating agents, said epitope-binding molecules being capable of specifically binding to an epitope of an epitope repeat array; wherein the nucleic acid system is capable of increasing IRAK-M expression in a target cell of a subject.

[0248] The present invention also provides the following fusion proteins: An aspect of the invention is a method for treating or preventing macular degeneration in a subject, comprising: (a) a nucleic acid binding molecule capable of binding to a target sequence in the promoter or regulatory sequence of the IRAK3 gene; and (b) one or more transcriptional activators; wherein the fusion protein is capable of increasing IRAK-M expression in a target cell of a subject.

[0249] Another aspect relates to a compound comprising: a) a nucleic acid binding molecule capable of binding to (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a target sequence in the IRAK3 gene; and b) one or more DNA demethylating agents; wherein the fusion protein is capable of increasing IRAK-M expression in a target cell of a subject.

[0250] The following system is also provided: Embodiments include a method for treating or preventing macular degeneration in a subject, comprising: a) a fusion protein, the fusion protein comprising (i) a nucleic acid binding molecule capable of binding to a target sequence in the promoter or regulatory sequence of the IRAK3 gene, and (ii) an epitope repeat array; and b) one or more epitope-binding molecules fused to one or more transcriptional activators, said epitope-binding molecules capable of specifically binding to an epitope of said epitope repeat array; wherein the system is capable of increasing IRAK-M expression in target cells of a subject.

[0251] Also provided are methods for treating or preventing macular degeneration in a subject, a) a fusion protein, the fusion protein comprising (i) a nucleic acid binding molecule capable of binding to (1) a target sequence in a promoter sequence of the IRAK3 gene, (2) a target sequence in a regulatory sequence of the IRAK3 gene, or (3) a target sequence in the IRAK3 gene, and (ii) an epitope repeat array; and b) one or more epitope-binding molecules fused to one or more DNA demethylating agents, said epitope-binding molecules being capable of specifically binding to an epitope of said epitope repeat array; wherein the system is capable of increasing IRAK-M expression in a target cell of a subject.

[0252] The fusion proteins and systems can be delivered to target cells as a complex. The delivery methods described for the gene editing systems can also be applied to the fusion proteins and systems described herein.

[0253] 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.

[0254] In some embodiments, the nucleic acid binding molecule is a transcription activator-like (TAL) effector (also known as a TALE) repeat array. Fusion proteins comprising a TAL effector repeat array and either (i) a transcription activator or (ii) a DNA demethylating agent can be used to increase endogenous expression of IRAK-M in target cells.

[0255] TALEs are proteins secreted by several β- and γ-proteobacteria. TALEs have a modular DNA-binding domain (DBD) consisting of a repeat sequence of residues. Each repeat region contains approximately 34 amino acids. Residues at positions 12 and 13 determine the nucleotide specificity and are known as the repeat variable diresidues (RVD). The RVD is highly variable and shows a strong correlation with specific nucleotide recognition.

[0256] TAL effector repeat domains can be engineered to each bind to one nucleotide of DNA, with specificity determined by the identity of two hypervariable residues. To construct a protein that can recognize a specific DNA sequence, repeats with different specificities are simply linked together in a TAL effector repeat array. Thus, TAL effector repeat arrays can be used to bind target sequences in the IRAK3 gene or the promoter / regulatory sequence(s) of the IRAK3 gene.

[0257] The TAL effector repeat array can be fused to either (i) a transcriptional activator or (ii) a DNA demethylating agent. Alternatively, the TAL effector repeat array can be fused to an epitope repeat array as described above.

[0258] In some embodiments, a nucleic acid is provided for use in a method for treating or preventing macular degeneration in a subject, the 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 sequence of the IRAK3 gene; and b) one or more transcriptional activators; wherein the fusion protein is capable of increasing IRAK-M expression in a target cell of a subject.

[0259] In some embodiments, for use in a method for treating or preventing macular degeneration in a subject, a) a TAL effector repeat sequence capable of binding to a target sequence in the promoter or regulatory sequence of the IRAK3 gene; and b) one or more transcriptional activators; A fusion protein comprising:

[0260] In some embodiments, a nucleic acid is provided for use in a method for treating or preventing macular degeneration in a subject, the 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 a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a target sequence in the IRAK3 gene; and b) one or more DNA demethylating agents; wherein the fusion protein is capable of increasing IRAK-M expression in a target cell of a subject.

[0261] In some embodiments, for use in a method for treating or preventing macular degeneration in a subject, a) a TAL effector repeat array capable of binding to (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a target sequence in the IRAK3 gene; and b) one or more DNA demethylating agents; A fusion protein comprising:

[0262] In some embodiments, the nucleic acid binding molecule is a zinc finger array. Fusion proteins comprising a zinc finger (ZNF) array and either (i) a transcriptional activator or (ii) a DNA demethylating agent can be used to increase endogenous IRAK-M expression.

[0263] The zinc finger motif is maintained by zinc ions coordinating cysteines and histidines in different combinations, allowing ZNFs to have the ability to interact with DNA and / or RNA. ZNFs can be engineered to change the DNA binding specificity of the zinc finger. Tandem repeats of zinc finger domains (and / or engineered zinc finger domains) can be used to target specific DNA (or RNA) sequences. Engineered zinc finger arrays can have 3-6 individual zinc finger motifs and can bind target sites ranging from 9 base pairs to 18 base pairs in length. Arrays with at least 6 zinc finger motifs are preferred as they can bind longer target sequences, increasing specificity.

[0264] 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 three zinc finger motifs. In some embodiments, the zinc finger array comprises at least six zinc finger motifs. The zinc finger array may bind to a target sequence of the IRAK3 gene or a promoter / regulatory sequence(s) of the IRAK3 gene.

[0265] In some embodiments, a nucleic acid is provided for use in a method for treating or preventing macular degeneration in a subject, the 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 sequence of the IRAK3 gene; and b) one or more transcriptional activators; wherein the fusion protein is capable of increasing IRAK-M expression in a target cell of a subject.

[0266] In some embodiments, for use in a method for treating or preventing macular degeneration in a subject, a) a zinc finger array capable of binding to a target sequence in the promoter or regulatory sequence of the IRAK3 gene; and b) one or more transcriptional activators; wherein the fusion protein is capable of increasing IRAK-M expression in a target cell of a subject.

[0267] In some embodiments, a nucleic acid is provided for use in a method for treating or preventing macular degeneration in a subject, the nucleic acid comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising: a) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a zinc finger array capable of binding to a target sequence in the IRAK3 gene; and b) one or more DNA demethylating agents; wherein the fusion protein is capable of increasing IRAK-M expression in a target cell of a subject.

[0268] In some embodiments, for use in a method for treating or preventing macular degeneration in a subject, a) (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a zinc finger array capable of binding to a target sequence in the IRAK3 gene; and b) one or more DNA demethylating agents; A fusion protein comprising:

[0269] The transcriptional activator can be any of the transcriptional activators described herein. Similarly, the DNA demethylating agent can be any of the DNA demethylating agents described herein.

[0270] In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the epitope-binding molecule is an antibody or an antibody-like molecule.

[0271] Viral delivery systems as described herein, or non-viral delivery systems as described herein, can be used to introduce nucleic acids encoding the fusion proteins described herein into target cells.

[0272] Small molecule drugs Small molecule and peptide agents can be used to increase endogenous expression of IRAK-M in target cells.

[0273] As used herein, the term small molecule refers to a low molecular weight organic compound. Small molecules can bind to specific biopolymers and act as effectors to change the activity or function of targets. Due to their small size, small molecules can have the advantage of being able to pass through cell membranes to reach intracellular targets.

[0274] An embodiment of the invention provides a small molecule for use in a method for treating or preventing macular degeneration in a subject, wherein the small molecule increases endogenous IRAK-M expression in a target cell of the subject.

[0275] Small molecules can be used to reduce DNA methylation in the promoter sequence of the IRAK3 gene or the IRAK3 gene itself, thereby increasing the accessibility of the IRAK3 gene or its promoter. Thus, in some embodiments, small molecules reduce DNA methylation in the promoter sequence of the IRAK3 gene. In some embodiments, small molecules reduce DNA methylation in the IRAK3 gene.

[0276] Examples of small molecules that can reduce DNA methylation in the promoter sequence of the IRAK3 gene or in the IRAK3 gene include EPZ-6438 and azacytidine. Geng et al., 2020, Communications Biology, vol. 3:306, describes the use of EPZ-6438 and azacytidine to induce IRAK-M expression in target cells by increasing IRAK-M transcripts. In some embodiments, the small molecule is EPZ-6438. In some embodiments, the small molecule is azacytidine.

[0277] Another small molecule that has been shown to increase endogenous IRAK-M expression is ibudilast. Oliveros et al., 2022, Brain.awac136. (doi:10.1093 / brain / awac136) describes that ibudilast increased IRAK3 transcripts. Ibudilast is a phosphodiesterase inhibitor and Toll-like receptor 4 (TLR4) antagonist, and is a multi-targeted drug, as it has also been shown to inhibit IRAK1 activity by increasing the expression of its negative regulator IRAK-M. Thus, in some embodiments, the small molecule is ibudilast.

[0278] Small molecules can increase the expression of IRAK-M by recruiting one or more polypeptides that promote transcription to the promoter of IRAK3. For example, Miyata et al., 2015, Nature Communications, vol. 6:6062, shows that glucocorticoids can upregulate IRAK-M by recruiting the glucocorticoid receptor (GR) to the IRAK-M promoter. Without wishing to be bound by theory, GR with p65 binding to the promoter can result in induction of IRAK-M transcription. In some embodiments, the small molecule is a glucocorticoid. In some embodiments, the small molecule is cortisol. In some embodiments, the small molecule is dexamethasone.

[0279] This small molecule can increase IRAK-M expression by decreasing the degradation of IRAK3 RNA transcripts. For example, Tong et al., 2021, Science Advances, Vol. 7, No. 18, reported that IRAK3 mRNA transcripts promote the degradation of IRAK3 mRNA by inhibiting IRAK-M expression. 6 The main mA modification is N6-adenosine methyltransferase-like 3 (METTL3), 6 Loss of the A "writer" can reduce the m6A modification of IRAK3 mRNA, resulting in reduced mRNA degradation and increased IRAK-M expression. In some embodiments, the small molecule is a METTL3 inhibitor. In some embodiments, the small molecule is STM2457 (Yankova et al. (2021) Nature, Vol. 593, pp. 597-601). In some embodiments, the small molecule is Cpd-564 (Wang et al., Science Translational Medicine, 2022, Vol. 14, No. 640). In some embodiments, the small molecule is UZH2 (Dolbois et al., J. Med. Chem. 2021, Vol. 64, No. 17, pp. 12738-12760).

[0280] Nucleic Acid Drugs A further aspect provides a nucleic acid for use in a method for treating or preventing macular degeneration in a subject, wherein the nucleic acid increases endogenous IRAK-M expression in a target cell in the subject.

[0281] As described herein, IRAK3 mRNA transcripts promote degradation of 6 Loss of major m6A "writers" such as the methyltransferase METTL3 reduces the m6A content of IRAK3. 6 A modification. Thus, nucleic acids targeting METTL3 can also be used to increase the expression of IRAK-M.

[0282] The nucleic acid can inhibit the expression of METTL3. The nucleic acid can bind to METTL3 mRNA. In some embodiments, the nucleic acid can hybridize to a target sequence in METTL3 mRNA. The nucleic acid can comprise a nucleic acid sequence that is at least partially complementary to a sequence in METTL3 mRNA. The nucleic acid can downregulate METTL3 expression, thereby increasing IRAK-M expression.

[0283] In some embodiments, the nucleic acid may be an inhibitory nucleic acid, such as an antisense or small interfering RNA, including but not limited to an shRNA or siRNA. In some embodiments, the nucleic acid is selected from the group consisting of siRNA, shRNA, miRNA, and ASO.

[0284] Depending on their origin, "small interfering RNA" (siRNA) or microRNA (miRNA) can be used to downregulate gene expression by binding to complementary RNA and inducing mRNA elimination (RNAi) or halting translation of mRNA into protein. siRNAs are derived by processing of long double-stranded RNAs and, when found in nature, are typically of exogenous origin. Microinterfering RNAs (miRNAs) are endogenously encoded small non-coding RNAs that are derived by processing of short hairpins. Both siRNAs and miRNAs can inhibit the translation of mRNAs with partially complementary target sequences without RNA cleavage and degrade mRNAs with fully complementary sequences.

[0285] Antisense oligonucleotide (ASO) is a preferably single-stranded oligonucleotide that targets and binds to a target oligonucleotide, such as mRNA, by complementary sequence binding.When the target oligonucleotide is mRNA, antisense binding to mRNA blocks the translation of mRNA and the expression of gene product.Antisense oligonucleotide can be designed to bind to sense genomic nucleic acid and inhibit transcription or promote the degradation of target nucleotide sequence.

[0286] Another alternative is the expression of short hairpin RNA molecules (shRNA) in cells. shRNAs are more stable than synthetic siRNAs. shRNAs consist of short inverted repeats separated by small loop sequences. One inverted repeat is complementary to the gene target. In cells, shRNAs are processed by DICER into siRNAs, which degrade the target gene mRNA and suppress expression. In embodiments, shRNAs are produced endogenously (intracellularly) by transcription from a vector. shRNAs can be produced intracellularly by transfecting cells with vectors encoding shRNA sequences under the control of an RNA polymerase III promoter, such as the human H1 or 7SK promoter, or an RNA polymerase II promoter. Alternatively, shRNAs can be synthesized exogenously (in vitro) by transcription from a vector. shRNAs can then be directly introduced into cells.

[0287] Examples of nucleic acids that decrease the expression of METTL3 can be found in Chinese Patent Application Publication No. 111676222A and Chinese Patent Application Publication No. CN114438085A. Peptide and Polypeptide Agents An agent may be a peptide or polypeptide. The term "peptide" is used herein to refer to a short chain of amino acids consisting of 40 or fewer amino acids linked by peptide bonds. The term "polypeptide" is used herein to refer to a larger biomolecule or macromolecule consisting of one or more longer chains of amino acid residues, each of which is greater than 40 amino acids in length.

