Treatment methods for eye disorders

Abstract

To provide a pharmaceutical composition for restoring lysosomal function of retinal pigment epithelial cells.SOLUTION: Provided is a pharmaceutical composition for restoring lysosomal function of retinal pigment epithelial (RPE) cells in a subject, comprising a polypeptide comprising βA1-crystallin or a nucleic acid encoding the polypeptide.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 898,405, filed September 10, 2019, and U.S. Provisional Patent Application No. 62 / 961,548, filed January 15, 2020, which are incorporated by reference.

[0002] Sequence Listing The nucleotide / amino acid sequence listing submitted concurrently with this application is hereby incorporated by reference in its entirety. [Background technology]

[0003] Age-related macular degeneration (AMD) is the world's leading cause of blindness among older adults. The number of people with AMD worldwide is projected to be 196 million in 2020 and increase to 288 million in 2040. More than 15 million Americans have AMD, with treatment costs exceeding $350 billion. The latest data suggests that by 2020, more than 3 million additional people in the United States will be diagnosed with the disease.

[0004] The vast majority of patients suffer from atrophic (dry) AMD. However, due to the relentless progression of the disease, patients will develop advanced disease within 10 years. The burden of dry AMD is increasing with the aging of the "baby boomer" generation. Despite this growing population of affected patients, no definitive treatment or prevention (other than the AREDS II formulation for moderate AMD) is currently available for dry AMD. Approximately 80-90% of patients with the disease suffer from dry AMD. Its cause is unknown, and dry AMD usually progresses more slowly than the wet type.

[0005] There are currently anti-VEGF therapies available for patients with wet AMD, such as AVASTIN™ (bevacizumab), LUCNETIS™ (ranibizumab injections), and EYLEA™ (aflibercept). However, the treatment regimens (which require monthly injections) are extremely expensive, do not work long-term, and are not effective for all patients. Summary of the Invention [Problem to be solved by the invention]

[0006] Given the large number of patients with dry AMD, there is a strong need for novel treatments that target early disease before vision is lost. [Means for solving the problem]

[0007] The present invention provides a method of inhibiting neutrophil activation in retinal pigment epithelial (RPE) cells, comprising administering an inhibitor of interferon (IFN) lambda to a subject in need thereof. The inhibitor can be a neutralizing anti-IFN lambda antibody, an antisense oligonucleotide, a small interfering ribonucleic acid (siRNA), a small hairpin RNA (shRNA), a non-antibody binding polypeptide, or a small chemical compound. The antibody can be an immunoglobulin, an antibody fragment (e.g., Fab, Fab', F(ab')2, F(ab'), F(ab), Fv, and scFv), and an antibody analog (e.g., affibody, repebody, affilin, DARPin, tetranectin, microbody, peptide aptamer, and avimer). When the inhibitor is an shRNA, the shRNA can target one or more of the nucleotide sequences consisting of the group consisting of SEQ ID NOs: 11-17. The shRNA can be contained in a vector (e.g., an AAV vector).

[0008] The present invention also provides a method for restoring lysosomal function of RPE cells, comprising administering to a subject in need thereof a polypeptide comprising βA1-crystallin or a nucleic acid encoding the polypeptide.

[0009] The present invention further provides a method for the production of RPE cells using a method comprising the steps of: + The present invention provides a method for rejuvenating V-ATPase (V-ATPase) activity in a subject in need thereof by modulating the assembly and disassembly of V-ATPase.

[0010] A subject (e.g., a human) may have age-related macular degeneration (AMD) or may be at risk of developing AMD, where AMD may be wet AMD or atrophic (dry) AMD, where atrophic (dry) AMD may include geographic atrophy.A subject may have Stargardt's macular retinal degeneration or may be at risk of developing Stargardt's macular retinal degeneration.A subject may have neurodegenerative disease, such as Alzheimer's disease or Parkinson's disease, or may be at risk of developing neurodegenerative disease.Alternatively or additionally, a subject may have diabetic retinopathy (DR) or may be at risk of developing DR.

[0011] In addition, the present invention provides a method of treating diabetic retinopathy in a subject, comprising the step of administering to the subject a polypeptide comprising βA1-crystallin or a nucleic acid encoding the polypeptide. [Brief description of the drawings]

[0012] [Figure 1] FIG. 11 is a tabular view of RNAseq analysis from RPE cells of 5-month-old and 10-month-old Cryba1 floxed and Cryba1 cKO mice showing expression of genes related to cGAS / STING. [Figure 2A]Figure 2 demonstrates activation of IFNλ in RPE cells forming Cryba1 cKO mice with aging. Figure 2A is a table of RNAseq analysis from RPE cells of 5- and 10-month-old Cryba1 floxed and Cryba1 cKO mice. Significant increase in RNA levels of neutrophil regulatory molecules such as CXCL1, CXCL0 and IFN family members, e.g., IFN type I (IFNα, IFNβ), type II (IFNγ) and type III (IFNλ), in retinal extracts from 10-month-old Cryba1 cKO mice compared to age-matched Crybafl / fl (controls). No such changes were observed in 5-month-old mice, and there was no difference in the expression of the various IFN receptors. For each gene, fragments per kilobase of transcript per million mapped reads (FPKM) are represented and expressed as log10 (counts per million). Figure 2B is a graph showing elevated levels of IFNλ in RPE-choroid tissue homogenates of 15-month-old Cryba1 cKO mice compared to age-matched Cryba1fl / fl controls as measured by ELISA. No changes were observed in 3-month-old mice. n=6 *P<0.05 (One-way ANOVA with Tukey's post-hoc test). [Figure 2B] This is a continuation of Figure 2A. [Diagram 3] FIG. 10: IFNλ drives neutrophil homing to the retina of NOD-SCID mice. Counts drawn from all groups demonstrated increased neutrophil numbers (cell counts) in NOD-SCID mice injected (intravenously) with IFNλ-treated WT neutrophils compared to untreated controls. Loss of LCN-2 in neutrophils (LCN-2− / −) showed reduced infiltration even after IFNλ exposure. n=1. Scale bar, 500 μm. [Figure 4-1]Figure 1 demonstrates that IFNλ-activated neutrophils cause retinal degeneration in vivo. Representative spectral OCT images of retinas from NOD-SCID mice subretinally injected with (i) vehicle (HBSS) or (ii) WT neutrophils revealed normal retinal structure. In contrast, mice injected with (iii) recombinant IFNλ (200 U / mL), (iv) WT neutrophils pretreated with either conditioned medium (1:1 dilution) from IFNλ-overexpressing mouse RPE cells, or (v) recombinant LCN-2 (10 pg / mL) showed obvious alterations in the ONL and INL layers (asterisks), accompanied by severe loss of the RPE and IS + OS layers (yellow arrowheads). Such alterations were not observed in mice injected with (vi) untreated neutrophils from LCN-2− / − mice or (vii-viii) IFNλ-exposed LCN-2− / − neutrophils. FIG. 5(ix) is a representative spider plot showing the thickness of the IS / OS + RPE layer using OCT images between experimental groups. n=10. *P<0.05 (One-way ANOVA and Tukey's post-hoc test). [Figure 4-2] This is a continuation of Figure 4-1. [Diagram 5] Mouse Cryba1 (βA3 / A1-crystallin) N-terminal sequence in wild-type (A), βA1-knockdown (B), and βA3-knockout mice. The wild-type sequence of Cryba1 shows dual ATG start sites for βA3- and βA1-crystallin (A). The βA1-knockdown mutant (B) shows that the insertion of CCACC (underlined) immediately before the first start site reinforces the Kozak consensus sequence, thereby making the first start codon stronger. The βA3-knockout mutant (C) shows an A to G (underlined) change that inactivates the first start site. A silent mutation (ACC to ACG, change underlined) was also introduced to prevent binding and re-cutting of the sequence by gRNA after homology-directed repair. [Figure 6]Figure 1 demonstrates that knockdown of βA1-crystallin induces inflammation in RPE cells. ELISA showing protein levels of various cytokines from RPE culture supernatants in WT (A), Cryba1 cKO (B), βA3-knockout (C) and (D). *P<0.5. [Figure 7] Figure 7 demonstrates that knockdown of βA1-crystallin causes oxidative damage in RPE cells. Figure 7A shows superoxide release from WT, Cryba1 cKO, βA3-knockout (A3 KO) and βA1-knockdown (A1 KD) RPE cells in culture. Figure 7B shows SOD2 expression by qPCR, which shows a significant increase in Cryba1 cKO and A1 KD cells compared to WT cells. *P<0.05. [Figure 8] FIG. 1 is a diagram of the vector map of scCAG_humCryba1. [Figure 9-1] FIG. 1 is a diagram of the nucleotide sequence of scCAG_humCryba1 (SEQ ID NO: 18). [Figure 9-2] This is a continuation of Figure 9-1. [Figure 9-3] This is a continuation of Figure 9-2. [Figure 10]Schematic demonstrating that βA3 / A1-crystallin binds to the V-ATPase in the lysosomal lumen. On the left, a possible interaction between βA3 / A1-crystallin and the V-ATPase in the lysosomal lumen is shown. In the schematic representation of the V-ATPase, the rotor (V1) and stator (V0) show only the ATP6V0A1 subunit of V0. On the right, an enlarged schematic of the V0a1 subunit is shown. The ATP6V0A1 N-terminus, cytoplasmic transmembrane connecting loop (cTMC), and C-terminal tail are distributed on the cytoplasmic side. The transmembrane helices are within the membrane (solid rectangle). The vacuolar-type transmembrane connecting loop (vTMC) is on the lumen side. Rat ATP6V0A1 / V0-ATPase (Uniprot #P25286) residue boundaries: N-terminus (1-388), cTMC1 (427-441), cTMC2 (556-573), cTMC3 (660-725), C-tail (811-838), vTMC1 (408-409), vTMC2 (472-535), vTMC3 (595-639), vTMC4 (751-771). The human and rat sequences are highly conserved (human ATP6V0A1 / V0-ATPase is Uniprot #Q93050), with only minor changes in residue boundaries compared to rat: N-terminus (1-388), cTMC1 (427-441), cTMC2 (555-572), cTMC3 (659-724), C-tail (810-837), vTMC1 (408-409), vTMC2 (472-534), vTMC3 (594-638), vTMC4 (750-770). [Figure 11-1] Figure 1 demonstrates that the Nuc1 rat model is a diabetic retinopathy model. The arrow points to degenerated vessels without any nuclei, a key marker of retinal vascular changes in diabetic retinopathy. As demonstrated in the top left image, degenerated vessels without nuclei were observed in Nuc1 rats but not in Sprague-Dawley (SD) rats. Addition of the toxin streptozotocin (STZ) to SD and Nuc1 rats also resulted in degenerated vessels without nuclei. The graph on the right demonstrates the number of acellular capillaries observed in rats. [Figure 11-2]This is a continuation of Figure 11-1. [Figure 12] Figure 1 demonstrates the presence of microaneurysms in the retina of diabetic Nuc1 rats. The arrows point to microaneurysms, a key marker of retinal vascular changes in diabetic retinopathy. No microaneurysms were observed in SD mice, even after administration of the toxin streptozotocin (STZ). [Figure 13-1] 1 demonstrates that βA3 / A1-crystallin is an uncompetitive inhibitor of PTP1B. Lineweaver-Burk plots show that Cryba1 treatment decreases the Km and Vmax of PTP1B activity. [Figure 13-2] This is a continuation of Figure 13-1. [Figure 14A] Figures 14A-D demonstrate altered PTP1B activity and expression in Nuc1 rat model and human diabetic retinopathy patients. Figure 14A shows PTP1B activity (U / mg protein) in wild-type and Nuc1 astrocytes in low and high glucose (HG) conditions. Figure 14B demonstrates elevated levels of PTP1B (pg / μg) in vitreous humor of human DR patients compared to controls. Figures 14C-14D demonstrate that samples from vitreous humor of human patients with progressive DR (PDR) showed lower levels of crystallins compared to PTP1B, which was not observed in normal samples (controls). *P<0.05, **P<0.01, ****p<0.0001; Mann Whitney test. [Figure 14B] This is a continuation of Figure 14A. [Figure 14C] This is a continuation of Figure 14B. [Figure 14D] This is a continuation of Figure 14C. [Figure 15A]Illustrates the βA1-crystallin construct for gene therapy. The sequence (SEQ ID NO: 19) corresponding to rat βA1-crystallin (starting with the second ATG) was cloned into the AAV2 transfer vector containing the rat Aldh1L1 promoter to ensure specific expression in astrocytes. The Cryba1-Al gene under the control of the Aldh1L1 promoter was delivered to Nuc1 rats by intravitreal injection. [Figure 15B] This is a continuation of Figure 15A. [Figure 15C] This is a continuation of Figure 15B. [Figure 16] 1 is a graph demonstrating that βA1-crystallin reduces aberrant PTP1B activity in Nuc1 astrocytes. Shown is PTP1B activity (U / mg protein) in wild-type and Nuc1 astrocytes under low and high glucose (HG) conditions, where addition of a vector encoding βA1-crystallin reduces aberrant PTP1B activity in Nuc1 astrocytes. [Figure 17-1] FIG. 13 demonstrates that βA1-crystallin rescues the DR phenotype in Nuc1. In the left image, black arrows point to acellular capillaries, black arrowheads point to acellular capillaries with vascular thinning, and empty arrows point to capillary loss. The graph on the right demonstrates the number of acellular capillaries observed in rats. Scale bar: 100 mm. [Figure 17-2] This is a continuation of Figure 17-1. [Figure 18A] Figure 18 demonstrates that βA1-crystallin rescues abnormal glucose metabolism in Nuc1 astrocytes. An increase in extracellular acidification rate (ECAR; Figure 18A) and a decrease in mitochondrial respiration (oxygen consumption rate or OCR; Figure 18B) were observed in Nuc1 astrocytes treated with high glucose (HG) (Figure 18B) compared to HG-exposed WT cells. Overexpression of βA1-crystallin in Nuc1 + HG cells rescued both ECAR and OCR levels. [Figure 18B] This is a continuation of Figure 18A. [Figure 19A]19A-19B demonstrate that βA1-crystallin reduces lactate production and mitochondrial oxidative stress in Nuc1 astrocytes under hyperglycemic conditions. Elevated lactate levels and mitochondrial reactive oxygen species (ROS) are key signs of metabolic alterations. The increase in lactate levels (FIG. 19A) and mitochondrial ROS (FIG. 19B) in HG-exposed Nuc1 astrocytes compared to controls (WT cells) was reduced after βA1-crystallin overexpression. [Figure 19B] This is a continuation of Figure 19A. [Figure 20A] 10A-10C demonstrate that a PTP1B inhibitor (MSI-1436) rescues retinal dysfunction in diabetic Nuc1 rats. Similar results were observed by intravitreally injecting βA1-crystallin vector in diabetic Nuc1 astrocytes. [Figure 20B] This is a continuation of Figure 20A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Age-related macular degeneration (AMD) is a growing problem as life expectancy increases worldwide. Inflammation clearly contributes to vision loss in AMD, but the mechanisms are controversial. We found that neutrophils are important in this inflammatory process. We observed neutrophil infiltration in the retina of both AMD patients and mouse models of an AMD-like phenotype. Such infiltration was experimentally confirmed using ribbon scanning confocal microscopy (RSCM) and dye-labeled normal neutrophils for IFNλ activation.

[0014] We developed a unique construct that overexpresses only βA1-crystallin among the leaky ribosomal proteins βA3 / A1-crystallin and packaged it into AAV2 with the hVMD2 promoter. Crystallin protein can rejuvenate lysosomal function of RPE cells and rescue AMD-like phenotypes. This concept has significance beyond AMD and can be used for other disorders, such as diabetic retinopathy, by targeting astrocytes.

[0015] In this regard, the present invention provides a method for restoring lysosomal function of RPE cells, comprising administering to a subject in need thereof a polypeptide comprising, consisting essentially of, or consisting of βA1-crystallin, or a nucleic acid encoding said polypeptide.

[0016] Without wishing to be bound by any theory, it is believed that administration of βA1-crystallin (i) reduces abnormal PTP1B activity, (ii) reduces abnormal glucose metabolism, (iii) reduces lactate production, (iv) reduces mitochondrial oxidative stress, and (v) rescues abnormal retinal function, thereby inhibiting, alleviating, and / or treating diabetic retinopathy in affected subjects (see Example 9). Thus, the present invention provides related methods (e.g., methods of reducing or inhibiting PTP1B activity, glucose metabolism, lactate production, and / or mitochondrial oxidative stress) comprising administering to a subject in need thereof a polypeptide comprising, consisting essentially of, or consisting of βA1-crystallin, or a nucleic acid encoding the polypeptide. In particular, the present invention provides a method of treating diabetic retinopathy in a subject comprising administering to the subject a polypeptide comprising, consisting essentially of, or consisting of βA1-crystallin, or a nucleic acid encoding the polypeptide.

[0017] The present invention also provides a method of inhibiting neutrophil activation in retinal pigment epithelial cells, comprising administering an inhibitor of IFNλ to a subject in need thereof.

[0018] The inhibitor may be any suitable inhibitor, such as a neutralizing anti-IFNλ antibody, an antisense oligonucleotide, a small interfering ribonucleic acid (siRNA), a small hairpin RNA (shRNA), a non-antibody binding polypeptide, or a small chemical compound.

[0019] antibody The method disclosed herein includes inhibiting IFNλ by administering one or more neutralizing antibodies directed against IFNλ. The antibody includes at least one antibody selected from immunoglobulins, antibody fragments, and antibody analogs. The antibody may include any naturally occurring immunoglobulin. The term "antibody fragment" includes an antibody fragment having a minimal unit of amino acid sequence that recognizes an antigen, and may include at least one antibody fragment selected from Fab, Fab', F(ab')2, F(ab'), F(ab), Fv, and scFv. The term "antibody analog" refers to an antibody that has a smaller protein molecular weight than an antibody fragment and recognizes and binds to an antigen. In various embodiments, the antibody analog may include at least one antibody analog selected from affibody, repebody, affilin, DARPin, tetranectin, microbody, peptide aptamer, and avimer. When an antibody analog is used, an antibody mimic may further be included. In various embodiments, examples of antibody mimics are affilin, affimer, affitin, anticalin, avimer, and monobody. Antibody mimics can be produced so that they target specific receptors or antigens, and the production methods available herein can include panning methods. The targeting agent of the antibody mimic has a smaller molecular mass and simpler structure than an antibody. The term "antibody" also refers to both monoclonal and polyclonal antibodies.

[0020] RNA interference The methods disclosed herein include inhibiting IFNλ by administering one or more nucleic acids selected from the group consisting of antisense oligonucleotides, small interfering ribonucleic acid (siRNA), and small hairpin RNA (shRNA).

[0021] RNA interference (RNAi) is a process in which dsRNA directs the homologous sequence-specific degradation of messenger RNA. In mammalian cells, RNAi can be activated by a nucleotide duplex of small interfering RNA (siRNA) without activating the host interferon response. The dsRNA can be an siRNA (containing two separate complementary RNA strands) or a short hairpin RNA (i.e., an RNA strand that forms a tight hairpin structure), either of which can be designed based on the sequence of the target gene. When the inhibitor is an shRNA, the shRNA can target one or more of the nucleotide sequences consisting of the group consisting of SEQ ID NOs: 11-17.