[0288] The peptides or polypeptides can be used to increase endogenous expression of IRAK-M in target cells. Thus, another aspect provides a peptide or polypeptide for use in a method for treating or preventing macular degeneration in a subject, wherein the peptide or polypeptide increases endogenous IRAK-M expression in a target cell of the subject. The agent may be a polypeptide.

[0289] For example, Zacharioudaki et al., 2009, The Journal of Immunology, vol. 182:6444-6451, describes the increase in IRAK-M expression using adiponectin (globular adiponectin (gAd)). Globular adiponectin has been shown to activate the Tpl2 / ERK and PI3K / Akt1 signaling pathways. In particular, Zacharioudaki reported that Tlp2 mediates adiponectin signaling to activate ERK1 / 2 and induce IRAK-M, and that activation of PI3K and its downstream effector Akt1 is also involved in the induction of IRAK-M expression. In some embodiments, the peptide or polypeptide activates ERK1 / 2 and / or activates PI3K and Akt1. In some embodiments, the peptide or polypeptide is adiponectin. In some embodiments, the peptide or polypeptide is globular adiponectin.

[0290] The small molecules, peptides, and polypeptides described herein can be introduced into target cells using any of the delivery methods described herein. Increased IRAK-M activity Some agents according to the invention may increase IRAK-M activity.

[0291] As described herein, IRAK-M is a negative regulator of the TLR / IL-1R-induced proinflammatory cascade. IRAK-M prevents the dissociation of IRAK-1 and IRAK-4 from MyD88 and the formation of the IRAK-1-TRAF6 complex. Thus, in some embodiments, the agent promotes binding of IRAK-M to IRAK-1 and / or IRAK-4. In some embodiments, the agent promotes binding of IRAK-M to MyD88.

[0292] Embodiments provide a small molecule for use in a method for treating or preventing macular degeneration in a subject, where the small molecule increases IRAK-M activity in target cells in the subject. Nguyen et al., 2022, Int. J. Mol. Sci. 2022, vol. 23, pp. 2552, reported that cyclic guanosine monophosphate (cGMP) can modulate IRAK-M activity without changing the expression level of IRAK-M. The authors report that the pseudokinase domain of IRAK-M contains a guanylate cyclase (GC) center that generates cGMP. cGMP then associates with IRAK-M and contributes to mediating its anti-inflammatory activity. Thus, IRAK-M activity can be increased by increasing cellular cGMP levels. Cellular cGMP levels can be increased by using nitric oxide donors (Nguyen et al., 2022). It is known that cGMP synthesis by guanylate cyclase (GC) is enhanced in response to nitric oxide (NO). Cellular cGMP levels can be increased by using riociguat. Riociguat has been described as a soluble guanylate cyclase stimulator (Lian et al., 2017, Drug Design, Development and Therapy: Vol. 11, pp. 1195-1207). In some embodiments, the small molecule can stimulate guanylate cyclase (GC). In some embodiments, the small molecule increases cellular cGMP. In some embodiments, the small molecule is a nitric oxide (NO) donor. In some embodiments, the small molecule is nitric oxide. In some embodiments, the small molecule is riociguat. Alternatively, the small molecule can be cGMP.

[0293] Another aspect provides a peptide or polypeptide for use in a method for treating or preventing macular degeneration in a subject, wherein the peptide or polypeptide increases IRAK-M activity in a target cell of the subject. The agent may be a polypeptide.

[0294] Examples of peptides / polypeptides are described in Taylor, J Neuroimmunol. 2005 May;162(0):43-50. Taylor 2005 describes that the neuropeptide alpha-melanocyte stimulating hormone (α-MSH) can promote binding of IRAK-M to IRAK-1. In some embodiments, the peptide or polypeptide promotes binding of IRAK-M to IRAK-1 and / or IRAK-4. In some embodiments, the peptide or polypeptide is α-MSH.

[0295] The small molecules, peptides, and polypeptides described herein can be introduced into target cells using any of the delivery methods described herein. Additional Therapeutic Agents Additional therapeutic agents can also be used to treat or prevent macular degeneration in subjects, along with or in combination with the agents described elsewhere in this specification.These additional therapeutic agents can target other signaling pathways or processes involved in macular degeneration.Therefore, the medical uses described herein can further include administering one or more additional therapeutic agents to subjects.

[0296] For example, complement activation is strongly implicated in AMD risk and pathogenesis, especially dry AMD risk and pathogenesis. Thus, the additional therapeutic agent can be an inhibitor of the complement system, such as a regulator, for example, complement factor H (CFH) or complement factor I (CFI). The additional therapeutic agent can be a biologic that inhibits C1q, C3, C5, complement factor B (CFB), or complement factor D (CFD). Examples of C3 inhibitors include Pegcetacoplan (Apellis) and NGM621 (NGM Bio). An example of a C5 inhibitor is Avacincaptad pegol (IVERIC Bio). An example of a CFD inhibitor is Lampalizumab (Novartis). Gene therapy can also be used, for example, GT005 (Gyroscope), which is a CFI gene therapy. It has been shown that some patients with AMD 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 the soluble form of CD59 (sCD59) in the target cells. One example is HMR59 (Hemera / J&J), a sCD59 gene therapy.

[0297] VEGF antagonists are also commonly used to treat AMD, especially wet AMD.Thus, in some embodiments, the additional therapeutic agent is an anti-VEGF therapeutic effector.Anti-VEGF therapeutic agents can include ranibizumab, aflibercept, bevacizumab, brolucizumab, or faricimab.

[0298] Inflammatory activation is involved in AMD, especially in patients with dry AMD. In some embodiments, the additional therapeutic agent is an inhibitor of the inflammatory pathway. Examples of inhibitors of the inflammatory pathway include anakinra and canakinumab. The additional therapeutic agent may be serine protease HtrA (gene name: HTRA1), which has been shown to be decreased in the retina of patients with dry AMD (Williams et al., PNAS 2021, Vol. 118, No. 30, e2103617118).

[0299] The additional therapeutic agent may be ciliary neurotrophic factor (CNTF), a neuroprotective factor. The additional therapeutic agent may be a mitochondrial targeted peptide antioxidant, which can inhibit oxidative stress associated with AMD, particularly dry AMD. Examples of mitochondrial targeted peptide antioxidants include SS-31 (also known as elamipretide) and SS-20 (Szeto. The AAPS Journal 2006; Vol. 8 (No. 2) Article 32).

[0300] The additional therapeutic agent may be a senescent cell-eliminating molecule. For example, the senescent cell-eliminating molecule may be an inhibitor of Bcl-xL protein. Bcl-xL protein has been found to be upregulated in senescent retinal cells and to avoid apoptosis (Crespo-Garcia et al., 2021, Cell Metabolism 33, 818-832).

[0301] The additional therapeutic agent can be a senescent cell inhibitory molecule that targets the SASP-proinflammatory signaling network. For example, the senescent cell inhibitory molecule can be a neutralizing antibody against either IL-1α or its receptor to reduce NF-κB transcriptional activity (Orjalo et al., PNAS, October 6, 2009; Vol. 106, No. 40: 17031-17036).

[0302] Additional therapeutic agents could be autophagy inducers that can promote autophagy and reduce inflammation, such as shRNAs for mTOR inhibition (Lee et al., Invest Ophthalmol Vis Sci. 2020;61(2):45).

[0303] An additional therapeutic agent may be pigment epithelium-derived factor (PEDF), a serpin superfamily protein and neuroprotective factor that has been found to significantly reduce expression levels in Bruch's membrane and RPE in patients with AMD (Bhutto et al., Exp Eye Res. 2006 January;82(1):99-110. doi:10.1016 / j.exer.2005.05.007) and diabetic retinopathy (DR) (Ogata et al., 2002, American Journal of Ophthalmology, 134(3):348-353).

[0304] The additional therapeutic agent can be a small molecule, a peptide, a polypeptide, an antibody or antibody-like fragment, or a nucleic acid (eg, shRNA). As used herein, "antibody" includes antibody fragments or derivatives, synthetic antibodies, or synthetic antibody fragments. Given today's state of the art in monoclonal antibody technology, antibodies against most antigens can be prepared. The antigen-binding portion can be a portion of an antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., a single chain Fv fragment [ScFv]). Suitable monoclonal antibodies against a selected antigen can be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies: Techniques and Applications", JGR Hurrell (CRC Press, 1982). Chimeric antibodies are discussed by Neuberger et al. (1988, 8th International RICMP7164916 Biotechnology Symposium Part 2, pp. 792-799).

[0305] The additional therapeutic agent can be administered simultaneously with an agent for increasing IRAK-M expression and / or increasing IRAK-M activity, as described herein. For example, a composition comprising (i) an agent for increasing IRAK-M expression and / or increasing IRAK-M activity, and (ii) an additional therapeutic agent, can be administered to a subject.

[0306] An additional therapeutic agent (eg, a peptide, polypeptide, antibody or antibody-like fragment, or an RNA molecule described herein) may be encoded by the nucleic acid sequence. When the agent for increasing IRAK-M expression and / or increasing IRAK-M activity is a nucleic acid, the nucleic acid may further comprise a nucleic acid sequence encoding an additional therapeutic agent. The nucleic acid may drive expression of the additional therapeutic agent. A nucleic acid comprising at least two nucleic acid sequences (e.g., one encoding IRAK-M and another encoding an additional therapeutic agent) may comprise a separate promoter for each nucleic acid sequence.

[0307] Alternatively, the nucleic acid sequence encoding a therapeutic agent can be delivered to a subject via a nucleic acid separate from the nucleic acid containing the nucleic acid sequence encoding an agent for increasing IRAK-M expression and / or increasing IRAK-M activity (i.e., two different nucleic acids).

[0308] The nucleic acid can be delivered to the target cell via a viral or non-viral delivery system. When the additional therapeutic agent is an antibody (or antibody-like molecule), the antibody (or Ab-like molecule) can be encoded in a single vector through the use of a 2A self-processing peptide sequence to express the heavy chain (HC) and light chain (LC) separately. Two promoters can be used to separate the IRAK-M from the HC and LC. Alternatively, each of the IRAK-M agents, the HC and LC, can be separated by a 2A self-processing (or self-cleaving) peptide. The use of nucleic acid sequences encoding 2A peptides is described in detail in Fuchs et al., 2016, PLOS ONE, DOI:10.1371 / journal.pone.0158009, and Lin and Balazs, Retrovirology (2018) 15:66. In some embodiments, the 2A sequence is a foot and mouth disease virus 2A sequence (F2A). In some embodiments, the 2A sequence is a picornavirus 2A sequence. In some embodiments, the 2A sequence is an Equine rhinitis A virus 2A sequence (E2A). In some embodiments, the 2A sequence is a Porcine Teschovirus-1 2A sequence (P2A). In some embodiments, the 2A sequence is a Thosea asigna virus 2A sequence (T2A). Without wishing to be bound by theory, it is believed that separation of the HC and LC (or any two sequences) occurs via a ribosomal skip mechanism that prevents the formation of a peptide bond during translation. A furin cleavage sequence can also be used to remove the 2A peptide during processing in the Golgi.

[0309] Pharmaceutical Compositions and Routes of Administration The agents described herein and additional therapeutic agents may be formulated into pharmaceutical compositions. Methods for administering gene therapy vectors are well known to those skilled in the art. The IRAK-M expression vector can be introduced systemically (e.g., intravenously or by injection). The IRAK-M expression vector can be introduced locally (i.e., directly into a specific tissue or organ, such as the liver). The IRAK-M expression vector can be introduced directly into the eye (e.g., by intraocular 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.

[0310] Administration may be peripheral, e.g., intravenous, cutaneous, subcutaneous, nasal, intramuscular, or intraperitoneal. Typically, however, in the context of the present 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 is by electroporation.

[0311] Typically, the retina can be accessed via three different routes: intravitreal, subretinal, and suprachoroidal. Subretinal injections are typically invasive surgical procedures in which a therapeutic composition is delivered between the photoreceptors and the RPE. This in vitro retinal method may require an operating room and is usually performed under general anesthesia. Intravitreal injections (IVI), on the other hand, do not require an operating room. Suprachoroidal injections are less invasive than subretinal injections and involve accessing the retina by injecting into the space between the choroid (which covers the RPE) and the sclera (Sahu B et al., Biomolecules 2021, vol. 11, p. 1135).

[0312] Administration is preferably a "prophylactically effective amount" or a "therapeutically effective amount", which is sufficient to show benefit to an individual. The actual amount administered, and rate and time-course of administration, will depend on the individual subject and the nature and severity of the condition.

[0313] In addition to one of the above substances, the pharmaceutical composition may contain a pharma- ceutically acceptable excipient, carrier, buffer, stabilizer, or other substance known to those skilled in the art. Such substances must be non-toxic and must not interfere with the efficacy of the active ingredient.

[0314] The nucleic acid-containing compositions of the invention can be stored and administered in a sterile, physiologically acceptable carrier, with the nucleic acid being dispersed together with any agent that aids in the introduction of DNA into cells. A variety of sterile solutions can be used to administer the composition, including water, PBS, ethanol, lipids, etc. The concentration of DNA is sufficient to provide a therapeutic dose and depends on the efficiency of delivery into the cells.

[0315] Gene therapy vectors must be manufactured in compliance with Good Manufacturing Practice (GMP) requirements that render the product suitable for administration to a patient. Disclosed herein are gene therapy vectors suitable for administration to a patient, including gene therapy vectors manufactured and tested in accordance with GMP requirements. Gene therapy vectors that receive regulatory approval must be tested for potency and identity, must be sterile, free of adventitious substances, and all components in the product (i.e., preservatives, diluents, adjuvants, etc.) must meet standards of purity, quality, and must not be harmful to the patient. For example, the nucleic acid preparation is shown to be free of mycoplasma. See, for example, Islam et al., 1997, An academic centre for gene therapy research and clinical grade manufacturing capability, Ann Med 29:579-583.