[0022] Antisense oligonucleotides refer to nucleic acid molecules that are complementary to a portion of a specific gene transcript that can hybridize to the transcript and block its translation. Antisense oligonucleotides may comprise RNA or DNA.

[0023] Non-antibody-binding polypeptides The methods disclosed herein include inhibiting IFNλ by administering one or more non-antibody binding polypeptides directed against IFNλ. Binding polypeptides can be chemically synthesized using known polypeptide synthesis methods or can be prepared and purified using recombinant techniques. Binding polypeptides are typically at least about 5 amino acids in length, or at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 1 The binding polypeptides may be 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 or more amino acids long, where such binding polypeptides are preferably capable of specifically binding to IFNλ. Binding polypeptides can be identified without undue experimentation using well-known techniques. In this regard, it is noted that techniques for screening polypeptide libraries for binding polypeptides capable of binding to a polypeptide target are well known in the art.

[0024] small molecule chemical compounds The methods disclosed herein include inhibiting IFNλ by administering one or more small molecule chemical compounds directed against IFNλ. The small molecule chemical compounds can be components of a combinatorial chemical library. A combinatorial chemical library is a collection of many different chemical compounds composed of smaller subunits or monomers. Combinatorial libraries come in a variety of sizes, ranging from hundreds to hundreds of thousands of different chemical compounds. A variety of library types also exist, including oligomer and polymer libraries composed of compounds such as carbohydrates, oligonucleotides, and small organic molecules. Such libraries have a variety of uses, including for immobilization and chromatographic separation of chemical compounds, and for identifying and / or characterizing ligands capable of binding acceptor molecules (e.g., IFNλ) or mediating a biological activity of interest (e.g., but not limited to, inhibition of neutrophil activation).

[0025] Various techniques are known in the art for synthesizing libraries of compounds on solid supports. The solid supports are usually polymeric objects with a surface that is functionalized to bind subunits or monomers to form the compounds of the library. The synthesis of a library usually involves multiple solid supports. To generate a combinatorial library, the solid supports are reacted with one or more subunits of the compounds and with one or more number of reagents in a carefully controlled, predetermined sequence of chemical reactions.

[0026] Small molecules can be identified and chemically synthesized using known methodologies. Small molecules are usually less than about 2000 daltons in size, or less than about 1500, 750, 500, 250 or 200 daltons in size, where such small molecules capable of binding, preferably specifically, to IFNλ can be identified without undue experimentation using well-known techniques. In this regard, it is noted that techniques for screening small molecule libraries for molecules capable of binding to a polypeptide target are well-known in the art. The small molecule can be, for example, an aldehyde, ketone, oxime, hydrazone, semicarbazone, carbazide, primary amine, secondary amine, tertiary amine, N-substituted hydrazine, hydrazide, alcohol, ether, thiol, thioether, disulfide, carboxylic acid, ester, amide, urea, carbamate, carbonate, ketal, thioketal, acetal, thioacetal, aryl halide, aryl sulfonate, alkyl halide, alkyl sulfonate, aromatic compound, heterocyclic compound, aniline, alkene, alkyne, diol, amino alcohol, oxazolidine, oxazoline, thiazolidine, thiazoline, enamine, sulfonamide, epoxide, aziridine, isocyanate, sulfonyl chloride, diazo compound, acid chloride, and the like.

[0027] Polypeptides comprising, consisting essentially of, or consisting of a polypeptide-containing IFN λ inhibitor (e.g., a neutralizing antibody or a non-antibody binding polypeptide) and βA1-crystallin can be prepared by any of a number of conventional techniques. In this regard, the polypeptide sequences may be synthetic, recombinant, isolated and / or purified.

[0028] The polypeptides of the present invention can be isolated or purified from recombinant sources. For example, a DNA fragment encoding the desired polypeptide can be subcloned into a suitable vector using well-known molecular genetic techniques. The fragment can be transcribed and the polypeptide can be subsequently translated in vitro. Commercially available kits can be used. Optionally, the polymerase chain reaction can be used in the manipulation of nucleic acids.

[0029] The polypeptides of the invention can also be synthesized using an automated peptide synthesizer, according to methods known in the art. Alternatively, the polypeptides can be synthesized using standard peptide synthesis techniques well known to those skilled in the art. In particular, the polypeptides can be synthesized using procedures of solid phase synthesis. If desired, the synthesis can be performed using an automated peptide synthesizer. Removal of the t-butyloxycarbonyl (t-BOC) or 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking groups and separation of the polypeptide from the resin can be performed, for example, by acid treatment at reduced temperature. The protein-containing mixture can then be extracted, for example with diethyl ether, to remove non-peptidic organic compounds, and the synthetic polypeptide can be extracted from the resin powder (e.g., with about 25% w / v acetic acid). Following synthesis of the polypeptide, further purification (e.g., using HPLC) can optionally be performed to remove incomplete proteins, polypeptides, peptides, or free amino acids. Amino acid analysis and / or HPLC analysis can be performed on the synthesized polypeptide to verify its identity. For other uses according to the invention, it may be preferable to produce the polypeptide as part of a larger fusion protein, by chemical conjugation or through genetic means as known to those skilled in the art. In this regard, the present invention also provides fusion proteins comprising a polypeptide and one or more other proteins having desired properties or functions, for example to facilitate separation, purification, analysis or stability of the fusion protein.

[0030] In addition to IFN lambda inhibitors, shRNA, siRNA and antisense oligonucleotides, the present invention provides nucleic acids encoding polypeptides or polypeptide-containing inhibitors (e.g., neutralizing antibodies or non-antibody binding polypeptides) comprising βA1-crystallin. As used herein, "nucleic acid" includes "polynucleotides", "oligonucleotides" and "nucleic acid molecules" and generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, synthetic or obtained (e.g., isolated and / or purified) from a natural source. The DNA or RNA polymer may contain natural, non-natural or modified nucleotides, and may contain natural, non-natural or modified internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, in place of the phosphodiesters found between nucleotides of unmodified oligonucleotides. It is generally preferred that the nucleic acid does not contain insertions, deletions, inversions and / or substitutions. However, in some cases, as discussed herein, it may be appropriate for the nucleic acid to contain one or more insertions, deletions, inversions and / or substitutions.

[0031] In one embodiment, the nucleic acid is recombinant. As used herein, the term "recombinant" refers to a molecule that is (i) constructed outside a living cell by joining a natural or synthetic nucleic acid segment to a nucleic acid molecule that can replicate in a living cell, or (ii) that results from replication as described in (i) above. For purposes of this specification, replication may be in vitro or in vivo.

[0032] The nucleic acids (e.g., DNA, RNA, cDNA, etc.) of the invention can be produced in any suitable manner, including but not limited to recombinant production and commercial synthesis. In this regard, the nucleic acid sequences can be synthetic, recombinant, isolated and / or purified.

[0033] The nucleic acids of the present invention can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art.See, for example, Green et al. (eds.), Molecular Cloning, A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, New York (2012).For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or using various modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosyl eosin, inosine, N 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -Substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocin, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention can be purchased from a company, for example, Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0034] The nucleic acid of the present invention can be provided as part of a construct that includes nucleic acid and elements that allow delivery of the nucleic acid to a cell and / or expression of the nucleic acid in a cell. For example, polynucleotide sequences corresponding to IFN lambda inhibitor shRNA, siRNA and antisense oligonucleotides, or polynucleotide sequences that code for polypeptides or polypeptide-containing inhibitors that include βA1-crystallin can be operably linked to expression control sequences. The expression control sequences operably linked to the coding sequence are ligated so that the expression of the coding sequence is carried out under conditions compatible with the expression control sequences. Expression control sequences include, but are not limited to, suitable promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of the gene that codes for the protein, splicing signals of introns, maintaining the correct reading frame of the gene to allow accurate translation of mRNA, and stop codons. Suitable promoters include, but are not limited to, hVMD2 promoter, Aldh1L1 promoter, SV40 early promoter, RSV promoter, adenovirus major late promoter, human CMV immediate early I promoter, poxvirus promoter, 30K promoter, I3 promoter, sE / L promoter, 7.5K promoter, 40K promoter, and C1 promoter.

[0035] The nucleic acids of the invention can be cloned or amplified by in vitro methods such as polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), self-sustained sequence replication system (3SR) and Qβ replicase amplification system (QB). For example, a polynucleotide encoding a polypeptide can be isolated by polymerase chain reaction of cDNA using primers based on the DNA sequence of the molecule. A variety of cloning and in vitro amplification methodologies are well known to those skilled in the art.

[0036] The present invention provides a vector comprising a nucleic acid of the present invention. The nucleic acid can be inserted into any suitable vector. The selection of vectors and methods for their construction are generally known in the art and are described in general technical references.

[0037] Suitable vectors include those designed for propagation and amplification or for expression or both. Examples of suitable vectors include, for example, plasmids, plasmid-liposome complexes, CELid vectors (see, e.g., Li et al., PLoS One., 8(8): e69879. doi: 10.1371 / journal.pone.0069879 (2013)) and viral vectors, such as parvovirus-based vectors (i.e., AAV vectors), retrovirus vectors, herpes simplex virus (HSV)-based vectors, adenovirus-based vectors, and poxvirus vectors. Any of these expression constructs can be prepared using standard recombinant DNA techniques.

[0038] In one aspect of the present invention, the vector is a viral vector, for example, an AAV vector.The AAV vector may be suitable for packaging into any AAV serotype or variant thereof that is suitable for administration to ocular cells (e.g., RPE cells).Examples of suitable AAV serotypes may include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, variants thereof, and engineered or newly synthesized AAV viruses, for example, AAV.7m8.

[0039] The AAV vector can be packaged into the capsid protein or fragment thereof of any of the AAV serotypes described herein. Preferably, the vector is packaged into an AAV2 or AAV8 capsid.

[0040] A suitable recombinant AAV can be produced by culturing a packaging cell that contains a nucleic acid sequence encoding an AAV serotype capsid protein or a fragment thereof, as defined herein; a functional rep gene; any of the vectors of the invention described herein; and sufficient helper functions to allow packaging of the vectors of the invention into AAV capsid proteins. The components required by the packaging cell to package the AAV vectors of the invention into AAV capsids can be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., the vectors of the invention, rep sequences, capsid sequences and / or helper functions) can be provided by a stable packaging cell that has been engineered to contain one or more of the required components using methods known to those of skill in the art.

[0041] In one embodiment of the invention, the AAV vector is self-complementary. Vectors that are self-complementary can advantageously overcome the rate-limiting step of second strand DNA synthesis and confer earlier initiation and stronger gene expression.

[0042] In one embodiment of the present invention, the vector is a recombinant expression vector. For the purposes of this specification, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that allows for the expression of an mRNA, protein, polypeptide or peptide by a host cell, where the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide or peptide, and the vector is contacted with a cell under conditions sufficient to express the mRNA, protein, polypeptide or peptide in the cell. The vector of the present invention is not naturally occurring in its entirety. However, parts of the vector may be naturally occurring. The recombinant expression vector of the present invention may contain any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially derived from natural sources, and may contain natural, non-natural or modified nucleotides. The recombinant expression vector may contain linkages between naturally occurring nucleotides, linkages between non-naturally occurring nucleotides or both types of linkages. Preferably, the non-naturally occurring or modified nucleotides or internucleotide linkages do not interfere with the transcription or replication of the vector.

[0043] The vectors of the invention can be prepared using standard recombinant DNA techniques, for example as described in Green et al., supra. Expression vector constructs, either circular or linear, can be prepared to contain a replication system functional in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColEl, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0044] In addition to the above nucleic acids, the vectors of the invention can include one or more nucleic acid sequences encoding one or more polypeptides for delivery and expression in a subject / host (e.g., a mammal, such as a mouse, rat, guinea pig, hamster, cat, dog, rabbit, pig, cow, horse, or primate (e.g., human)).

[0045] The vectors of the present invention may contain one or more marker genes to allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance, resistance to e.g. antibiotics, heavy metals, etc., complementation in auxotrophic hosts to confer prototrophy, etc. Suitable marker genes for the expression vectors of the present invention include, by way of example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0046] The vector of the present invention can further comprise a regulatory sequence that allows one or more of transcription, translation and expression of the nucleic acid contained in the vector in a cell transfected with the vector or infected with a virus containing the vector. For example, an "operably linked" sequence includes both a regulatory sequence that is contiguous with the nucleic acid of interest (e.g., a nucleotide sequence encoding βA1-crystallin) and a regulatory sequence that acts in trans or at a distance to control the nucleotide sequence (e.g., a nucleotide sequence encoding βA1-crystallin polypeptide).

[0047] Regulatory sequences may include appropriate transcription initiation, termination, promoter and enhancer sequences; RNA processing signals, such as splicing and polyadenylation (polyA) signal sequences; sequences that stabilize the mRNA in the cytoplasm; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, optionally, sequences that enhance secretion of the encoded product. PolyA signal sequences may be synthetic or derived from a number of suitable species, including, for example, SV-40, human and bovine. Figures 8 and 9 provide exemplary vector maps of vectors containing the nucleotide sequence of βA1-crystallin (scCAG_humCryba1).

[0048] If the vector is intended for administration to a host (e.g., a human), the vector (e.g., AAV) preferably has low replication efficiency in the target cell (e.g., about 1 progeny or less per cell, or more preferably 0.1 progeny or less per cell is produced). Replication efficiency can be readily determined by empirically determining the viral titer following infection of a target cell.

[0049] The polypeptides, nucleic acids, vectors, or inhibitors (e.g., small molecules) of the invention can be formulated as a composition (e.g., a pharmaceutical composition) comprising the polypeptide, nucleic acid, vector, or inhibitor and a carrier (e.g., a pharma- ceutical or physiologically acceptable carrier). Additionally, the polypeptides, nucleic acids, vectors, inhibitors, or compositions of the invention can be used in the methods described herein, either alone or as part of a pharmaceutical preparation.

[0050] A composition (e.g., a pharmaceutical composition) can include more than one polypeptide, nucleic acid, vector, inhibitor (e.g., small molecule) or composition of the invention. Alternatively, or in addition, the composition can include one or more other pharma- ceutically active agents or drugs. Examples of such other pharma- ceutical active agents or drugs that may be suitable for use in the pharmaceutical compositions include lampalizumab (anti-complement factor D; Genentech) for patients with geographic atrophy secondary to AMD; brolicizumab (pan-isoform ant-VEGF-A; Novartis) for wet AMD; OPT-302 (soluble VEGF-C / D receptor; Ophthea) for wet AMD and diabetic retinal edema (DME); PanOptica's topical VEGF inhibitors for wet AMD; pegprelanib (DNA aptamer that binds PDGF isoforms; Ophtotech / Novartis) optionally in combination with LUCENTIS™ (ranibizumab injection); linucumab (anti-PDGF receptor; Regeneron) optionally co-formulated with EYLEA™ (aflibercept); DE-120 (anti-PDGF / VEGF bispecific; Santen); borolanib (an oral RTK inhibitor that inhibits the kinase activity of pDGF and VEGF; Tyrogenex); nebacumab (anti-angiopoietin 2; Regeneron) optionally in combination with EYLEA™ (aflibercept); RG-7716 (anti-angiopoietin 2 / VEGF bispecific; Chugai); ARP-1536 (anti-VE PTP; Akebia / Aeripo); ICON-1 (chimeric protein that binds tissue factor; Iconic Therapeutic); and carotuximab (anti-endogin; Tracon / Santen). Additionally or alternatively, the composition may include an inhibitor of PTP1B.

[0051] The carrier can be any of those conventionally used, limited only by physicochemical considerations such as solubility and lack of reactivity with the active compound, and only by the route of administration. Pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients and diluents, are known to those skilled in the art and are generally readily available. Pharmaceutically acceptable carriers are preferably those that are chemically inert to the active agent and have no adverse side effects or toxicity under the conditions of use.

[0052] The choice of carrier will be determined in part by the particular polypeptide, nucleic acid, vector, inhibitor (e.g., small molecule) of the invention or composition thereof, as well as other active agents or drugs being used, as well as by the particular method used to administer the polypeptide, nucleic acid, vector, inhibitor or composition thereof.

[0053] The polypeptides, nucleic acids, vectors, inhibitors (e.g., small molecules) of the present invention or compositions thereof can be administered to a subject by any method. For example, the polypeptides, nucleic acids or vectors can be introduced into a cell (e.g., in a subject) by any of a variety of techniques, for example, by contacting the cell with the nucleic acid or vector as part of a construct, as described herein, that allows for delivery and expression of the nucleic acid. Specific protocols for introducing and expressing nucleic acids in cells are known in the art.

[0054] Any suitable dose of the polypeptide, nucleic acid, vector, inhibitor (e.g., small molecule) or composition thereof of the present invention can be administered to a subject. The appropriate dose will vary depending on factors such as the subject's age, weight, height, sex, general condition, previous medical history, and disease progression, and can be determined by a clinician. The amount or dose should be sufficient to achieve a desired biological response, e.g., a therapeutic or prophylactic response, in the subject over a clinically reasonable time frame.

[0055] For example, the polypeptide can be administered at a dose of about 0.05 mg to about 10 mg (e.g., 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, and ranges thereof) per vaccination of a host (e.g., a mammal, e.g., a human), preferably at a dose of about 0.1 mg to about 5 mg per vaccination. Several doses (e.g., 1, 2, 3, 4, 5, 6 or more) may be provided (e.g., over a period of weeks or months).

[0056] The dosing period can be appropriately determined according to the progress of treatment.In an embodiment, the dosing period can include less than 1 year, less than 9 months, less than 8 months, less than 7 months, less than 6 months, less than 5 months, less than 4 months, less than 3 months, less than 2 months, or less than 1 month.In another embodiment, the dosing period can include 3 doses per day, 2 doses per day, or 1 dose per day during the length of the dosing period.

[0057] If the vector is a viral vector, a suitable dose may include approximately 1×10 5 ~Approx. 1×10 12 (For example, 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , and ranges therebetween) plaque forming units (pfu), although lower or higher doses may be administered to the host.

[0058] The polypeptides, nucleic acids, vectors, inhibitors (e.g., small molecules) or compositions thereof of the present invention can be administered to a subject by a variety of routes, including, but not limited to, topical, subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intravenous, subretinal, and intravitreal injection. In one embodiment, the polypeptides, nucleic acids, vectors, or compositions can be administered directly (e.g., administered locally) by direct injection into the eye by subretinal or intravitreal injection, or by topical application (e.g., as eye drops). When multiple doses are given, administration can be performed at one or more sites on a subject, and a single dose can be administered by dividing the single dose into equal amounts for administration at one, two, three, four, or more sites on an individual.

[0059] The present invention further provides a method for the production of RPE cells using a method comprising the steps of: + The present invention provides a method for rejuvenating the V-ATPase (V-ATPase) activity by modulating the assembly and disassembly of V-ATPase in a subject in need thereof. The assembly and disassembly of V-ATPase holoenzyme can be modulated by any suitable means, including targeting specific residues or peptides of V-ATPase (e.g., residues that interact with βA3 / A1-crystallin). Figure 10 provides a schematic showing βA3 / A1-crystallin binding to V-ATPase in the lysosomal lumen and listing residue boundaries for a portion of the rat V-ATPase holoenzyme. The rat and human V-ATPase sequences are highly conserved. Modulating the assembly and disassembly of V-ATPase holoenzyme (e.g., by targeting specific residues of V-ATPase for mutation) can rescue the correct function of RPE in disorders, such as dry AMD.