[0316] Pharmaceutical compositions can be prepared using pharma- ceutically acceptable "carriers" consisting of materials that are considered safe and effective. The term "carriers" refers to diluents, binders, lubricants, and disintegrants. Those skilled in the art are familiar with such pharmaceutical carriers and methods of formulating pharmaceutical compositions using such carriers. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", e.g., physiologically acceptable and typically do not cause allergic or similar adverse reactions, such as stomach upset, when administered to humans. In some embodiments, the term refers to molecular entities and compositions that have been approved by a US federal or state government regulatory agency as a GRAS listing under sections 204(s) and 409 of the Federal Food, Drug, and Cosmetic Act, which are subject to premarket review and approval by the FDA or similar listing, the United States Pharmacopeia, or other generally accepted pharmacopeia for use in animals, more specifically in humans.

[0317] The pharmaceutical compositions provided herein may include one or more excipients, such as solvents, dissolution enhancers, suspending agents, buffers, isotonicity agents, antioxidants, or antimicrobial preservatives. When used, the excipients of the composition do not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredient. Thus, those skilled in the art will understand that a composition is provided in which there is no incompatibility between any of the components of the dosage form. The excipients may be selected from the group consisting of buffers, solubilizers, isotonicity agents, chelating agents, antioxidants, antimicrobial agents, and preservatives.

[0318] The compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially depending on the condition to be treated. The terms "treatment", "treat" or "treating" as used herein refer to reducing the severity of a disease or condition, reducing the duration of a disease; improving or eliminating one or more symptoms associated with a disease or condition, or providing a beneficial effect to a subject with a disease or condition. The term also encompasses the prevention of a disease or condition or a symptom thereof. "Prevention" is known in the art to mean reducing or reducing the occurrence or severity of a particular disease outcome. For example, delaying the progression of cancer in a subject.

[0319] As used herein, the term "subject" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). In many embodiments, the subject is a human. A subject may be a patient, referring to a human presenting to a health care provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." In some embodiments, the subject is a human. A subject may be affected or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder. In some embodiments, the subject is affected or may be affected by a retinal disease, particularly macular degeneration.

[0320] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in a particular form, or as means for performing a disclosed function, or as a method or process for obtaining a disclosed result, can, where appropriate, be utilized separately or in any combination of such features to realize the invention in diverse forms thereof.

[0321] While the present invention has been described in connection with exemplary embodiments, as noted above, many equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the exemplary embodiments of the present invention described above are considered to be illustrative and not limiting. Various modifications to the described embodiments may be made without departing from the spirit and scope of the present invention.

[0322] For the avoidance of doubt, the theoretical explanations provided herein are provided for the purpose of enhancing the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0323] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Throughout this specification, including the claims which follow, unless the context requires otherwise, words such as "comprise" and "include" and variations such as "comprises," "comprising" and "including" are understood to imply the inclusion of stated integers or steps or groups of integers or steps but not the exclusion of other integers or steps or groups of integers or steps.

[0324] It should 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 use of the antecedent "about," it will be understood that the particular value forms another embodiment. The term "about" in reference to numerical values ​​is arbitrary and may mean, for example, ±10%. EXAMPLES

[0325] Example 1 Dry age-related macular degeneration (AMD) represents a large unmet clinical need - currently incurable, affecting hundreds of millions of people and placing a significant burden on society and healthcare budgets. Low-grade chronic inflammation plays a key role in AMD progression, but the molecular mechanisms that initiate dysregulation in the immune response and promote pro-degenerative cues remain poorly understood.

[0326] As shown below, expression of an anti-inflammatory molecule named interleukin-1 receptor-associated kinase (IRAK)-M was identified in both human and mouse retinal pigment epithelium (RPE). IRAK-M expression levels were decreased with age and oxidative stress, and were also reduced in AMD donor eyes compared to age-matched controls. IRAK-M-deficient mice exhibited an AMD-like phenotype at an early age and were more susceptible to various oxidative insults than wild-type mice. Mechanistically, lack of IRAK-M disrupted RPE cell homeostasis and function, as evidenced by altered mitochondrial metabolism, accelerated cellular senescence, and elevated inflammatory cytokine production. Conversely, increased IRAK-M expression in RPE cells protects against oxidative or immune stress. The data reveal an underlying mechanism of neurodegeneration in the eye due to a proinflammatory process that is further exacerbated by aging and oxidative stress. Thus, IRAK-M is a critical immunoregulatory molecule in maintaining the metabolic health and function of the RPE.

[0327] Materials and Methods mouse Irak3 - / - Mice were obtained from Jaxo Laboratory (B6.129S1-Irak3tm1Flv / J, stock #007016). Although not previously reported (90), we found an Rd8 mutation in the Crb1 gene in this strain using established PCR genotyping protocols (90). Therefore, mice were backcrossed to C57BL / 6J (wild type or WT, Charles River Laboratories, Portishead, UK) to broodstock the Rd8-negative Irak3 - / -Genotypes were established (not shown). Breeding colonies were maintained at the Animal Services Unit at the University of Bristol. All mice were housed in the University's animal care facility in accordance with Home Office regulations. Animal procedures conformed to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Methods were performed in accordance with the approved University of Bristol institutional guidelines and all experimental protocols under Home Office Project Licence 30 / 2745 and PP9783504 were approved by the University of Bristol Ethical Review Group.

[0328] Human ocular tissue and sample processing Human donor eye or ocular tissue surplus for corneal transplantation (without documented ocular disease) were obtained from the National Health Service (NHS) Blood and Transplant Services after Research Ethics Committee approval (20 / LO / 336) and experiments were performed in accordance with the Declaration of Helsinki and in compliance with UK legislation. Age and sex of human eye samples are shown in the figures. For immunohistochemistry, tissues were fixed in 4% formaldehyde in PBS, embedded in optimal cutting temperature compound, frozen in dry ice and subsequently stored at -80°C before preparation of frozen sections. For Western blotting, dissected RPE / choroid tissues were disrupted in 400 μL Pierce® RIPA buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific, Paisley, UK) for protein extraction.

[0329] Data Mining Potential datasets for analyzing AMD-related changes were selected according to the availability of RNA-seq data processed through NCBI GEO datasets, using the search terms "AMD retina" and "AMD RPE". We used the RNA-seq dataset from Kim et al. in retina and RPE-Choroid-Scleral (RCS) Homo Sapiens tissues (GEO accession GSE99248). It includes both antisense and sense transcriptome data for seven donor eyes with no documented ocular disease (age range 83-92y, three females and five males), and eight AMD donor eyes (age range 83-95y, five females and two males). The AMD eyes were characterized at various stages, including two early AMD, one dry AMD with RPE atrophy, three late dry AMD, and one late wet AMD. The dataset samples were classified according to histological type (either retina or RCS) and phenotype (AMD or normal); normal samples corresponding to each histological type were used as controls. Geometric means were calculated for each group, fold changes were calculated for each gene, and p-values ​​were calculated using an unpaired two-tailed t-test. Genes with significant p-values ​​(<0.05) were compiled into gene lists for analysis (fold change >2 for upregulation, <0.5 for downregulation). The same procedure was performed for both mRNA and antisense datasets. The compiled gene lists were uploaded into Metascape online analysis resource (91) and path enrichment analysis was performed. Gene sets whose members were significantly over-represented among the input gene lists were recorded. Heatmaps of significantly enriched clusters were generated.

[0330] antibody Rabbit polyclonal anti-IRAK-M, rabbit polyclonal anti-c-Fos (Phospho T325), rabbit monoclonal anti-c-Fos antibody, rabbit polyclonal anti-HMGB1, rabbit monoclonal anti-c-Jun, rabbit monoclonal anti-c-Jun (Phosphor S63), rabbit monoclonal anti-p21, rabbit monoclonal anti-taminin B1, mouse monoclonal anti-RPE65, mouse monoclonal anti-rhodopsin and goat polyclonal anti-8-hydroxyguanosine were all purchased from Abcam (Cambridge, UK). Rabbit polyclonal anti-ZO-1 was obtained from Thermo Fisher Scientific. Rat monoclonal anti-CD11b (M1 / 70) was obtained from BD Biosciences (Wokingham, UK). Secondary antibodies used for western blotting, such as HRP-conjugated goat anti-rabbit and anti-mouse IgG, were obtained from New England Biolabs (Hitchin, UK). Secondary antibodies used for immunostaining, including Alexa Fluor 488-goat anti-rabbit or anti-mouse IgG, Alexa Fluor 488-rabbit anti-rat IgG, Alexa Fluor 555-goat anti-rabbit IgG, and Alexa Fluor 488-donkey anti-goat IgG, were obtained from Thermo Fisher Scientific.

[0331] RPE cell lines, primary cells, iPSC-derived RPE cells and treatments Human RPE cell line ARPE-19 (American Type Culture Collection) and mouse RPE cell line B6-RPE07 (a gift from Professor Heping Xu, Belfast) (92) were maintained in DMEM medium supplemented with 10% FBS, 1% L-glutamine, 1 mM sodium pyruvate, 60 μM 2-mercaptoethanol, and 1% penicillin / streptomycin (complete medium) at 37°C in a 5% CO2 atmosphere. Cells were passaged at a 1:5 split ratio using trypsin / EDTA and allowed to recover in complete medium for 2 days before processing.

[0332] Mouse primary RPE cells were isolated and cultured as previously described ( 79 ). Briefly, WT or Irak3 - / - Eyes from mice were enucleated and cleaned using angled scissors to ensure no connective tissue remained. After removing the cornea and lens, the eyes were incubated in hyaluronidase for 45 min at 37°C and in Hank's Balanced Salt Solution (HBSS) containing 10 mM HEPES for an additional 30 min, after which the retina was removed by dissection. After incubation in trypsin / EDTA for 45 min at 37°C, the eyecups were transferred to HBSS containing 20% ​​heat-inactivated FBS and gently shaken to detach the RPE. The RPE sheets were incubated in trypsin / EDTA for an additional 1 min to form a single cell suspension. The obtained RPE cells were resuspended in αMEM basal medium supplemented with 1% N1 medium supplement, 1% L-glutamine, 1% penicillin-streptomycin, 1% non-essential amino acid solution (NEAA), 20 μg / l hydrocortisone, 250 mg / l taurine, 0.013 μg / l triiodothyronine, and 5% FBS. The purity of the cells was confirmed by immunoblotting for RPE65 and rhodopsin as we previously described (23). The cells were plated at 25,000 / cm on laminin-precoated Seahorse XF cell culture plates (Agilent Technologies, Santa Clara, California, USA) or 8-well chamber slides (Corning GmbH, Wiesbaden, Germany). 2 After the first 72 h of incubation, serum was removed from the medium. The medium was changed twice a week. Cells were used for experiments 7–10 days after isolation.

[0333] Primary human RPE cells (H-RPE) were purchased from Lonza (Slough, UK). Cells were maintained in RPE basal medium supplemented with 2% L-glutamine, 0.5% FGF-B and 0.1% GA-1000 and subcultured using trypsin / EDTA at a ratio of 1:3 for up to four passages. H-RPE cells were grown at 10,000 cells / cm. 2The cells were seeded in 24-well plates at a density of 1000 x 1000. After overnight incubation, the cells were used for induction of oxidative stress.

[0334] Human fibroblast-induced pluripotent stem cells (iPSCs) derived from healthy donors were a kind gift from Professor Peter Coffey at UCL (78). iPSC colonies were cultured on Matrigel hESC-qualified matrix (BD Biosciences, Wokingham, UK) in E8 (Thermo Fisher Scientific). Once 80% confluent, the medium was changed to differentiation medium containing Knockout-DMEM, 20% Knockout serum replacement, 1% non-essential amino acids (NEAA), 1% Glutamax and 0.2% 2-mercaptoethanol (all from Thermo Fisher Scientific). Cultures were fed twice weekly for at least 8 weeks until pigmented foci were observed. These pigmented foci were manually isolated and plated at 50,000 cells / cm on Matrigel-precoated 96-well plates or Seahorse XF plates. 2 The cells were seeded at a density of 100x100x100cm, subjected to oxidative or immune stress, and their metabolic functions were analyzed.

[0335] Cells from different sources were treated with different concentrations of paraquat (PQ), hydrogen peroxide (HO) or lipopolysaccharide (LPS, all from Sigma-Aldrich, Poole, UK) continuously for up to 72 h, or repeatedly exposed to PQ or HO for 2 h each day for a total of 7 days (21). In some embodiments, RPE cells were pretreated with a c-Jun inhibitor (SP600125, Sigma-Aldrich) or a c-Fos inhibitor (T-5224, Cambridge Bioscience, Cambridge, UK) for 2 h before the addition of PQ.

[0336] immunohistochemistry To investigate the expression pattern of IRAK-M in human and mouse retina, human ocular tissue from a 20-year-old donor (no documented ocular disease) or enucleated eyes from 8-week-old WT mice were fixed in 4% (for human tissue) or 2% (for mouse tissue) paraformaldehyde (PFA) before 12 μm-thick frozen sections were prepared on a cryostat. Sections were permeabilized with 0.1% Triton X-100 and blocked with 10% normal donkey serum, 5% BSA + 0.3 M glycine, followed by incubation overnight at 4°C with rabbit anti-IRAK-M (1:1000) and mouse anti-rhodopsin (1:500) or mouse anti-RPE65 (1:100). After washing, sections were incubated with donkey anti-rabbit IgG conjugated with Alexa Fluor 555 and donkey anti-mouse IgG conjugated with Alexa Fluor 647 (both 1:1000). DAPI counterstaining was used to reveal nuclei in the sections. Tissues were washed, mounted in Vectashield antifade medium, and examined by confocal microscopy.

[0337] To prepare mouse retina and RPE / choroid whole mounts, enucleated eyes were first fixed in 2% PFA overnight. After dissection of the eyes, retina and RPE / choroid tissues were blocked and permeabilized in 10% normal goat serum, 5% BSA, 0.3 M glycine with 0.3% Triton X-100 in PBS for 2 h, followed by incubation with rat anti-CD11b (1:200) in 1% BSA with 0.15% Triton X-100 at 4°C overnight. After extensive washing, samples were further incubated with Alexa Fluor 488-goat anti-rat IgG (1:400). Tissues were counterstained with DAPI and flat-mounted for observation by confocal microscopy.