[0060] In one embodiment, the subject has age-related macular degeneration (AMD) or is at risk of developing AMD. The AMD can be wet AMD or atrophic (dry) AMD. The subject can have geographic atrophy secondary to AMD. Therefore, the present invention also provides a method for treating or preventing AMD in a subject.

[0061] In one embodiment, the subject has or is at risk of developing Stargardt's macular retinal degeneration. Accordingly, the present invention also provides methods of treating or preventing Stargardt's macular retinal degeneration.

[0062] In another embodiment, the subject has or is at risk of developing a neurodegenerative disease, such as Alzheimer's disease or Parkinson's disease. Thus, the present invention also provides methods of treating or preventing a neurodegenerative disease, such as Alzheimer's disease or Parkinson's disease, in a subject.

[0063] In a further embodiment, the subject has or is at risk of developing diabetic retinopathy (DR). The subject may also have or be at risk of developing diabetic macular edema (DME). Thus, the present invention also provides a method for treating or preventing DR or DME in a subject.

[0064] The administration of the polypeptides, nucleic acids, vectors, inhibitors (e.g., small molecules) or compositions of the present invention may be "prophylactic" or "therapeutic". When provided prophylactically, the polypeptides, nucleic acids, vectors, inhibitors (e.g., small molecules) or compositions of the present invention are provided prior to the subject's diagnosis of AMD, neurodegenerative disease, DR and / or DME. For example, subjects at risk of developing AMD, Stargardt's macular retinal degeneration, neurodegenerative disease, DR and / or DME are a preferred group of patients to be treated prophylactically. The prophylactic administration of the polypeptides, nucleic acids, vectors, inhibitors (e.g., small molecules) or compositions of the present invention prevents, improves, or delays AMD, Stargardt's macular retinal degeneration, neurodegenerative disease, DR and / or DME. When provided therapeutically, the polypeptides, nucleic acids, vectors or compositions of the present invention are provided at the time of or after the diagnosis of AMD, Stargardt's macular retinal degeneration, neurodegenerative disease, DR and / or DME.

[0065] If the subject has already been diagnosed with AMD, Stargardt's macular retinal degeneration, neurodegenerative disease, DR or DME, the polypeptides, nucleic acids, vectors or compositions thereof may be administered in combination with other therapeutic treatments, such as, for example, lampalizumab (anti-complement factor D; Genentech); brolicizumab (pan-isoform ant-VEGF-A; Novartis); OPT-302 (soluble VEGF-C / D receptor; Ophthea); PanOptica's topical VEGF inhibitors; pegpleranib (DNA aptamer that binds PDGF isoforms; Ophtotech / Novartis); LUCENTIS™ (ranibizumab injection); linucumab (anti-PDGF receptor; Regeneron); EYLEA™ (aflibercept); DE-120 (anti-PDGF / VEGF bispecific; Santen); borolanib (oral RTK inhibitor that inhibits the kinase activity of pDGF and VEGF; Tyrogenex); nebacumab (anti-angiopoietin 2; Regeneron); It may be administered in conjunction with RG-7716 (anti-angiopoietin 2 / VEGF bispecific; Chugai); ARP-1536 (anti-VE PTP; Akebia / Aeripo); ICON-1 (chimeric protein that binds tissue factor; Iconic Therapeutic); and / or carotuximab (anti-endogin; Tracon / Santen).

[0066] The terms "treat" and "prevent" as used herein do not necessarily imply 100% or complete treatment or prevention, rather there are various degrees of treatment or prevention that one of skill in the art would recognize as having potential efficacy or therapeutic effect.

[0067] There are a variety of suitable formulations of pharmaceutical compositions for the method of the present invention. Any suitable carrier can be used within the context of the present invention, and such carriers are well known in the art. The selection of the carrier will be determined in part by the specific site where the composition is administered (e.g., ocular cells, RPE cells, photoreceptor cells, rods and cones) and the specific method used to administer the composition. The pharmaceutical composition can be optionally sterile or can be sterile with the exception of one or more adeno-associated viral vectors.

[0068] Suitable formulations for the pharmaceutical compositions of the invention include aqueous and non-aqueous solutions, isotonic sterile solutions, which may contain antioxidants, buffers and bacteriostatic agents. Suitable formulations for the pharmaceutical compositions also include aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers and preservatives. The formulations may be provided in unit-dose or multi-dose sealed containers, for example in ampoules or vials, or may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example water, immediately prior to use. Extemporaneous solutions and suspensions may be prepared from sterile powders, granules and tablets. Preferably, the carrier is a buffered saline solution. More preferably, the pharmaceutical compositions for use in the methods of the invention are formulated to protect the polypeptide, nucleic acid or vector from damage prior to administration. For example, the pharmaceutical compositions may be formulated to reduce loss of the polypeptide, nucleic acid or vector on the devices used to prepare, store or administer the polypeptide, nucleic acid or vector of the invention, for example on glassware, syringes or needles. The pharmaceutical composition can be formulated to reduce the light and / or temperature sensitivity of the polypeptide, nucleic acid or vector. To this end, the pharmaceutical composition preferably comprises a pharma- ceutically acceptable liquid carrier, such as those described above, and a stabilizer selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof. The use of such compositions can extend the shelf life of the polypeptide, nucleic acid, vector, or inhibitor (e.g., small molecule), facilitate administration, and improve the efficiency of the methods of the invention.

[0069] Pharmaceutical compositions can also be formulated to enhance transduction / transfection efficiency of the polypeptides, nucleic acids, vectors, or inhibitors (e.g., small molecules) of the invention. In addition, one of skill in the art will appreciate that pharmaceutical compositions can include other therapeutic or biologically active agents.

[0070] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0071] [Example 1] This example provides the materials and methods for the following examples.

[0072] antibody PE / Cy7-tagged CD45 (Cat# 103114), APC-tagged Ly6C (Cat# 128016), FITC-tagged CD66b (Cat# 555724), V450-tagged Ly6G (Cat# 560603), Alexa fluor 700-tagged CD11b (Cat# 557960), anti-human PE / Cy7-tagged CD45 (Cat# 560178), and anti-human CD34 antibodies (Cat# 343602) were purchased from BD Biosciences (USA), and anti-human PE-tagged IL-28AR antibody (Cat# 337804) was purchased from Biolegend (USA). Anti-neutrophil elastase (Cat# ab68672), anti-GRO alpha (CXCL1) (Cat# ab86436), anti-STAT1 (phosphor S727) (Cat# abl09461), anti-IL28 receptor alpha or IL28R1 (Cat# ab224395), anti-histone H3 citronylation (Cat# ab219407), VCAM1 (Cat# abl34047), CD34 (Cat# 8158), and IL28 + IL29 (Cat# abl91426) antibodies were purchased from Abeam (USA). Anti-ICAM-1 (Cat# SC-107), anti-STAT1 (Cat# 9172T), anti-AKT (Cat# 4685S), anti-AKT2 (Cat# 2964S) and anti-DAB2 (Cat# 12906S) were purchased from Cell Signaling Technologies (USA).Other antibodies used included: Alexa fluor 488-tagged β1 Integrin (Santa Cruz Biotechnology, USA; Cat# sc-374429 AF488), anti-IL-28A / IFNλ2 (Antibodies online, USA; Cat# ABIN357173), anti-Ly6G (Antibodies online, USA; Cat# ABIN1854937), IL-29 antibody (Biorbyt, USA; Cat# orb6201), anti-IFNα (Thermo Fisher, USA; Cat# 221001), anti-myeloperoxidase / MPO (R&D Systems, USA; Cat# AF3667-SP), anti-LCN-2 (EMD Milipore; Cat# AB2267) and anti-actin (Sigma Aldrich, USA; Cat# A2066).

[0073] animal Both male and female βA3 / A1-crystallin conditional knockout mice (Cryba1 cKO C57Bl / 6J mice) and LCN-2 KO C57Bl / 6J mice were generated as previously described (Valapala et al., Aging Cell, 13: 1091-1094 (2014); and Berger et al., Proc. Natl. Acad. Sci. USA, 103: 1834-1839 (2006)). NOD-SCID mice (NOD.CB17-Prkdescid / J; 4-5 weeks old) were purchased from Jackson Laboratory (USA). All animal experiments were performed in accordance with the Guide for the Care and Use of Animals (National Academy Press) and approved by the University of Pittsburgh Animal Care and Use Committee.

[0074] Human Eye Diagnosis and classification of AMD in human donor eyes was performed as previously described (Ghosh et al., J. Pathol., 241: 583-588 (2017)). For immunostaining, human donor eyes were obtained from the National Disease Research Interchange (NDRI; Philadelphia, Pennsylvania, USA) within 12–35 h of death. Donor eyes from five subjects with early dry AMD (age range 79–95 years; mean age 85.8 years) and three older controls with no evidence of macular disease (age range 77–89 years; mean age 82.5 years) were studied (Ghosh et al., supra). This study adhered to the norms of the Declaration of Helsinki for research involving human tissue. For immunophenotyping and quantification of soluble factors in human peripheral blood and aqueous humor experiments, samples were collected from human donors and reported to Narayana Nethralaya (Bangalore, India). All subjects underwent ophthalmologic examination, including visual acuity and retinal examination. Patients with early AMD were diagnosed by fundus imaging, Amsler grid test and OCT imaging if deemed necessary and classified according to the Age-Related Eye Disease Study Research Group (AREDS). Subjects with coexisting glaucoma or other degenerative retinal disorders were excluded. The control group consisted of individuals without any history of AMD, diabetes, cardiovascular disorders or retinal diseases. Blood samples of 4-6 mL were collected in EDTA tubes by venipuncture from 18 control and 43 AMD subjects. Aqueous humor samples (approximately 50 μL) were collected from subjects undergoing cataract surgery (n=7 controls, n=6 AMD) by anterior parapuncture under sterile conditions. Within this group, early AMD subjects were identified by the presence of drusen and RPE abnormalities with characteristics of retinal pigmentary changes according to the AREDS classification, if surgery was not contraindicated. All collected samples were immediately stored in a biorepository until further processing. All patient samples and relevant clinical information were collected after approval by the Narayana Nethralaya Institutional Review Board (IRB) and with written informed consent from the patients.

[0075] immunostaining Freshly enucleated eyes were fixed in 2% paraformaldehyde (PFA) for 10 min, then the anterior part (cornea, lens, and attached iris pigment epithelium) was removed. The resulting posterior eye cup was fixed in 2% PFA for 1 h at room temperature for either frozen sections or RPE / retina flat mounts. For frozen sections, the eye cup was dehydrated through gradient sucrose solutions and embedded in OCT, whereas for RPE / retina flat mounts, the eye cup was quartered like a petal structure before tissue removal. The resulting eye cup was further cut into 8 pieces radially from the optic nerve head to the periphery. Immunostaining on human / mouse retina sections or on retina / RPE flat mounts was performed by using appropriate primary antibodies (1:100) and incubated overnight at 4°C. RPE / retina flat mounts or human or mouse retina sections were washed three times with 1×TBS and then stained with appropriate secondary antibodies (1:300) containing 1 μg / mL DAPI (Sigma Aldrich, USA) for 2 h at room temperature in the dark. Tissue sections or flat mounts were washed six times with 1×TBS. Tissues were mounted on cover slips with DAKO mounting medium and then visualized under a confocal microscope (Zeiss LSM710, Switzerland).

[0076] Quantification of soluble factors Peripheral venous blood was obtained by venipuncture (n = 43 AMD patients and n = 18 control subjects) and aqueous humor (AH) was collected by anterior chamber paracentesis in AMD patients (n = 6) and control subjects (n = 7) from subjects undergoing cataract surgery. IFNα, IFNβ, IFNγ, IFNλ1-3, VEGF and CXCL1 levels were measured in plasma and AH by bead-based multiplex ELISA (BioLegend, Inc, USA) using a flow cytometer (BD FACS Canto II, FACS DIVA software, BD Biosciences, USA). Absolute concentrations of each analyte were calculated based on standard curves using LEGENDplex™ software (Biolegend, Inc, USA).

[0077] Immunophenotyping Cells from peripheral blood (n = 43 AMD patients and n = 18 control subjects) were labeled with fluorochrome-conjugated anti-human antibodies specific for leukocytes (APC-Cy7-tagged CD45), neutrophils (FITC-tagged CD66b) and IFN lambda receptor (PE-tagged IL-28R1) for 45 min at room temperature. Red blood cells from peripheral blood samples were lysed with 1x BD lysis buffer for 10 min, washed and resuspended in 1x phosphate-buffered saline prior to flow cytometry (BD FACS Canto II, FACS DIVA software, BD Biosciences, USA)-based acquisition and analysis. Data were analyzed using FCS Express 6 Flow Research Edition software. Leukocyte populations were analyzed using SSC / CD45 + Neutrophils were identified by manual gating using the CD45 profile. Subsequent gating was performed with SSC / CD66bFITC to identify neutrophils. The activation state of neutrophils was determined based on CD66b cell surface expression. + CD66b high The cells were classified as activated neutrophils and expressed CD45 + CD66b low CD45 was defined as inactive neutrophils. + CD66b high / low IL-28R1 + IFNλ receptor positive neutrophils were indicated by: The percentage of positive cell events for each staining panel was calculated.

[0078] Isolation and culture of RPE Mouse RPE were isolated from control C57BL / 6J mice (3 weeks old, n = 9; Jackson Laboratories, USA) and cultured by enucleating the eyes, then washing twice with DMEM containing high glucose and incubating with 2% (wt / vol) Dispase in DMEM (Roche, 10269638001) for 45 min at 37°C. The eyes were then washed twice with growth medium made of DMEM (high glucose) containing 10% FCS, 1% penicillin / streptomycin, 2.5 mM L-glutamine and 1x MEM non-essential amino acids (Gibco, Invitrogen, 11095). An incision was made around the ora serrata of each eye and the anterior part was excised. The resulting posterior eye cup was placed in growth medium for 20 min at 37°C to initiate the separation of the neural retina from the RPE. The neural retina was removed and an intact sheet of RPE cells was peeled off from the underlying Bruch's membrane and transferred to a sterile 60 mm culture dish containing fresh growth medium. The RPE sheet was washed three times with growth medium, then twice with calcium- and magnesium-free HBSS, and then triturated briefly using a fine-tipped Pasteur pipette. The RPE cells were centrifuged at 200 g for 5 min and cultured in transwell plates in growth medium.

[0079] IFNλ overexpression in cultured RPE cells pLV-C-IL28A-GFPSpark and control vectors were purchased from Sino Biological Inc. (Beijing, China, Cat# MG51305-ACGLN). Primary mouse RPE cells (in monolayer; 90% confluent) were transfected with the respective vectors using X-tremeGENE transfection reagent (Roche, Switzerland) according to the manufacturer's instructions. Transfection efficiency was estimated by assessing the levels of IL-28A / IFNλ released (in cell-free supernatant) from overexpressing transfected RPE cells versus control vector transfected cells by ELISA. A minimum of a three-fold increase in IL-28A / IFNλ levels was deemed appropriate for further experiments with conditioned media.

[0080] Neutrophil isolation and culture WT mice and LCN-2 - / - Neutrophils from mice were isolated by centrifugation of bone marrow cells flushed from femurs and tibias, and following isolation, purified on a discontinuous Percoll gradient to isolate neutrophils using Ca 2+ and Mg 2+ 10 x 10 per ml in free HBSS 6 Supplement with 20 mM HEPES and resuspend at a density of 3 x 10 per ml at 37 °C. 6 Cells were cultured at a density of 100x and subsequently stimulated with either recombinant IFNλ or conditioned medium from RPE cells overexpressing IFNλ.

[0081] pHrodo phagocytosis assay Neutrophils in culture were incubated with fluorescently tagged particles (pHrodo™ Red E. coli BioParticles™ Conjugate for Phagocytosis Assay Kit, Thermo Fisher, USA, Cat# P35361) and flow cytometric assessment of the percentage of cells that had phagocytosed pHrodo particles (phagocytes) was performed by following the manufacturer's protocol.

[0082] Integrin β1 shRNA transfection Integrin β1 shRNA lentivirus (Cat# sc-60044-V) and control shRNA (Cat# sc-108080) particles were purchased from Santa Cruz Biotechnology (USA). Mouse bone marrow-derived neutrophils (5 × 10 in HBSS containing 20 mM HEPES) were cultured at 10 °C for 1 h. 6 Cells / mL) were plated and then transfected with integrin β1 shRNA lentivirus or control shRNA particles for 8 h according to the manufacturer's protocol.

[0083] Rapid neutrophil adhesion assay LCN-2 - / -Mouse bone marrow-derived neutrophils (5 × 10 in HBSS containing 20 mM HEPES) from mice and WT mice, respectively 6 Neutrophils transfected with 1000 cells / mL, or either control shRNA or integrin β1 shRNA, were subjected to rapid adhesion assay. Glass-bottom 35 mm plates were coated with human fibrinogen (20 μg / well in endotoxin-free PBS) for 16 h at 4°C. All experimental conditions (10 per well) were 5 5 × 10 per mL in PBS with 10% FCS, 1 mM CaCl2 / MgCl2, pH 7.2 6 ) were added, incubated at 37°C for 10 min, then fixed in 1.5% glutaraldehyde on ice for 60 min, and then counted by computer-assisted counting.

[0084] Neutrophil extravasation assay LCN-2 - / - Neutrophils (5 × 10 in HBSS containing 20 mM HEPES medium) from mice and WT mice, respectively 6 Cell migration was assessed by using transwell plates using neutrophils transfected with either control shRNA or integrin β1 shRNA (cells / mL). Neutrophils were plated at 5 × 10 per ml in transwell inserts. 6 Cells were plated, exposed to various experimental conditions, and cultured at 37° C. Cells at the bottom of the transwells were fixed with 1.5% glutaraldehyde for 60 min, stained with Giemsa, and then counted by computer-assisted counting.

[0085] Estimation of the percentage of neutrophils in the mouse retina Mouse retinas were dissected from enucleated eyes, digested with 0.05% collagenase D (Roche, Switzerland, Cat# 11088858001) for 30 min at 37° C., torn with blunt end forceps, pipetted to release cells, passed through a 70 μm cell strainer, and centrifuged at 1,300 g for 20 min at 4° C. After staining with anti-Ly6G, anti-Ly6C, anti-CD11b, and anti-CD45 antibodies at a concentration of 1 μg / mL for 90 min at room temperature, the entire pellet was used to assess the % neutrophils by flow cytometry.

[0086] Intracellular reactive oxygen species (ROS) Flow cytometry was performed to incubate cells (1 × 10 ) from each experimental group with 2′,7′-dichlorofluorescin diacetate (DCFDA, Sigma Aldrich, USA, Cat# D6883-50MG) (25 μg / ml) for 30 min at 37 °C. 6 Intracellular ROS in neutrophils was assessed by staining DCFDA (cells). Excess DCFDA was washed away and cells were resuspended in PBS. The ROS content of cells was measured by flow cytometer.