[0338] Cell apoptosis in retina and RPE / choroid whole mounts was determined by TUNEL staining using the in situ cell death detection kit, TMR Red (Roche Diagnostics, Burgess Hill, UK), as previously described ( 14 ).

[0339] Human iPSC-derived RPE cell cultures were fixed with 2% PFA and permeabilized with 0.1% Triton X-100. After blocking with 10% normal goat serum, cells were incubated with polyclonal rabbit anti-ZO-1 (1:200) overnight at 4°C, followed by labeling with goat anti-rabbit conjugated with Alexa Fluor 488 (1:400). Nuclei were detected with DAPI.

[0340] Western blot Protein extracts from tissues or cells were prepared using Pierce® RIPA lysis buffer containing Halt™ protease and phosphatase inhibitors (Thermo Fisher Scientific). Protein concentrations were measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). 5–15 μg of protein for each sample was mixed with Tris-glycine SDS sample buffer (1:2) and reducing reagent (1:10, Thermo Fisher Scientific) followed by denaturation at 80°C for 2 min. After separation using Novex™ 4–20% Tris-glycine minigels (Thermo Fisher Scientific), proteins were transferred to PVDF membranes (Thermo Fisher Scientific) and then blocked with 5% w / v milk in Tris-buffered saline (TBS) + Tween 20 (TBS-T; 0.1% v / v). Blots were incubated overnight at 4°C with primary antibodies against IRAK-M (1:2000), c-Jun (1:1000), phosphor-c-Jun (1:1000), c-Fos (1:1000), phosphor-c-Fos (1:1000), p21 (1:1000), lamin B1 (1:1000) or β-actin (1:2000). After extensive washing, blots were incubated with the appropriate secondary antibodies, anti-rabbit HRP (1:2000) or anti-mouse HRP (1:2000; both from Cell Signalling Technologies, London, UK). Chemiluminescence was detected by Amersham ECL reagent (Sigma-Aldrich) and developed using Hyperfilm™ ECL film (Sigma-Aldrich) and X-developer.

[0341] Multiplexed cytokine arrays and enzyme immunoassays (EIAs) Supernatants of ARPE-19 or BMMΦ cultures, or mouse sera prepared from lateral tail vein sampling, were examined for concentrations of inflammatory cytokines using the LEGENDplex human or mouse cytokine array kit (BioLegend, London, UK) according to the manufacturer's instructions. Concentrations of HMGB1 in ARPE-19 cell culture supernatants were determined by direct EIA using polyclonal anti-HMGB1 (Abcam) as per the manufacturer's protocol.

[0342] Seahorse metabolic analysis Seahorse XFp or XFe96 cell culture miniplates, sensor cartridges with utility plates, and all reagents for Mito stress testing were obtained from Agilent Technologies. Different RPE cells were incubated in Seahorse XF DMEM (pH 7.4) containing 25 mM glucose, 1 mM pyruvate, and 2 mM glutamine for 45 min in a 37°C incubator without CO2 (23). Oligomycin (ATPase inhibitor, 1 μM), FCCP (protonophore uncoupler, 0.5 μM), and antimycin A / rotenone (electron transport inhibitor, 1 μM) were injected at the indicated locations, and oxygen consumption rate (OCR, pmol O2 / min) and extracellular acidification rate (ECAR, mpH / min) were measured in real time. Measurement rates were normalized by total protein content analyzed using the BCA assay. Metabolic parameters were calculated using the following formulas: non-mitochondrial respiration (minimum OCR after antimycin A / rotenone injection), basal respiration (difference between OCR before oligomycin and non-mitochondrial respiration), maximum respiration (difference between maximum OCR and non-mitochondrial respiration after FCCP injection), H + (Proton) leak (difference between minimum OCR and non-mitochondrial respiration after oligomycin injection), ATP production (difference between OCR before oligomycin injection and minimum OCR after oligomycin), spare respiratory capacity (difference between maximum respiration and basal respiration), glycolysis (maximum EACR before oligomycin injection), maximum glycolytic capacity (maximum ECAR after oligomycin injection), and glycolytic reserve (difference between maximum glycolytic capacity and glycolysis) (93).

[0343] Senescence-associated β-galactosidase staining Primary mouse RPE cells cultured in laminin-precoated 8-well chamber slides (Corning GmbH, Wiesbaden, Germany) were treated with 0.25 mM PQ or H2O2 for 2 h daily for a total of 7 days for induction of senescence (21). A fluorescence-based live cell senescence β-galactosidase (SA-β-Gal) assay kit (Enzo Life Sciences, Exeter, UK) was used to quantify cellular senescence according to the manufacturer's instructions. Briefly, cells were incubated with the pretreatment solution for 2 h at 37 °C, followed by addition of SA-β-Gal substrate solution (1:200). After a further 4 h of incubation, cells were rinsed extensively with PBS and captured via the GFP channel (480 nm excitation, 520 nm emission) on an Evos FL fluorescence microscope.

[0344] Chromatin immunoprecipitation (ChIP) A one-step ChIP kit (Abcam) was used to identify whether the AP-1 subunits c-Jun and c-Fos are transcription factors for IRAK-M in RPE cells. Subconfluent ARPE-19 cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by adding chromatin lysis buffer and subsequently disrupted with a Dounce homogenizer. The lysate was sonicated to shear DNA to an average length of 300–500 bp. Crosslinked chromatin fragments (1 × 10 6 Cells (from the NIH-derived IgG ...

[0345] Cytotoxicity assay At various time points, RPE cell culture supernatants were harvested and chemical-induced RPE cytotoxicity was assessed using a lactate dehydrogenase (LDH) detection kit (Abcam) according to the manufacturer's instructions. Released LDH activity was normalized to the LDH value of RPE lysates (100% cytotoxicity).

[0346] Detection of autophagic flux The formation of autophagosomes and autolysosomes in RPE cells was monitored through LC3B protein localization using the Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The RFP-GFP-LC3B Sensor Kit uses the high transduction efficiency and minimal toxicity of BacMam 2.0 expression technology and allows the detection of LC3B-positive, neutral pH autophagosomes in green fluorescence (GFP) and LC3B-positive acidic pH autolysosomes in red fluorescence (RFP). The RFP and GFP genes contained in this chimera are TagRFP and Emerald GFP, respectively. Briefly, ARPE-19 cells were transduced overnight with a mixture of TagRFP-LC3B and Emerald GFP-LC3B at an MOI of 30 in cell culture medium, followed by the addition of chemicals for 24 hours. LC3B-positive fluorescent puncta (autophagosomes in green, autolysosomes in red) were analyzed using a fluorescence microscope and quantified using Image J.

[0347] Quantitative RT-PCR (QRT-PCR) Total RNA was isolated using TRIzol Reagent (Thermo Fisher Scientific). 1 μg of total RNA was treated with RQ1 RNase-free DNase and then reverse transcribed using ImProm-IITM Reverse Transcription System (Promega). cDNA was amplified using PowerUp SYBR® Green PCR Master Mix Reagent (Thermo Fisher Scientific) on a QuantStudio Real-Time PCR System. Primer sequences were designed using Primer-BLAST (NCBI): mouse Irak3, forward 5'-GACCAGCTCCAACCCAAACT (SEQ ID NO: 36), reverse 5'-GCCACCGCCGGTCATATTTA (SEQ ID NO: 37); human Irak3, forward 5'-CCCACTCCCTTGGCACATTC (SEQ ID NO: 38), reverse 5'-AGCATGGTTGAACGTTGTGC (SEQ ID NO: 39); mouse Irak1, forward 5'-CAGAGGTGGA ACAGCTATCAAG (SEQ ID NO: 40), reverse 5'-CATTGGGCAAGAAGCCATAAAC (SEQ ID NO: 41); mouse Irak4, forward 5'-AAAGGACAGGACATCCGTAATG (SEQ ID NO: 42), reverse 5'-TCGCTGGACTCTACACTTCT (SEQ ID NO: 43); mouse Rps29, forward 5'-ACGGTCTGATCCGCAAATAC (SEQ ID NO: 44), reverse 5'-ATCCATTCAAGGTCGCTTAGTC (SEQ ID NO: 45).

[0348] In vivo induction of retinal degeneration WT or Irak3 - / -Retinal degeneration in mice was induced by paraquat (PQ, a pro-oxidant) or light-induced oxidative damage as previously described (22,35,36). Six- to eight-week-old mice were anesthetized with an intraperitoneal injection of 200 μl of Vetelar (ketamine hydrochloride 100 mg / ml, Pfizer, Sandwich, UK) and Rompum (xylazine hydrochloride 20 mg / ml, Bayer, Newbury, UK) mixed in a ratio of 0.6:1:84 with sterile water. Pupils were dilated with 1% tropicamide and 2.5% phenylephrine (both Chauvin, Essex, UK). Then, one drop of Viscotears (Novartis, London, UK) was applied to cover the ocular surface before the following procedure. For PQ-induced retinal degeneration ( 22 , 36 ), administration of PQ (0.375–1.5 mM, Sigma-Aldrich, Poole, UK) or PBS in the contralateral eye was delivered by a 2-μl intravitreal injection performed under a surgical microscope.

[0349] For light-induced retinal degeneration (LIRD) (35), intense light delivered by a fundus camera was delivered to the retina through a Nikon D80 digital camera connected to an endoscope with a 5-cm-long telescope. The mouse was positioned using the stage control so that the cornea was in contact with the edge of the telescope and the optic disc at the center of the fundus image. One eye was provided with a single exposure for 20 min at an intensity of 100 klux. Light intensity was measured periodically with a light meter to ensure isoluminance. The contralateral eye served as a control without light challenge.

[0350] Optical Coherence Tomography (OCT) At selected time points during the lifespan or after induction of retinal degeneration in mice, pupils were dilated and animals were anesthetized for clinical evaluation. OCT scans, as well as brightfield and fluorescent fundus images, were captured using a Micron IV retinal imaging microscope (Phoenix Research Laboratories, Pleasanton, CA). Prior to imaging, the Micron IV CCD and OCT were calibrated according to the manufacturer's protocol. Gain was set at +3 dB and FPS at 15, or +12 dB and 2 for brightfield and GFP fluorescent images, respectively. OCT images were used to assess retinal structure and thickness using ImageJ (47).

[0351] Plasmid transfection and siRNA To activate IRAK-M or c-Jun expression in ARPE-19 cells, cells were seeded in 48-well plates and allowed to reach 70-80% confluence. CRISPR activating plasmids (Santa Cruz Biotechnology) were used to upregulate the expression of endogenous gene expression. For each transfection, 0.3 μg of plasmid DNA was mixed with 1.5 μl of Lipofectamine 3000 and 1 μl of P3000 reagent and left for 15 min. The transfection complex was then added to the cells and left with reduced serum (1% FBS) for 48 h, followed by Western blot analysis of protein expression. A non-targeting CRISPR plasmid (Santa Cruz Biotechnology) served as a negative control.

[0352] To induce stable expression of exogenous IRAK-M gene in B6-RPE07 cells, 70% confluent cells in 48-well plates were transfected with 0.3 μg of control pUNO1 plasmid (catalog no. punol-mcs) or pUNO1 plasmid carrying human IRAK-M (catalog no. punol-hirakm) or mouse IRAK-M (catalog no. punol-mirakm) using Lipofectamine 3000 as described above. All plasmids were obtained from Invitrogen, Toulouse, France. Two days after transfection, the medium was replaced with selection medium containing 10 μg / ml blasticidin. Stable transfectants were selected and expanded for 3 weeks, and stable IRAK-M expression was determined by qRT-PCR or Western blot.

[0353] To knockdown IRAK-M expression in ARPE-19 cells, siRNA specific for human IRAK-M (Santa Cruz Biotechnology, Heidelberg, Germany) was utilized according to the manufacturer's instructions. siRNA was mixed with Lipofectamine 3000 reagent (Thermo Fisher Scientific) to form a transfection complex and then added to the RPE medium at a final concentration of 40 nM. Non-silencing siRNA was used as a negative control. IRAK-M expression was determined by Western blot 48 h after transfection.

[0354] statistics Results are presented as mean ± standard deviation (SD). Statistical analysis was performed using unpaired two-tailed Student's t-test between two groups. One-way analysis of variance was used to test for normal distribution and homogeneity of variance and to compare between more than two groups. Two-way analysis of variance was used to evaluate the correlation of two independent variables on the dependent variable, followed by Kruskal-Wallis test with Bonferroni correction for post hoc comparisons. Differences between groups were considered significant at P < 0.05. Statistical analysis was performed using GraphPad Prism 7.0.

[0355] result IRAK-M is expressed primarily by the RPE in the retina Previously, IRAK-M transcripts were shown to be expressed in a mouse RPE cell line (B6-RPE07) (13). To identify tissue expression patterns in the retina, we performed immunohistochemistry on human retinal sections from a young donor eye (20 years old) without documented ocular disease, showing strong immunopositivity for IRAK-M localized in the RPE layer (stained with anti-RPE65 and counterstained with anti-rhodopsin and DAPI) (Figures 1A and 1B). Weak immunopositivity for IRAK-M was observed in the ganglion cell layer (GCL), inner plexiform layer (IPL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor outer segments (POS), and within the choroid (Figures 1A and 1B). Negative controls in which the primary antibody was omitted showed no significant fluorescent signal. Consistent with human samples, mouse retinal sections (8 weeks old) showed expression of IRAK-M primarily in the RPE within the retina and choroid (FIGS. 1C and 1D).

[0356] IRAK-M expression is decreased in aged RPE and AMD Dysregulated inflammation is typical with age, a major risk factor for AMD. Hallmarks of immune activation in the RPE, including lipofuscin and drusen formation and inflammasome activation, are evident during AMD progression (32). We hypothesized that IRAK-M, a key immune regulator, changes expression in age and AMD.

[0357] In human RPE / choroid protein lysates taken from donor eyes (all without documented ocular disease), Western blot analysis showed a significant decrease in IRAK-M expression in older samples (76–84 years; a time when AMD prevalence increases sharply (33)) compared to younger ages (20–22 years) (Figure 1E and Figure 9). A decrease was observed in middle-aged (52–55 years) compared to younger individuals, but this was not statistically significant (Figure 1E). Similarly, 19-month-old mice showed a decrease in IRAK-M expression in the RPE compared to young (2–3 months, Figure 1F) or 13-month-old mice.