[0087] Estimated intracellular and extracellular expression of integrin β1 WT mice and LCN-2 - / - Freshly cultured bone marrow-derived neutrophils from mice were incubated with Alexa fluor 488-tagged β1-integrin (Santa Cruz Biotechnology, USA) antibody at a concentration of 1 μg / mL in PBS containing 1% BSA for 1 h, and cell surface expression of integrin β1 (FITC fluorescence) was assessed in these cells as previously described (Teckchandani et al., J. Cell Biol., 186, 99-111 (2009)). For intracellular expression of integrin β1, cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min at 25°C, followed by incubation with anti-integrin β1 antibody at a concentration of 1 μg / mL in PBS containing 1% BSA for 1 h. Cells were analyzed by flow cytometry.

[0088] SDS-PAGE and Western blot analysis SDS-PAGE and Western blot analyses were performed by suspending and sonicating cell or tissue samples in RIPA lysis buffer (Millipore, Billerica, MA, 20-188) supplemented with 1% protease and phosphatase inhibitors (Sigma). Samples were placed on ice for 20 min and then centrifuged at 13,000 g for 20 min at 4°C. Supernatants were subjected to protein assessment with a BCA kit (Thermo Fisher, USA). 12 μg of protein was used per sample, mixed with 4× protein sample buffer (Invitrogen, Carlsbad, CA) containing 5% 2-mercaptoethanol (Sigma Aldrich, USA) and heated at 100°C for 10 min. Samples were loaded onto 4–12% Bis-Tris Nu-PAGE gels (Invitrogen) and electrophoresis was performed in MES buffer (Novex, Waltham, MA, USA). Proteins were transferred to nitrocellulose membranes and blocked with 5% skim milk (Biorad, USA) or 5% BSA (Sigma, for phosphorylated proteins). Primary antibodies were used at a dilution of 1:1000, while all secondary antibodies were used at a dilution of 1:3000.

[0089] Preparation of recombinant lipocalin-2 (LCN-2) protein Full-length LCN-2 cDNA was synthesized by GeneScipt (USA). It was subcloned into pET28a vector at Ndel and Xhol restriction sites. The construct was transformed into E. coli DH5-α cells for amplification and E. coli Rosetta for expression. A single colony was grown overnight as mother culture. 10% of the mother culture was inoculated, grown to 0.8-1.0 OD and induced with 0.5 mM IPTG for 2 h at 37 °C. The cells were then pelleted by centrifugation in a microfuge at 6000 rpm for 10 min at 4 °C and resuspended in 10% volume of 20 mM Tris pH 8.0 containing 300 mM NaCl and 10% glycerol. The mixture was sonicated for 6 cycles, on and off for 30 s each, then centrifuged at 12000 rpm for 30 min at 4 °C. The supernatant fraction was passed through a Nickel NTA (BioVision, USA) column according to the manufacturer's protocol. The column was washed twice with 20 mM Tris pH 8.0 containing 300 mM NaCl, 10% glycerol and 20 mM imidazole at 10 times the bed volume. The protein was eluted in multiple fractions with 20 mM Tris pH 8.0, 300 mM NaCl, 10% glycerol and 300 mM imidazole at approximately 5 times the bed volume. The protein was dialyzed overnight in 1×PBS and 50% glycerol at 4° C., and then polished with Sephacryl S-300 (GE Healthcare, USA, GE17-0599-10). The filter (0.25 micron) sterilized protein was stored in aliquots at −20° C.

[0090] Protein-protein interactions Human Proteome Microarray 2.0 analysis was performed as a paid service by CDI NextGen Proteomics (MD, USA). Recombinant LCN-2 was analyzed for protein-protein interaction profiling on HuProtTM v3.1 Human Proteome Arrays, samples were probed on array plates at 1 μg / mL with data analyzed using GenePix software. Hit identification was assessed as the ratio of the median foreground of each protein probe on the microarray to the median of the surrounding background, followed by normalization to the median of all adjacent probes within a 9 × 9 × 9 window size and expressed as the significance of the difference (Z score) of the probe binding signal from random noise. In this study, a cutoff Z score of 6 was used for the analysis of triplicates. Only protein interactions with Z scores above 6 were considered.

[0091] Enzyme-linked immunosorbent assay (ELISA) RPE-choroid complexes from freshly enucleated mouse eyes were kept on ice and then homogenized in 300 μL of complete extraction buffer (Abcam, USA, Cat# ab193970). The homogenized tissue was used to perform ELISA in 96-well microtiter plates coated with tissue lysate and incubated overnight at 4°C. The plates were blocked with 5% BSA for 2 hours. After washing, 50 μl of the appropriate primary antibody diluted 1:1000 was added to each well and incubated for 2 hours at room temperature. Bound cytokines were detected with secondary IgG peroxidase (Sigma Aldrich, USA). Color was developed with TMB substrate solution (BD Pharmingen, USA). The reaction was stopped with 2N H2SO4 solution and absorbance was measured at 450 nm using a microplate reader.

[0092] Clearing and imaging of whole eyes Whole mouse eyes from animals injected with red CMTPX-labeled neutrophils were fixed overnight in 4% paraformaldehyde. Eyes were subjected to BA:BB clearing by a series of PBS:ethanol gradients to dehydrate the organs, followed by clearing in a 1:2 mixture of benzyl alcohol (Sigma, 305197) and benzyl benzoate (Sigma, B6630) as previously described (Waxman et al., Invest. Ophthalmol. Vis. Sci., 59: 2371-2380 (2018) and Dodt et al., Nat. Methods, 4: 331-336 (2007)). After the specimens were visually clear, they were mounted in BA:BB solution between coverslips.

[0093] Each eye was scanned using an RS-G4 ribbon-scanning confocal (Caliber ID) with a 20x / 1.00 Glyc (CFI90 20XC, Nikon) set to a correction color of 1.50. Linear interpolation of 561 nm laser excitation (iChrome-MLE-LFA, Toptica) was set at between 15-30% power from the top to the bottom of the z-stack. Emission was detected using a 630 / 69 bandpass filter with PMT settings of HV, 85; offset, 5. Voxels were measured (0.395 × 0.395 × 5.33 μm). Each sample required approximately 3.5 hours of total acquisition time. Images were collected at 16-bit pixel depth and comprised approximately 65 GB per eye. Images were collected as ribbons, stitched and assembled using a custom algorithm in MATLAB v2017b. Each data set was converted to Nikon ND2 format and deconvolved using a custom NIS-Elements application configured for the Richardson-Lucy algorithm, line-scan confocal, high image noise level, and a 0.76 μm pinhole. The deconvolved images were then converted to IMS format and loaded into Imaris 9.2.1 (Bitplane). A Gaussian filter with a filter width of 0.395 was applied prior to analysis in Imaris.

[0094] Neutrophils were quantified using the spot count function in Imaris Surpass. Spots were quantified across the entire image and then manually edited to keep only those spots that were within the retina and Schlemm's canal. Finally, all remaining spots were volume filtered to fit the approximate diameter of neutrophils, between 728 and 5800 μm. 3 Any structures not falling within the range (11-22 μm in diameter) were excluded, as well as structures incorrectly selected during spot counting. Imaris surpass spot count parameters were the same for all datasets: region growing enabled = true; estimated diameter = 10.0 μm; background subtraction = true; "quality" above 190; region growing type = local contrast; region growing manual threshold = 139.744; region growing diameter = diameter from volume.

[0095] RNAseq analysis 5-month-old and 10-month-old Cryba1, respectively. fl / fl RPE-choroids from enucleated eyes from mice and Cryba1 cKO mice (n = 4) were subjected to total RNA isolation as previously described (Broggi et al., Nat. Immunol., 18: 1084-1093 (2017)). RNA sequencing was performed using approximately 30ng / μL of total RNA as a paid service from DNA Link (USA). All sequence reads were matched to the reference genome (NCBI37 / mm) using the RNAseq mapping algorithm included in CLC Genomics Workbench. 9 ) The maximum number of mismatches allowed for mapping was set to 2. To estimate gene expression levels and analyze differentially expressed genes between different groups, RPKM was calculated.

[0096] Co-immunoprecipitation To evaluate the association between LCN-2 / Dab2 and also between Dab2 / integrin β1 in the various experimental conditions, cultured neutrophils from the various experimental groups were subjected to co-immunoprecipitation (Co-IP) using the Pierce™ Co-Immunoprecipitation Kit (Thermo Fisher, USA, Cat# 26149). Cells were sonicated in IP Lysis / Wash Buffer (supplied in the kit) supplemented with 1% protease inhibitors (Sigma Aldrich, USA). Total lysates were processed with the kit according to the instructions. Seventy micrograms of lysate from each group were subjected to immunoprecipitation overnight at 4°C with 10 μg of immobilized LCN-2 and dab2 antibodies, respectively. As a negative control, normal rabbit IgG (Santa Cruz, USA) was used. After elution, samples were loaded on SDS-PAGE (15 μg per well) and Western blotted.

[0097] Subretinal injection of neutrophils and OCT in NOD-SCID mice NOD-SCID mice (NOD.CB17-Prkdescid / J, Jackson Laboratories, USA, male, 4-5 weeks old) were used in this study. A large sample size (n=10) was taken to nullify any experimental abnormalities. Mice were anesthetized and given subretinal injections of neutrophils or recombinant LCN-2 protein from the various experimental groups as previously described (Maruotti et al., Proc. Natl. Acad. Sci. USA, 112: 10950-10955 (2015)). Seven days after the procedure, NOD-SCID mice were anesthetized by intraperitoneal injection of a ketamine and xylazine mixture and then subjected to fundus imaging with OCT analysis using a Bioptigen Envisu R2210 system. OCT images were analyzed in optical sections (100 sections per retina) from each eye ranging from -2.0 to +2.0 mm relative to the optic nerve head (ONH) using Diver 2.4 software (Bioptigen) with the FIJI-ImageJ (NIH) plugin provided with the instrument. After the experiment, animals were euthanized with CO2 gas and eyes were harvested for further experiments.