[0358] We performed data mining on an RNA-seq dataset containing both sense and antisense transcripts (GEO accession no. GSE99248) (34). Over 1000 genes were altered at the mRNA level, and over 3000 genes were altered at the antisense RNA level in AMD-derived RPE / choroid / sclera complexes compared to age-matched normal controls. AMD-associated genes are involved in various biological processes, with top GO clusters related to immune response, cytoskeleton reorganization, extracellular matrix organization, regulation of MAPK cascades, lipid metabolism, and cell apoptosis (Figure 10). By focusing on IRAK family genes, we found that only the levels of IRAK3 mRNA in AMD RPE / choroid / sclera showed a significant decrease compared to age-matched normal controls (Figure 1G). Meanwhile, antisense RNA specific to IRAK3 showed no significant difference (Figure 1G). Neither mRNA nor antisense RNA for other IRAK family members (IRAK1, IRAK2 and IRAK4) showed any differences between AMD and controls (FIG. 11).

[0359] Oxidative stress mimics the age-related decline in IRAK-M levels in the RPE Age-related accumulation of oxidative stress in the RPE is a recognized contributor to the progression of AMD. To examine whether further oxidative stress could accelerate the decline in IRAK-M expression, we induced oxidative insult both in vitro and in vivo.

[0360] In vitro human RPE cell line (ARPE-19) was treated for up to 72 h with different concentrations of paraquat (PQ), a potent and stable chemical that induces primarily mitochondrial ROS. LDH cytotoxicity assays showed dose-dependent cytotoxicity caused by PQ after 72 h (Figure 2A). To clarify the potential regulation of IRAK-M expression by oxidative stress without inducing cell death, we tested a subtoxic dose (0.25 mM) and observed that it resulted in a significant decrease in IRAK-M protein levels after 72 h (Figure 2B). The decrease in IRAK-M was accompanied by increased secretion of HMGB1, IL-18, and GM-CSF, and a decrease in IL-11, as determined by EIA, multiplex cytokine array (Figure 2C). Similarly, human iPSC-derived RPE (Figures 2D and 2E) and primary human RPE cells (Figures 2F and 2G) downregulated IRAK-M expression following 72-hour treatment with subtoxic doses of PQ (0.25-0.5 mM).

[0361] In vivo, retinal oxidative damage was induced in 8-week-old C57BL / 6J WT mice by fundus camera-directed light exposure (100 kLux, 20 min) (35) or intravitreal administration of PQ (2 μl at 1.5 mM) (36). Western blot analysis showed that IRAK-M expression in RPE lysates was significantly attenuated 7 days after light induction in both models (Figures 3A and 3D). Fundus examination and OCT photographs obtained on day 14 showed a basal appearance of white spots (red arrows, Figures 3B and 3E) indicative of accumulated microglia / macrophages inside the ONL (37), along with outer retinal thinning indicative of cell loss in the light challenge model (Figure 3C), and reduced thickness of the outer and inner retina in the PQ model (Figure 3F).

[0362] IRAK-M expression in RPE cells is regulated by AP-1 AP-1 transcription factor is one of the downstream effectors of the TLR / IL-1R-mediated signaling pathway. AP-1 has been shown to regulate IRAK-M in monocytes and lung epithelial cells, acting as an inhibitory loop (38). To examine whether AP-1 regulates IRAK-M in RPE, we performed ChIP in ARPE-19 using antibodies against c-Jun and c-Fos (AP-1 subunits) for immunoprecipitation. The results showed occupancy of c-Jun and c-Fos at the IRAK-M promoter in untreated cells, which was more pronounced in cells treated with LPS for 24 h (39) (Figure 4A). In parallel with the expression of IRAK-M, the expression level of c-Jun was decreased in an age-dependent manner in aged mouse RPE (Figure 4B). The decrease began at 13 months, earlier than the changes observed in IRAK-M (Figure 4B vs. Figure 1F). Data mining of an RNA-seq dataset in AMD (GSE99248) showed a non-significant decrease (P=0.12) in JUN mRNA levels in AMD RPE / choroid / sclera compared with age-matched normal tissues (Figure 12).

[0363] To examine whether oxidative stress also affects AP-1 activity or expression in RPE, we treated ARPE-19 with PQ and demonstrated downregulation of both c-Jun and c-Fos phosphorylation after 72 h, while total c-Jun and c-Fos remained the same (Figure 4C). Two AP-1 inhibitors, SP600125 (for c-Jun) and T5224 (for c-Fos), reduced the expression of IRAK-M in ARPE-19 (Figure 4D). Conversely, by increasing c-Jun expression via a CRISPR / Cas9 activation plasmid, IRAK-M expression was upregulated (Figure 4E). Furthermore, treatment with AP-1 inhibitors sensitized ARPE-19 to PQ-induced cytotoxicity (Figure 4F), similar to the deleterious effects of IRAK-M siRNA (Figure 4G). We also performed experiments to test whether oxidative stress-induced cell damage could be inhibited by enhancing c-Jun expression using a CRISPR / Cas9-based activation plasmid, which effectively activated the c-Jun promoter to enhance expression. However, overexpression of c-Jun itself tended to be cytotoxic (P>0.05) and did not protect cells from oxidative treatment (Figure 13A). This is probably not surprising because of the diverse functions of c-Jun / AP-1 signaling in stress responses and apoptosis (40, 41).

[0364] IRAK-M-deficient mice exhibit advanced AMD-like pathology Having demonstrated a decrease in IRAK-M expression in aging and AMD, we asked whether the absence of IRAK-M might accelerate retinal aging and pathology. - / -Mice (without Rd8 mutation) were followed for 15 months using fundus examination and OCT. At 2-5 months of age, the frequency of retinas exhibiting various numbers of fundus white spots increased sharply from 22.7% (5 of 22 eyes) to 50% (15 of 30 eyes) (Figures 5A and 5C). The incidence of abnormal retina increased continuously, reaching 78.6% (11 of 14 eyes) at 15 months (Figure 5C). We also noticed that white spots developed in these affected retinas with age (Figure 5B). In comparison, WT mice retained a normal retinal appearance (i.e., no progression of white spots) at 12 months, but a substantial incidence of WT retinas exhibited white spots at 19-21 months (60% or 6 out of 10, Figure 5C). Notably, fundus examination was not applied for WT mice aged 12-19 months due to the limited availability of aged mice. However, this finding (Fig. 5C) was not supported by Irak3 - / - This clearly indicates that the onset of retinal abnormalities in mice is much earlier than in the WT. The early appearance of white spots was associated with outer retinal lesions on OCT imaging (Figure 5D).

[0365] Irak3 - / - Alongside the early clinical changes in the mice, we observed increased numbers of CD11b + Myeloid cell populations (Figure 6A) and CD11b in the subretinal space + We observed the presence of increased cell numbers (Figure 6B), as well as an increase in apoptotic cells within the retina and RPE / choroid tissues (Figure 6C). No difference in retinal thickness was observed between WT and KO mice at 5 months, but the outer retina of KO mice was thinner at 12–13 months (Figure 6D). In parallel, serum cytokine concentrations in KO mice at 12–13 months were increased compared to WT mice (significant increases in TNF-α, MCP-1, and IL-10; Figure 6E).

[0366] Given the age-dependent increase in retinal pathology in the absence of IRAK-M, we next explored whether additional oxidative stress exaggerates the pathology. Acute retinal oxidative stress was induced by light induction or PQ administration in adult WT and Irak3 mice. - / -PQ was induced in WT animals (8 weeks of age). KO mice showed amplified retinal damage compared to WT, especially a greater decrease in outer and inner retinal thickness after light challenge (Figure 6F), and a further decrease in inner retinal thickness with PQ administration (Figure 6G).

[0367] Loss of IRAK-M disrupts RPE cell homeostasis Within the retinal "metabolic ecosystem," RPE primarily utilizes mitochondria-dependent oxidative phosphorylation (OXPHOS) for energy synthesis and transports glucose to the outer retina, particularly photoreceptors that rely primarily on aerobic glycolysis (42). To elucidate the metabolic mechanisms involved in IRAK-M deficiency-induced retinal degeneration, we investigated RPE cell metabolism and aging using primary mouse RPE cells. Cells lacking IRAK-M showed reduced levels of mitochondrial basal respiration (BR) and ATP production compared to WT cells as assessed by OCR analysis (Figure 7A), whereas ECAR examination revealed no significant differences in basal glycolysis (BG) and maximum glycolytic capacity (MGC) between genotypes (Figure 7B). The data infer a role for IRAK-M to maintain mitochondrial function in RPE cells. In support, Irak3 - / - RPE cells were more susceptible to oxidative stressor (PQ or H2O2)-induced sensing, as indicated by increased SA-β-Gal activity (Figure 7C), enhanced expression of the cyclin-dependent kinase inhibitor p21CIP1, decreased expression of the nuclear lamina protein LB1 (Figure 7D), and induced secretion of IL-6, a known SASP-cytokine released by the RPE (16, 21) (Figure 7E). - / - Basal secretion levels of the proinflammatory cytokine HMGB1 in RPE cells were significantly higher than in WT cells, but only in the absence of oxidative stressors (Figure 7F).

[0368] Overexpression of IRAK-M protects RPE cells Given the data supporting the role of IRAK-M in maintaining RPE function and health in the presence of oxidative stress, we wished to examine whether overexpression of IRAK-M could protect RPE cells. We increased the expression of native IRAK-M in human iPSC-RPE cells by transfection of a CRISPR / Cas9-based activation plasmid (Figure 13B). After 48 hours of transfection, cells were treated with H2O2 or LPS for an additional 24 hours. OCR analysis showed that basal and maximal mitochondrial respiration was maintained by IRAK-M overexpression but was impaired in control transfected cells following either oxidative or immune stress (Figure 8A). Untreated IRAK-M-overexpressing iPSC-RPE cells showed lower maximal glycolytic activity than control plasmid-transfected cells, but the levels did not change when stressed with H2O2 or LPS (Figure 8B). In contrast, glycolytic activity in control cells was significantly decreased by both H2O2 and LPS (Figure 8B). The lower levels of glycolysis in resting iPSC-RPE overexpressing IRAK-M suggest a lower dependency on glucose for energy metabolism, which may be beneficial for photoreceptors that are primarily dependent on glycolysis (42).

[0369] Using ARPE-19 cells, enhancing IRAK-M expression by CRISPR / Cas9 also partially reversed the LPS-induced decrease in maximal mitochondrial respiration (Figure 14A and B), partially supporting our findings in iPSC-RPE (Figure 8A). However, ARPE-19 was able to maintain glycolytic activity after H2O2 or LPS exposure in control cells, and enhancing IRAK-M expression increased glycolysis in response to H2O2 (Figure 14C). Furthermore, overexpression of IRAK-M in ARPE-19 induced the generation of autophagosomes (LC3B-GFP) and autolysosomes (LC3B-RFP) upon H2O2 or LPS treatment, and autophagic flux was upregulated, as shown by the tandem sensor RFP-GFP-LC3B kit (Figure 14D). Furthermore, ARPE-19 senescence induced by a subtoxic dose of PQ (0.25 mM) was inhibited by overexpression of IRAK-M, as evidenced by reduced SA-β-Gal activity and HMGB1 secretion (Figure S4E and S4F). Finally, a toxic dose of PQ (1 mM), which induces marked LDH release (Figure 2A), was significantly inhibited by increasing IRAK-M expression (Figure S4G).

[0370] Gene therapy approaches to deliver human genes to mouse eyes are crucial for preclinical evaluation (43-45). Here, we created stably transfected RPE cell lines from the parental mouse B6-RPE07 cell line using the pUNO1 vector (see Materials and Methods). The newly established cell lines were maintained and subcultured in blasticidin-containing medium and stably and robustly expressed human or mouse IRAK-M mRNA (Figure 15A). Expression of IRAK1 and IRAK4 mRNA was not affected, indicating the specificity of gene delivery. Next, NF-κB activity assays showed a decrease in the DNA-binding activity of nuclear NF-κB in human IRAK-M-expressing mouse cells after acute LPS stimulation (30 min), which was similar to the inhibitory effect of mouse IRAK-M overexpression (Figure 15B), confirming the functionality of human IRAK-M in suppressing the TLR / NF-κB signaling cascade in mouse RPE cells. Since stable transfection allows long-term studies of mechanisms and outcomes of gene regulation and pharmacological studies, we maintained post-confluence cell monolayers in serum-free conditions for up to 5 days. Cell viability of RPE cells expressing human IRAK-M was dramatically maintained, whereas control cells showed increased toxicity between days 3 and 5 (Figure 8C). When freshly confluent cells (day 0) were treated with PQ (0.125 mM) or LPS (40 ng / ml) for 3 days, transduction with human IRAK-M significantly inhibited stress-induced cytotoxicity (Figure 8D). To exclude the potential implication of endogenous mouse IRAK-M in the cellular responsiveness observed above, we transduced adult Irak3 - / - Transient transfections were performed on primary RPE cells isolated from mice. In this regard, the Seahorse metabolic flux assay was applied to investigate metabolic changes in response to a brief treatment with H2O2 (24 h, Fig. 8E and F). Supporting the findings from human iPSC-RPE cells transfected with CRISPR / Cas9 activating plasmids (Fig. 8A and B), primary Irak3 cells were upregulated in mitochondrial cells, in contrast to control transfections, which showed a marked decrease in mitochondrial activity. - / -Maximal mitochondrial respiration in RPE cells was preserved by human IRAK-M transduction after H2O2 treatment (Figure 8E). H2O2-induced oxidative stress was upregulated by Irak3. - / - It did not significantly alter the glycolytic activity of RPE cells (Figure 8F). Stable expression of human IRAK-M in transfected B6-RPE07 cells suppressed the production of proinflammatory cytokines under stress, including LPS- or PQ-induced GM-CSF and LPS-induced MCP-1 (Figure 8G). This set of data using a cellular model implies that human IRAK-M functions in mouse RPE, facilitating in vivo evaluation and clinical translation.

[0371] overview AMD is a progressive, polygenic, and multifactorial eye disease. Herein, we reveal a molecular mechanism that highlights IRAK-M in modulating responses to stressors and maintaining cellular health that may prevent progressive degeneration and cell loss.

[0372] A decrease in IRAK-M levels was observed with aging or after oxidative stress, each of which, alone or in combination, triggered an unchecked immune response in the RPE (Figures 1-3). The RPE is important for nourishing the retina throughout the visual cycle and maintaining photoreceptor function. Constant exposure to damage caused by high metabolic rate, light exposure, heterophagy and free radical formation renders RPE health vulnerable to age-related defects and mitochondrial dysfunction (8, 46, 47). As an early clinical indication and risk determinant of AMD, drusen contain a comparable protein profile as degenerated RPE and are therefore considered the main source of releasing drusen components via exosomes (48, 49).

[0373] Dynamic crosstalk between signaling cascades and intact feedback systems ensures immune homeostasis at the level of tissues (autophagy or inflammasome activation) or in local immune networks (parainflammation) (50), where immunosuppression is a crucial control mechanism. RPE cells are actively involved in the activation and suppression of local innate and adaptive immunity through the expression of many immune molecules and act as a gateway for leukocyte trafficking throughout the body (27, 51, 52). Apart from known immunosuppressive factors produced by the RPE, such as anti-inflammatory cytokines (TGF-β, IL-11, and IFN-β), chemokine CX3CL1 (fractalkine), IL-1R antagonist (IL-1Ra), IL-1R2 (CD121b), and membrane glycoprotein CD200 (13, 27, 53-55), we identified IRAK-M as a key intracellular anti-inflammatory molecule localized in the retina and expressed primarily by the RPE (Figure 1A-D). Thus, our findings complement and expand our understanding of the essential role of the RPE in immune regulation in the posterior segment of the eye (51).

[0374] Oxidative stress induces TLR-mediated inflammatory responses directly by ROS (e.g., H2O2) or indirectly by autocrine or paracrine secretion of oxidative damage products (e.g., HMGB1) (25). This has been previously verified (56) and confirmed in the present study (Figure 2C). Other oxidative damage includes DNA breaks, mitochondrial damage, and impaired intracellular RPE processing pathways (autophagy, phagolysosome, and protein trafficking). At least 10 functional TLRs (TLR1-10) have been identified in humans, of which TLR1-7, 9, and 10 are found in the RPE (57). Transduced by TLR / IL-1R and serving as a first-order inhibitor of NF-κB / AP-1-mediated inflammatory responses, IRAK-M expression is regulated by numerous endogenous or exogenous factors, including adiponectin, TGF-β1, GM-CSF, and cell surface or intracellular molecules, including TREM-1 and PI3K (30). For example, acute alcohol intake increases IRAK-M in human monocytes, whereas chronic alcohol exposure leads to reduced expression of IRAK-M and hypersensitivity to LPS, which is associated with hyperactivation of NF-κB and increased TNF-α secretion (58). Reduced IRAK-M concentrations in monocytes and adipose tissue of obese subjects constitute causative factors of systemic inflammation and mitochondrial stress (31). We demonstrate that aging and oxidative stress cause downregulation of IRAK-M in RPE and subsequent degenerative cytokine responses (Figures 1E, 1F, 2B, 2D, 2F, 3A, 3D). In support, transcriptome data mining suggests that expression levels of IRAK-M are further silenced during AMD compared to age-matched controls, where other IRAK family members show no significant changes in AMD (Figure 1G, Figure S1), suggesting that IRAK-M may act as a precursor molecule for AMD degeneration.

[0375] We show that IRAK-M in RPE is regulated by AP-1 (Figures 4A, 4D, 4E) and acts as a negative feedback control of inflammation. AP-1 is a dimeric transcription factor assembled from Jun and Fos family proteins, of which c-Jun and c-Fos are among the most important regulators of genes involved in cell function, proliferation, differentiation, apoptosis, and immunity (59). The decrease in AP-1 transcriptional activity has been linked to tissue and cellular senescence (60, 61), in contrast to NF-κB, whose activity is increased in aged tissues and in age-related conditions such as Alzheimer's disease, diabetes, and osteoporosis (61, 62). In mice, depletion of c-Jun, but not c-Fos, causes embryonic death (63). We found that c-Jun expression or activity declined in parallel with IRAK-M reduction during aging or under oxidative stress (Figures 4B, 4C). This is consistent with earlier studies that showed reduced AP-1 activity or reduced AP-1 subunit expression in aged rodent tissues (64). Interestingly, the decrease in c-Jun phosphorylation did not occur until 72 h later (Figure 16A). This may be consistent with previous reports that AP-1 transcription upon oxidative stress increased within 24 h in RPE cells (65, 66), implying that time is required for oxidative stress to accumulate during aging (60, 64). Notably, inhibition of AP-1 activity sensitized RPE cells to oxidative damage (Figure 4F), whereas overexpression of c-Jun failed to protect (Figure 13A). This is likely due to the diverse functions of c-Jun / AP-1 signaling in cellular responses and apoptosis (40, 41).

[0376] Dysregulation of TLR-mediated signaling components has emerged as a key mediator in the initiation and progression of inflammation-associated degenerative diseases (67). There is increasing evidence of aberrant IRAK-M expression or IRAK signaling in human diseases such as chronic alcoholic liver disease, inflammatory bowel disease, insulin resistance, and features of metabolic syndrome (28, 30, 31). Knocking out IRAK-M in mice disrupts systemic or local immune activity, resulting in susceptibility to endotoxic shock, autoimmune diabetes, osteoporosis, and neurovascular injury (29, 30, 68-70). The present inventors have demonstrated that Irak3 is a key regulator of Irak signaling in the pathogenesis of inflammatory diseases such as chronic alcoholic liver disease, inflammatory bowel disease, insulin resistance, and metabolic syndrome. - / - Mice spontaneously develop AMD-like characteristics of retinal pathology and show increased cell death as early as 5 months of age (Figures 5A-D, 6A-C). They are more susceptible to oxidative insults, as shown in two different retinal degeneration models (Figures 6F and 6G), suggesting a convergence of pathways of immune dysregulation and excessive oxidative stress. Moreover, the absence of IRAK-M in RPE cells of different origins (iPSC-derived, primary cells or cell lines) leads to disrupted cellular homeostasis evidenced by reduced mitochondrial energetics, enhanced cellular senescence and enhanced SASP cytokine secretion (Figures 7A, 7C-F).

[0377] A gene therapy approach to deliver and restore IRAK-M expression in the RPE may have clinical implications for preventing AMD progression at an early stage. Retinal degeneration in AMD is the collective result of abnormalities in inflammation, mitochondrial function, lipid metabolism, autophagy, and cellular senescence (8, 16, 71, 72), where immune modulators have emerged as interplaying scripts, promising to break the vicious cycle (16, 73). Numerous nonsteroidal anti-inflammatory drugs (NSAIDs), neutralizing antibodies against IL-1α or IL-1R, and anti-inflammatory IL-10 have been shown to regulate cell metabolism and autophagy (74-77). We have recently developed an immune-mediated insidious retinal degeneration model (Cfh + / -Using a high-fat diet (17, 23), we found a protective effect of IL-33 in the retina. In this study, increasing IRAK-M in RPE via boosting endogenous gene expression or exogenous gene delivery helped maintain cell function (mitochondrial activity and autophagy) and inhibit senescence / SASP, thereby promoting cell survival (Figures 8A, 8C, 8D, 8E and Figures 14B, 14D, 14E-G), implying that IRAK-M is a master regulator of the immune regulatory hierarchy in RPE cells and AMD pathogenesis. Notably, we found that increasing IRAK-M expression was more protective against oxidative and / or immune stress in human iPSC-RPE cells and mouse primary RPE cells compared to ARPE-19 cells (Figures 8A and E and Figure 14B). Furthermore, the glycolytic response in these RPE cell cultures was distinct, with increased IRAK-M expression not altering glycolysis in human iPSC-RPE (Figure 8B) and mouse primary RPE (Figure 8F) but inducing glycolysis in ARPE-19 (Figure 14C). Despite variability between cell models (78, 79), the results demonstrate a protective role of IRAK-M expression in RPE mitochondrial health, which is essential for RPE with its high metabolic demands (42).

[0378] Experimental approaches have been used to introduce human genes, such as RPE65 and NADH dehydrogenase subunit 4 (ND4), into mouse models for preclinical evaluation (43–45). Human and mouse IRAK-M have been shown to have comparable cellular expression and functional similarity in terms of signaling activity (29, 80). The IRAK-M gene is located on chromosome 12 in humans (Uniprot ID Q9Y616, 596 amino acids (aa) in length) and on chromosome 10 in mice (Q8K4B2, 609 aa), respectively. Regardless of species, the full length of IRAK-M contains a death domain (DD, 41–106 aa for both human and mouse IRAK-M), involved in binding to other IRAK family members, a pseudokinase domain (165–452 aa for human and 178–463 aa for mouse), and an unstructured C-terminal domain with a TRAF6-binding motif (81). We performed a BLAST search and revealed that human IRAK-M shares 74.55% aa sequence identity with the mouse homologue, where there is 91.67% identity in the DD sequence and 83.22% identity in the pseudokinase domain sequence. A dot plot of the BLAST sequence alignment showed close similarity in domain sequences between human and mouse IRAK-M (Figure 16B). Furthermore, in vitro functional studies using newly created stable transfectant cell lines and primary cells confirmed the functionality of human IRAK-M gene delivery in mouse RPE cells (Figure 8C-F).

[0379] Gene therapy approaches that induce sustained therapeutic transgene expression have been utilized to treat chronic diseases (82, 83) where long-lasting pathological clues cannot be eliminated by other solutions (84). The eye is an ideal organ for gene therapy due to its ease of access and compartmentalization, relative immune privilege, and small size to reduce the required viral load. Ocular gene therapy has been successfully applied to a variety of diseases (84, 85). There are two ongoing phase I gene therapy trials for dry AMD, including GT005, which induces complement factor I (CFI) expression (86) and HMR59 (AAVCAGsCD59), which expresses C59 to block the formation of the membrane attack complex (MAC) (87). Because our data clearly demonstrated that an age-related oxidative stress-induced decline in IRAK-M in immune activity is the root cause of AMD, our ongoing research is to explore novel approaches of targeted immunotherapy to restore RPE health and function by increasing IRAK-M expression using viral gene therapy. We utilize subretinal administration of AAV2 vectors, the best-characterized AAV serotype in clinical trials, to treat RPE-associated eye diseases with sustained gene expression and no adverse side effects (43, 88, 89). Proof of concept is tested through the increase of IRAK-M in RPE in a light-induced retinal degeneration model (LIRD) using Irak3- / - mice. Future studies are also required to evaluate tissue and humoral responses in mice following delivery of the human IRAK-M transgene. With continuously advancing safer viral vector technology, our results will enable further developments toward effective gene therapy to treat dry AMD. The findings may pave the way for the development of gene therapies not only for AMD but also for other age-related diseases in which chronic inflammation plays an important role.

[0380] Example 2 Materials and Methods Immunohistochemistry of human ocular sections Paraffin-embedded human ocular sections from AMD and non-AMD subjects were obtained from Lions Gift of Sight (Minnesota, USA) after research ethics board approval (20 / LO / 336) and experiments were performed in accordance with the UK law, in accordance with the Declaration of Helsinki. Slides were deparaffinized and rehydrated, followed by antigen retrieval with citrate buffer (pH 6.0) at 90°C for 20 min. After three washes with PBS and blocking and permeabilization with 5% normal goat serum (NGS), 5% BSA and 0.1% Triton X-100, specimens were incubated overnight at 4°C with rabbit anti-IRAK-M antibody (1:250, catalogue ab8116, Abcam, Cambridge, UK). The secondary antibody, biotinylated goat anti-rabbit IgG (1:1000, Thermo Fisher Scientific, Paisley, UK), was visualized by the avidin-biotin-alkaline phosphatase complex (ABC-AP) method (Vectastain ABC-AP kit, 2Bscientific, Upper Heyford, UK) using Vector Red substrate (2Bscientific). Slides were then counterstained with hematoxylin, dehydrated and mounted with Histomount medium. Lack of staining when the primary antibody was omitted was used as a negative control. IHC images of the macular and peripheral retina were taken using an Evos XL Core microscope (Thermo Fisher Scientific). Images were processed using the Colour Deconvolution plugin in Fiji to separate hematoxylin (blue), IRAK-M (AP-red) and dye (brown). ROIs for pigmented RPE were carefully identified in the pigmented / brown image that was copied and pasted into the same area of ​​the AP-red image. The mean staining intensity of IRAK-M for RPE was measured using Fiji. ROIs for retina or choroid were selected based on nuclear staining (blue).

[0381] subretinal injection Eight-week-old male C57BL / 6J mice (Charles River Laboratories, Portishead, UK) or 2- to 4-month-old Irak3 - / -Mice were anesthetized using an intraperitoneal injection of 200 μl of Vetelar (ketamine hydrochloride 100 mg / ml, Pfizer, Sandwich, UK) and Rompun (xylazine hydrochloride 20 mg / ml, Bayer, Newbury, UK) mixed with sterile water in the ratio 0.6:1:84. Pupils were dilated using 1% tropicamide and 2.5% phenylephrine (both Chauvin, Essex, UK). One drop of Viscotears (Novartis, London, UK) was then applied to cover the ocular surface before the following procedure. All transscleral subretinal injections used were in a volume of 2 μL at the indicated titers and were delivered using a surgical microscope and a 33G needle on a microsyringe under direct visualization (Hamilton Company, Reno, NV, USA). 1% chloramphenicol ointment (Martindale Pharma, Wooburn Green, UK) was applied topically immediately after injection.

[0382] Light-induced retinal degeneration (LIRD) Two weeks after subretinal administration of the AAV vector, the mice were subjected to LIRD as described in Example 1.

[0383] Optical Coherence Tomography (OCT) As described in Example 1. Quantitative RT-PCR (QRT-PCR) Gene expression of exogenous human IRAK3 and endogenous mouse IRAK3 was analyzed using QRT-PCR as described in our application. The primer sequences were: mouse IRAK3, forward 5'-GACCAGCTCCAACCCAAACT (SEQ ID NO: 36), reverse 5'-GCCACCGCCGGTCATATTTA (SEQ ID NO: 37); human IRAK3, forward 5'-CCCACTCCCTTGGCACATTC (SEQ ID NO: 38), reverse 5'-AGCATGGTTGAACGTTGTGC (SEQ ID NO: 39); mouse RPS29, forward 5'-ACGGTCTGATCCGCAAATAC (SEQ ID NO: 44), reverse 5'-ATCCATTCAAGGTCGCTTAGTC (SEQ ID NO: 45).

[0384] Mouse retina sections and fluorescent staining for IRAK-M, mitochondria and apoptosis To assess whether subretinal administration of AAV2 vectors increases expression of human IRAK-M in the mouse retina, eyes were enucleated 2 weeks after injection and fixed in 2% paraformaldehyde (PFA) before preparing frozen sections. Sections were permeabilized with 0.1% Triton X-100 and blocked with 10% normal donkey serum, 5% BSA + 0.3M glycine, followed by overnight incubation at 4°C with either rabbit anti-human IRAK-M (1:500, catalogue HPA043097, Merck, Gillingham, UK) or rabbit anti-IRAK-M (1:500, catalogue ab8116, Abcam). After washing, sections were incubated with donkey anti-rabbit IgG conjugated with Alexa Fluor 488 (1:1000, Thermo Fisher Scientific). DAPI counterstaining was used to show nuclei in the sections. Tissues were washed, mounted in Vectashield antifade medium, and examined by confocal microscopy.

[0385] MitoView Green is a mitochondrial membrane potential-insensitive dye that can be used for mitochondrial staining in live and formaldehyde-fixed cells. The fluorescence of cells stained with this dye is directly proportional to the mitochondrial content. Fixed mouse retinal sections were washed in PBS and incubated with 100 nM MitoView Green for 30 min at RT. After washing and counterstaining with DAPI, the samples were mounted for observation under a confocal microscope.

[0386] Retinal cell apoptosis was determined by TUNEL staining using an in situ cell death detection kit (Roche Diagnostics, Burgess Hill, UK) according to the manufacturer's instructions.

[0387] LDH cytotoxicity assay As described in Example 1. statistics Results are presented as mean ± SD. Two-way ANOVA was used to assess the correlation of two independent variables on the dependent variable, followed by Kruskal-Wallis test with Bonferroni correction for post-hoc comparisons. Unpaired two-tailed Student's t-test was performed between the two groups. Differences between groups were considered significant at P < 0.05. Statistical analysis was performed using GraphPad Prism 7.0.

[0388] result Histological analysis demonstrates decreased IRAK-M expression in the RPE in aging and AMD To determine the tissue spatial expression of IRAK-M protein in relation to aging and AMD, we performed IHC analysis on paraffin-embedded retinal sections retrieved from two "young" (30 and 59 years old) and five "old" (76-97 years old) individuals with no history of AMD and from eleven AMD patients (76-95 years old). In the young samples, IRAK-M was present across distinct layers of the inner and outer retina, RPE and choroid (data not shown, subject (Ctr) 59 years old). In aged control or AMD samples, the pattern and intensity of signal immunopositivity varied, with increased signal in, for example, the outer plexiform layer (OPL) / outer nuclear layer (ONL) (data not shown, Ctr 97 y), nerve fiber layer (NFL) (data not shown, early AMD 95y) or inner nuclear layer (INL) / ONL / inner segment (IS) (data not shown, mild AMD 76 y). Color deconvolution allowed us to separate IRAK-M immunopositivity from RPE pigment and to quantify IRAK-M expression in the RPE, as well as to analyze the choroid and retina. We confirmed that macular IRAK-M expression was significantly reduced in both the aged RPE and choroid (Figure 17). Furthermore, in AMD patients, IRAK-M expression was lower in the macular RPE, but not in the macular choroid, compared to age-matched non-AMD subjects (Figure 17). Decreased IRAK-M expression in the peripheral RPE, choroid, and retina was only evident in the choroid of elderly subjects compared to young subjects (Figure 17). Furthermore, nonspecific staining of Bruch's membrane (BM) for hematoxylin and IRAK-M was noted in AMD samples (data not shown), which was also observed in negative control staining. Enhanced BM was not evident in control eyes, which is consistent with the finding that BM is significantly thickened in AMD (101).

[0389] Subretinal administration induces AAV2-mediated transgene expression of human IRAK-M in mouse RPE To date, AAV2 is the best-characterized AAV serotype in clinical trials for treating RPE-associated eye diseases (88). To identify dose-dependent transduction effects, 1 × 10 12Or 2 x 10 11 Two microliters of AAV2 encoding EGFP under the control of the constitutive cytomegalovirus (CMV) promoter (AAV2.CMV.EGFP) was delivered to mouse eyes via the subretinal route at 1000 ng / ml. High doses (1 × 10 in 2 μl) were 12 gc / ml, or 2 x 10 9 gc / eye) at a low dose (2 × 10 11 gc / ml or 4 x 10 8 gc / eye) more significantly induced EGFP expression 2-11 weeks after injection (Figure 18). High-dose administration of AAV2.CMV.hIRAK3 resulted in higher hIRAK3 mRNA expression in the RPE / choroid 2 weeks after injection compared with null AAV2.CMV (Figure 19A). AAV-induced hIRAK3 mRNA expression in the retina was lower compared with that in the RPE / choroid. Notably, endogenous mouse IRAK3 mRNA expression in both the RPE / choroid and retinal tissues was unchanged after introduction of exogenous hIRAK3 (Figure 19A). Induced hIRAK-M protein expression was detected in the RPE and confirmed by immunohistochemistry using two independent IRAK-M antibodies (Figure 19B).

[0390] IRAK-M gene therapy suppresses light-induced retinal degeneration To assess the protective effect of IRAK-M transgene expression in vivo, 2 × 10 9Light-induced retinal degeneration (LIRD) was applied 2 weeks after subretinal injection of AAV2.CMV.hIRAK3 or null AAV2.CMV in gc / eyes. Optical coherence tomography (OCT) was used to detect retinal pathology in response to treatment. In control eyes that were not light-challenged, transduction of hIRAK3 did not alter the gross morphology in OCT sections 4 weeks after injection (Figure 20A). In the LIRD model, only the outer retinal thickness was reduced, whereas the inner retinal thickness was not changed as expected in this model. Light exposure of null AAV2-injected eyes resulted in a decrease in the outer retinal thickness indicative of PR loss. The protective effect of AAV2.CMV.hIRAK3 treatment from PR damage was striking, as demonstrated by the suppression of light-induced outer retinal thinning (Figure 20B). Concurrent with the preservation of retinal thickness by IRAK-M gene therapy, a reduction in light-induced TUNEL-positive apoptosis in the retina was observed (Figure 21).

[0391] The retina is one of the most energy-demanding organs in the body. Along with aerobic glycolysis, mitochondrial activity in retinal cells, such as the PR, is essential for tissue function and normal vision (102). Within the retina, mitochondria are abundantly distributed in ganglion cells (GCs), the inner plexiform layer (IPL), the outer plexiform layer (OPL), and the inner segment (IS) of the PR (103). In the LIRD model, mitochondrial staining with MitoView green dye on retinal sections demonstrated mitochondrial damage in PR cells due to light damage (Figure 22). This damage was significantly reversed by AAV2-mediated IRAK-M gene delivery, as mitochondria in GL, IPL, and OPL were less affected in the light damage model (Figure 22).

[0392] IRAK-M gene therapy is Irak3 - / - Preventing age-related retinal degeneration in mice Irak3 - / - Based on the finding that young Irak3 mice develop signs of retinal degeneration earlier than WT controls, we investigated whether AAV-mediated IRAK-M expansion could prevent retinal pathology caused by IRAK-M deficiency.- / - (2–4 months of age) were transfected with AAV2.CMV.hIRAK3 or null AAV2.CMV (2 × 10 9 gc / eye) was administered subretinal. After subretinal delivery of the IRAK3 transgene, not only was the age-dependent appearance of retinal spots significantly suppressed during aging (Figure 23A and B), but the number of retinal spots in aged KO mice (8-10 months old) was also significantly reduced in the retina (Figure 23C), more prominently in the virus-injected side (Figure 23D). The therapeutic effect of IRAK3 gene therapy was further demonstrated by suppressing the thinning of the outer retina in KO mice (8-10 months old) (Figure 24).

[0393] Comparison of different promoters for efficient IRAK3 gene transfer The promoter of a viral vector is a major regulatory element that determines the efficiency and specificity of transgene expression. We compared the transduction efficiency of human IRAK-M gene transfer in mouse B6-RPE07 cells using AAV2 under the control of five different promoters, including the universal CMV promoter, the RPE-specific Bestrophin 1 (Best1) promoter, and three putative endogenous (Endo) promoters of the human IRAK3 gene. As shown in Figure 25, three fragments (Ensembl ID: ENSG00000090376) in front of the first exon of the human IRAK3 gene were selected as putative Endo promoters. The 0.88 kb fragment (Endo1) is our predicted "core promoter" that contains a CpG island and H3K methylation marks. The 1.36 kb fragment (Endo2) is predicted to be the maximum promoter size that the AAV backbone CMV.GFP.WPRE.io2 can accommodate for a given IRAK3 gene size plus the WPRE and io2 elements. The 1.6 kb fragment (Endo3) is approximately the maximum promoter size of an AAV vector that can generally accommodate a given IRAK3 gene size. Figure 26A shows comparable transgene expression induced by different AAV2 vectors with CMV, Best1 or Endo3 (1.6 kb) promoters, while the other two endogenous promoters with shorter sequences were less efficient but were nevertheless able to drive robust expression. Notably, human IRAK3 gene transfer did not affect the expression of the endogenous mouse IRAK3 gene (Figure 26B) or induce cell death (Figure 26C). After determining the transduction efficiency of different promoters, we next evaluated the protective effect of promoter-driven AAV2.hIRAK3 in human RPE cells (ARPE-19) in response to oxidative stress. The data demonstrate that AAV2.CMV.hIRAK3, AAV2.Best1.hIRAK3, and AAV2.Endo3.hIRAK3 can all significantly prevent paraquat-induced cell death (Figure 26D).In the LIRD model, AAVs with ubiquitous, RPE-specific, or native promoters also demonstrated the ability to protect retinal degeneration against light damage (Figure 26E).

[0394] AAV5 transduces IRAK-M gene expression Except for AAV2, which is the main serotype used in ongoing ocular gene therapy trials, other AAV capsid types, such as AAV5, are also being intensively studied. Here, we show that AAV5.CMV.hIRAK3 transduction in mouse RPE cells enhances human IRAK3 gene expression in a dose-dependent manner (Figure 27A) without affecting the expression of endogenous mouse IRAK3 (Figure 27B). Therefore, demonstration of other AAV capsid serotypes can be used in the context of the present invention.

[0395] overview As an in vivo proof of concept (POC), the proposed gene therapy to increase IRAK-M expression is effective and we demonstrate prevention of AMD-like phenotypes in two animal models, light-induced retinal damage (LIRD) in wild-type mice and age-related retinal damage in IRAK-M KO mice.

[0396] Apart from mechanism-based functional studies, IHC staining of human donor eyes revealed a significant downregulation of IRAK-M in macular RPE with aging, which is even lower in older adults with AMD. Whether a more rapid decline in IRAK-M triggers AMD initiation or whether AMD accelerates the decline in IRAK-M expression remains to be identified. Nevertheless, our data point to an age-related oxidative stress-induced decline in IRAK-M expression as the root of AMD, so increasing IRAK-M expression by gene therapy holds the potential to prevent or delay the progression from early to late dry AMD, which is currently unattainable.

[0397] The AAV2 vector we utilize for subretinal administration is the best-characterized AAV serotype to date in clinical trials for treating RPE-associated eye diseases with sustained gene expression and no observed adverse side effects (88, 89, 43). Among other AAV capsid variants, AAV5 has the known advantages of low seroprevalence and high transduction efficiency. A gene therapy product using AAV5 in X-linked retinitis pigmentosa (XLRP) treatment has recently entered phase 3 clinical trials (Clinical Trials.gov Identifier: NCT04671433). In our studies, AAV5 demonstrated the ability to deliver targeted human IRAK3 expression in mouse RPE, expanding our vehicle options for more comprehensive studies.

[0398] Another determinant of transduction specificity and efficiency is the promoter used to drive expression of the target gene. In addition to CMV, all three promoters, the RPE-specific Best1 promoter and the endogenous human IRAK3 gene (Endo1, 2, and 3), achieved robust transgene expression.

[0399] The two main strategies currently being investigated for the treatment of dry AMD are stem cell replacement by RPE transplantation (105) and immunomodulation (106). Clinical trials along these two groups of strategies are ongoing at different stages. Other therapeutic innovations focused on immunomodulation include gene therapy targeting the complement signaling cascade, such as the gene therapy trial GT005, which induces complement factor I (CFI) expression (107), and HMR59 (AAVCAGsCD59) (108), which expresses C59 to prevent the formation of the membrane attack complex (MAC). The present invention simultaneously targets both cellular bioenergetic health, inflammation, and oxidative stress by restoring IRAK-M expression, restoring homeostatic control and treating AMD.

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Claims

1. 1. A pharmaceutical composition comprising a nucleic acid for use in a method for treating or preventing macular degeneration in a subject, comprising: The nucleic acid comprises a nucleic acid sequence encoding IRAK-M and is capable of driving expression of IRAK-M in a target cell. The pharmaceutical composition.

2. The pharmaceutical composition of claim 1 , wherein a promoter is operably linked to the nucleic acid sequence.

3. The promoter is selected from the group consisting of a CMV promoter, a Best1 promoter, and a native promoter of IRAK3, or a functional fragment thereof; or, the promoter is selected from the group consisting of CAG promoter, mini-CAG (SV40 intron) promoter, SV40 promoter, CB7 promoter, smCBA promoter, CBh promoter, MeCP2 promoter, shCMV promoter, CMVd2 promoter, SV40 mini-promoter, SCP3 promoter, EF1-α promoter, PGK promoter, GAPDH promoter, and UbC promoter; The pharmaceutical composition of claim 2.

4. (i) autophagic flux is maintained or increased in target cells containing nucleic acid compared to comparable cells not containing nucleic acid; (ii) mitochondrial activity is maintained or increased in target cells containing nucleic acid compared to comparable cells not containing nucleic acid; and / or (iii) the production of proinflammatory cytokines is reduced in target cells containing nucleic acid compared to comparable target cells not containing nucleic acid; The pharmaceutical composition of claim 1.

5. (a) the nucleic acid is delivered to the target cell via a viral vector; and / or (b) the nucleic acid is a viral vector genome; The pharmaceutical composition of claim 1.

6. (a) The nucleic acid is delivered to the target cell via a viral vector, 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 poxvirus vector, an anellovirus vector, and an alphavirus vector; and / or (b) the nucleic acid is a viral vector genome, 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 poxvirus vector genome, anellovirus vector genome, and an alphavirus vector genome; The pharmaceutical composition according to claim 5.

7. (a) The macular degeneration is age-related macular degeneration (AMD), and / or (b) the target cell is a cell of the retina or choroid; The pharmaceutical composition of claim 1.

8. (a) The age-related macular degeneration is dry AMD. and / or (b) the target cells are cells of the RPE; The pharmaceutical composition of claim 7.

9. 10. The pharmaceutical composition of claim 1, wherein the nucleic acid is administered to a subject intraocularly, intravitreally, subretinally, suprachoroidally, or periocularly.

10. The pharmaceutical composition of claim 9 , wherein the nucleic acid is administered by subretinal injection.

11. the nucleic acid sequence encodes a polypeptide comprising an amino acid sequence having at least 60% sequence identity with the amino acid sequence of SEQ ID NO:1; and / or the nucleic acid sequence encodes a polypeptide capable of preventing dissociation of IRAK-1 and / or IRAK-4 from MyD88 in a target cell; The pharmaceutical composition according to any one of claims 1 to 10.

12. 1. A pharmaceutical composition comprising a vector virion for use in a method for treating or preventing macular degeneration in a subject, comprising: the vector virion comprises a nucleic acid comprising a nucleic acid sequence encoding IRAK-M, the nucleic acid being capable of driving expression of IRAK-M in a target cell; The pharmaceutical composition.

13. 13. The pharmaceutical composition of claim 12, wherein a promoter is operably linked to the nucleic acid sequence.

14. The promoter is selected from the group consisting of a CMV promoter, a Best1 promoter, and a native promoter of IRAK3, or a functional fragment thereof; or, the promoter is selected from the group consisting of CAG promoter, mini-CAG (SV40 intron) promoter, SV40 promoter, CB7 promoter, smCBA promoter, CBh promoter, MeCP2 promoter, shCMV promoter, CMVd2 promoter, SV40 mini-promoter, SCP3 promoter, EF1-α promoter, PGK promoter, GAPDH promoter, and UbC promoter; The pharmaceutical composition of claim 13.

15. (i) autophagic flux is maintained or increased in target cells containing vector virions compared to comparable cells that do not contain vector virions; (ii) mitochondrial activity is maintained or increased in target cells containing the vector virions compared to comparable cells not containing the vector virions; and / or (iii) the production of proinflammatory cytokines is reduced in target cells containing vector virions compared to equivalent target cells that do not contain vector virions; The pharmaceutical composition of claim 12.

16. 13. The pharmaceutical composition of claim 12, wherein the nucleic acid is suitable for integration into the genome of a target cell by an RNA-guided endonuclease system.

17. 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, poxvirus, anellovirus, and alphavirus; The pharmaceutical composition of claim 12.

18. 18. The pharmaceutical composition of claim 17, wherein the vector virion is an adeno-associated virus (AAV).

19. 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); 19. The pharmaceutical composition of claim 18.

20. (a) The macular degeneration is age-related macular degeneration (AMD), and / or (b) the target cell is a cell of the retina or choroid; The pharmaceutical composition of claim 12.

21. (a) The age-related macular degeneration is dry AMD. and / or (b) the target cells are cells of the RPE; 21. The pharmaceutical composition of claim 20.

22. 13. The pharmaceutical composition of claim 12, wherein the vector virion is administered to a subject intraocularly, intravitreally, subretinally, suprachoroidally, or periocularly.

23. 23. The pharmaceutical composition of claim 22, wherein the vector virion is administered by subretinal injection.

24. the nucleic acid sequence encodes a polypeptide comprising an amino acid sequence having at least 60% sequence identity with the amino acid sequence of SEQ ID NO:1; and / or the nucleic acid sequence encodes a polypeptide capable of preventing dissociation of IRAK-1 and / or IRAK-4 from MyD88 in a target cell; The pharmaceutical composition according to any one of claims 12 to 23.

25. A pharmaceutical composition comprising an IRAK-M polypeptide for use in a method for treating or preventing macular degeneration in a subject.

26. (a) the IRAK-M polypeptide comprises an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO:1; (b) the IRAK-M polypeptide is capable of preventing the dissociation of IRAK-1 and / or IRAK-4 from MyD88 in the target cell; and / or (c) the IRAK-M polypeptide further comprises a cell-penetrating peptide (CPP); 26. The pharmaceutical composition of claim 25.

27. (i) autophagic flux is maintained or increased in target cells containing an IRAK-M polypeptide compared to comparable cells that do not contain an IRAK-M polypeptide; (ii) mitochondrial activity is maintained or increased in target cells containing an IRAK-M polypeptide compared to comparable cells that do not contain an IRAK-M polypeptide; and / or (iii) the production of proinflammatory cytokines is reduced in target cells containing an IRAK-M polypeptide compared to comparable target cells that do not contain an IRAK-M polypeptide; 27. The pharmaceutical composition of claim 25 or claim 26.

28. For use in a method for treating or preventing macular degeneration in a subject, 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 IRAK-M; 1. A pharmaceutical composition comprising a nucleic acid system comprising one or more nucleic acids, comprising: the nucleic acid sequence encoding IRAK-M is capable of driving expression of IRAK-M in a target cell of interest, and the nucleic acid system is suitable for directed insertion of the nucleic acid sequence encoding IRAK-M at an insertion site in the genome of the target cell; The pharmaceutical composition.

29. For use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease fused to one or more transcriptional activators; and, b) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the promoter or regulatory sequence of the IRAK3 gene and is capable of directing the RNA-guided endonuclease to the target sequence; 1. A pharmaceutical composition comprising a nucleic acid system comprising one or more nucleic acids, comprising: the nucleic acid system increases IRAK-M expression in target cells of a subject; The pharmaceutical composition.

30. For use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease; and, b) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in a promoter or regulatory sequence of the IRAK3 gene and that is capable of directing the RNA-guided endonuclease to the target sequence, wherein the guide RNA further comprises an aptamer that is capable of specifically binding to a transcriptional activator; 1. A pharmaceutical composition comprising a nucleic acid system comprising one or more nucleic acids, comprising: the nucleic acid system increases IRAK-M expression in target cells of a subject; The pharmaceutical composition.

31. For use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated 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 that is complementary to a target sequence in a promoter or regulatory sequence of the IRAK3 gene and that is capable of directing the RNA-guided endonuclease to the target sequence, wherein the guide RNA further comprises an RNA aptamer that is capable of specifically binding to an RNA-binding protein; 1. A pharmaceutical composition comprising a nucleic acid system comprising one or more nucleic acids, comprising: the nucleic acid system increases IRAK-M expression in target cells of a subject; The pharmaceutical composition.

32. (i) The RNA aptamer is capable of binding to an RNA-binding protein dimer; (ii) the RNA-binding protein is MS2; and / or (iii) the inactivated RNA-guided endonuclease is fused to an additional transcriptional activator; 32. The pharmaceutical composition of claim 31.

33. For use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease fused to an epitope repeat array comprising one or more epitopes; b) one or more nucleic acid sequences encoding epitope-binding molecules fused to one or more transcriptional activators, said epitope-binding molecules being capable of specifically binding to an epitope of the epitope repeat array; and, c) a nucleic acid sequence encoding a guide RNA that is complementary to a target sequence in the promoter or regulatory sequence of the IRAK3 gene and that is capable of directing the RNA-guided endonuclease to the target sequence; 1. A pharmaceutical composition comprising a nucleic acid system comprising one or more nucleic acids comprising the nucleic acid system increases IRAK-M expression in target cells of a subject; The pharmaceutical composition.

34. the epitope-binding molecule comprises a nuclear localization sequence (NLS); and / or the epitope-binding molecule is an antibody or antibody-like molecule; 34. The pharmaceutical composition of claim 33.

35. For use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease fused to one or more DNA demethylating agents; and, b) (i) a target sequence in the promoter sequence of the IRAK3 gene, (ii) a target sequence in the regulatory sequence of the IRAK3 gene, or (iii) a nucleic acid sequence that is complementary to a target sequence in the IRAK3 gene and encodes a guide RNA capable of directing the RNA-guided endonuclease to the target sequence; 1. A pharmaceutical composition comprising a nucleic acid system comprising one or more nucleic acids, comprising: the nucleic acid system increases IRAK-M expression in target cells of a subject; The pharmaceutical composition.

36. The pharmaceutical composition described in claim 35, wherein the DNA demethylating agent is TET1 or lysine-specific demethylase 1.

37. For use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease; and, b) a nucleic acid sequence that is complementary to (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a target sequence in the IRAK3 gene and encodes a guide RNA capable of directing the RNA-guided endonuclease to the target sequence, wherein the guide RNA further comprises an aptamer capable of specifically binding to a DNA demethylating agent; 1. A pharmaceutical composition comprising a nucleic acid system comprising one or more nucleic acids, comprising: the nucleic acid system increases IRAK-M expression in target cells of a subject; The pharmaceutical composition.

38. The pharmaceutical composition described in claim 37, wherein the DNA demethylating agent is TET1 or lysine-specific demethylase 1.

39. For use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided 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 that is complementary to (i) a target sequence in a promoter sequence of the IRAK3 gene, (ii) a target sequence in a regulatory sequence of the IRAK3 gene, or (iii) a target sequence in the IRAK3 gene and encodes a guide RNA capable of directing the RNA-guided endonuclease to the target sequence, wherein the guide RNA further comprises an RNA aptamer capable of specifically binding to an RNA-binding protein; 1. A pharmaceutical composition comprising a nucleic acid system comprising one or more nucleic acids, comprising: the nucleic acid system increases IRAK-M expression in target cells of a subject; The pharmaceutical composition.

40. The pharmaceutical composition described in claim 39, wherein the DNA demethylating agent is TET1 or lysine-specific demethylase 1.

41. (i) The RNA aptamer is capable of binding to an RNA-binding protein dimer; (ii) the RNA-binding protein is MS2; and / or (iii) the inactivated RNA-guided endonuclease is fused to an additional DNA demethylating agent; 40. The pharmaceutical composition of claim 39.

42. For use in a method for treating or preventing macular degeneration in a subject, a) a nucleic acid sequence encoding an inactivated RNA-guided endonuclease fused to an epitope repeat array comprising one or more epitopes; b) one or more nucleic acid sequences encoding epitope-binding molecules fused to one or more DNA demethylating agents, wherein said epitope-binding molecules are capable of specifically binding to an epitope of the epitope repeat array; and, c) (i) a target sequence in the promoter sequence of the IRAK3 gene, (ii) a target sequence in the regulatory sequence of the IRAK3 gene, or (iii) a nucleic acid sequence that is complementary to a target sequence in the IRAK3 gene and encodes a guide RNA capable of directing the RNA-guided endonuclease to the target sequence; 1. A pharmaceutical composition comprising a nucleic acid system comprising one or more nucleic acids, comprising: the nucleic acid system increases IRAK-M expression in target cells of a subject; The pharmaceutical composition.

43. the epitope-binding molecule comprises a nuclear localization sequence (NLS); and / or the epitope-binding molecule is an antibody or antibody-like molecule; 43. The pharmaceutical composition of claim 42.

44. 43. The pharmaceutical composition of claim 42, wherein the DNA demethylating agent is TET1 or lysine-specific demethylase 1.

45. The pharmaceutical composition according to any one of claims 28 to 40, wherein the one or more nucleic acids are one or more viral vector genomes.

46. 46. ​​The pharmaceutical composition of claim 45, wherein the one or more viral vector genomes are one or more adeno-associated viral vector genomes.

47. 1. A pharmaceutical composition comprising a nucleic acid comprising a nucleic acid sequence encoding a fusion protein for use in a method for treating or preventing macular degeneration in a subject, comprising: the fusion protein (a) a nucleic acid binding molecule capable of binding to a target sequence in the promoter or regulatory sequence of the IRAK3 gene; and, (b) one or more transcriptional activators; Including, The fusion protein is capable of increasing IRAK-M expression in target cells of a subject. The pharmaceutical composition.

48. 48. The pharmaceutical composition of claim 47, wherein the transcriptional activator is the transactivation domain VP64.

49. 1. A pharmaceutical composition comprising a nucleic acid comprising a nucleic acid sequence encoding a fusion protein for use in a method for treating or preventing macular degeneration in a subject, comprising: the fusion protein a) a nucleic acid binding molecule capable of binding to (i) a target sequence in the promoter sequence of the IRAK3 gene, (ii) a target sequence in the regulatory sequence of the IRAK3 gene, or (iii) a target sequence in the IRAK3 gene; and, b) one or more DNA demethylating agents; Including, The fusion protein is capable of increasing IRAK-M expression in target cells of a subject. The pharmaceutical composition.

50. 50. The pharmaceutical composition of claim 49, wherein the DNA demethylating agent is TET1 or lysine-specific demethylase 1.

51. (a) the nucleic acid binding molecule is a TAL effector repeat array or a zinc finger array; (b) the nucleic acid is delivered to the target cell via a viral vector; and / or (c) the nucleic acid is a viral vector genome; The pharmaceutical composition according to any one of claims 47 to 50.

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