[0098] Hematoxylin and eosin staining Eyes from NOD-SCID mice were fixed in 2.5% glutaraldehyde followed by formalin, transferred to graded ethanol, dehydrated, and subsequently embedded in methyl methacrylate. 1 μm sections were cut, stained with hematoxylin and eosin, and viewed under a light microscope.

[0099] Statistics and reproducibility Statistical analysis was performed using Microsoft Excel and GraphPad Prism6 software for Windows using one-way ANOVA. Group means were compared using Tukey's post hoc test with significance set at P<0.05. For experiments with human samples, comparisons between control and AMD groups were performed by Mann-Whitney test with significance set at p<0.05, and data distribution was determined by Shapiro-Wilk normality test. Center and edge lines of box plots indicate median and interquartile range, respectively, and whiskers indicate the most extreme data points. Analyses were performed in triplicate for technical purposes. Results are presented as mean ± standard deviation (SD).

[0100] [Example 2] This example demonstrates neutrophil infiltration in AMD and in a mouse model.

[0101] Similar to human AMD, Cryba1 cKO mice exhibit immune cell infiltration into the retina with age. Flow cytometric analysis of the total retinal cell population from the posterior eyecup was performed using CD45 high CD11b + This was performed by gating on cells (monocytes, macrophages and neutrophils). high CD11b + Neutrophils (Ly6C high Ly6G +The relative numbers of CD45 (cells positive for CD45) were determined by simultaneously labeling the cells with appropriate antibodies as previously described (Wu et al., Nat. Commun., 9: 4777 (2018)). high CD11b + Ly6C high Ly6G + Neutrophils were not increased in 2-month-old Cryba1 cKO retinas by 4 months, but they were involved in the development of an AMD-like phenotype in this mouse model. fl / fl It increased nearly threefold compared to control retinas and continued to increase with age, as seen in 13-month-old Cryba1 cKO retinas relative to aged control mice. Furthermore, immunofluorescence analysis of retinal flat mounts from Cryba1 cKO mice revealed a significant increase in intraretinal Ly6G expression compared to age-matched controls. + The number of Ly6G cells was increased in RPE flat mounts. + An increase in subretinal neutrophils, as determined by staining, was also observed in Cryba1 cKO mice compared with age-matched controls.

[0102] The percentage of neutrophils and their activation state in human early dry AMD were studied by phenotyping cells in peripheral blood by flow cytometry using an appropriate gating strategy. + CD66b in the (white blood cell) population + An increased proportion of neutrophils was observed in the peripheral blood of AMD patients compared to control subjects. Furthermore, activated neutrophils (CD45 + CD66b high ) was observed in peripheral blood, whereas inactive neutrophils (CD45 + CD66b low There was no change in the number of IFN λ receptor (IL-28R1)-positive leukocytes (CD45 + IL-28R1 + A significant increase in the total number of IL-28R1 + Activated neutrophils (CD66b high) significantly increased total neutrophil (CD66b + Immunolocalization studies showed that CD66b cells were significantly higher in human tissue sections from normal and AMD samples. + The presence of neutrophils.

[0103] Increased neutrophil numbers occur in the retinas of human AMD patients compared to age-matched control subjects (Gosh et al., J. Pathol., 241: 583-588 (2017)). However, IL28R1 + Expression of CD66b was only observed in retinal sections from AMD patients. + was evident for neutrophils but not controls, indicating that activated neutrophils are directed toward the retina only in early AMD patients.

[0104] These results suggest that IL-28R1 + This indicates a greater tendency for activated neutrophils to be guided towards the eye, giving a likely scenario for the role of IFNλ-mediated signaling in these infiltrating neutrophils. Once in the area of ​​inflammation, neutrophils release neutrophil extracellular traps (NETs), which can damage host tissues in immune-mediated diseases. Indeed, eyes with early dry AMD showed elevated staining for NET markers, myeloperoxidase (MPO), neutrophil elastase and citrullinated histone H3, compared to age-matched control eyes.

[0105] Taken together, these results support the notion that neutrophil infiltration into the retina is enhanced during the early dry AMD process.

[0106] [Example 3] This example identifies factors that promote neutrophil infiltration into the retina.

[0107] To identify soluble factors, including cytokines and chemokines, released from the retina that can promote neutrophil infiltration, we performed RNAseq analysis on retinal tissues from 5- and 10-month-old Cryba1 cKO and floxed control mice. Activation of genes related to the cGAS / STING pathway was observed (Figure 1). In addition, significant elevations in the levels of IFNs, including IFNα, IFNγ, and IFNλ, as well as CXCL1 and CXCL9, were observed in aged Cryba1 cKO retinas compared to controls (Figure 2A). ELISA was performed to further confirm these results (Figure 2B).

[0108] Immunofluorescence studies were performed to identify which cell types express IFNλ in the retina. Elevated levels of IFNλ were observed in RPE cells of human dry AMD patients compared to age-matched controls. Western analysis confirmed increased protein of IFNλ and CXCL1 in human AMD RPE / choroid lysates compared to controls. In addition, elevated levels of IFNα and IFNλ1 were observed in plasma and AH of early AMD patients compared to controls, whereas levels of IFNλ2 / 3 were not different in AMD patients compared to controls. Plasma levels of IFNγ showed a significant increase in AMD patients compared to controls, whereas no such change was seen in AH. IFNβ and VEGF levels in plasma and AH of AMD patients were not different from controls.

[0109] These results therefore support a proinflammatory environment in the eye, a condition likely involving IFNλ, secreted from diseased RPE and thereby inducing an inflammatory response. Therefore, we hypothesized that elevated levels of IFNλ may be a key factor promoting neutrophil activation and infiltration into the retina, since the IFNλ receptor (IL28R1) is expressed on circulating neutrophils.

[0110] In addition to soluble factors, neutrophils also require adhesion molecules for their transmigration to sites of injury. Neutrophils adhere to endothelial cells when their integrins interact with members of the endothelial cell immunoglobulin superfamily, such as ICAM-1 and VCAM-1, two important adhesion molecules on endothelial cells, which allow neutrophils to transmigrate into diseased or damaged tissues. Increased levels of ICAM-1 as well as VCAM-1 were observed in the retinas of aged Cryba1 cKO mice and human early dry AMD patients, respectively, compared to age-matched controls.

[0111] [Example 4] This example demonstrates that IFNλ mediates LCN-2 expression and neutrophil activation.

[0112] It has been previously reported that IFN-lambda activates the phosphorylation and nuclear translocation (activation) of STAT1 (Maher et al., Cancer Bio. Ther., 7, 1109-1115 (2008)). In the early AMD process, STAT1 activation is crucial for the expression of the lcn2 gene (Ghosh et al., J. Pathol., 241, 583-588 (2017)). LCN-2 is an adipokine and is known to be important for neutrophil activation and innate immune function. Indeed, binding of NF-kappa B and STAT1 to the promoter of LCN-2 leads to pathogenesis (Ghosh et al., supra, and Zhao et al., Mol. Metab., 2: 161-170 (2013)).

[0113] Murine bone marrow-derived neutrophils cultured with either recombinant IFNλ or conditioned medium from primary RPE cells overexpressing IFNλ, simulating the elevated IFNλ levels observed in the RPE of human AMD patients, exhibited elevated levels of LCN-2 and phosphorylated STAT1. Furthermore, IFNλ-exposed neutrophils showed significant increases in reactive oxygen species (ROS) levels and phagocytosis. Enhanced NET formation was evident from widespread distribution of extracellular nuclear material (stained with DAPI) that showed elevated staining for myeloperoxidase (MPO) and citrullinated histone H3, known markers of NETs.

[0114] Thus, the data support that IFNλ not only induces STAT1-mediated LCN-2 expression, but also enhances neutrophil activation.

[0115] [Example 5] This example demonstrates that LCN-2-activated neutrophils cause retinal degeneration.

[0116] Ribbon scanning confocal microscopy (RSCM) was applied as a means to rapidly image red CMTPX-tagged neutrophils within the whole eye of NOD-SCID immunodeficient mice to verify the transmigration of activated neutrophils. - / - Bone marrow-derived neutrophils from (knockout) mice, WT neutrophils treated with IFNλ, or LCN-2 - / - IFNλ-treated neutrophils from mice were injected intravenously into mice. To demonstrate homing of activated neutrophils to specific regions of the eye, RSCM combined with benzyl alcohol benzyl benzoate (BABB) clearing of NOD-SCID mouse eyes was performed. The clearing procedure ensured a constant refractive index throughout the eye, thereby making the tissue transparent and allowing image acquisition throughout the entire depth of the organ.

[0117] NOD-SCID mice administered red CMTX-tagged WT neutrophils showed little ocular infiltration, and similarly, ocularly infiltrated LCN-2- / - The data clearly suggest that neutrophils are predominantly directed toward the choroid under both conditions, but that WT neutrophils are not as abundant as LCN-2 neutrophils, likely due to the lack of stimulation from IFNλ. - / - Due to disrupted migratory signaling axes in mice, these cells are unable to cross the intraocular compartment in significant numbers through the blood-retinal or blood-aqueous barriers.

[0118] Interestingly, red CMTPX-tagged neutrophils treated with IFNλ showed a notable number of neutrophils infiltrating the eye, mostly into the retina, compared to controls (Figure 3). During the early stages of AMD, neutrophils migrate from the peripheral blood to the intraocular compartment in response to a chemotactic cue identified as IFNλ. In addition, IFNλ-treated LCN-2 - / - NOD-SCID mice injected with neutrophils were significantly more sensitive to untreated LCN-2 - / - Compared with neutrophil-injected mice, the retina showed significantly fewer infiltrating cells, demonstrating that neutrophil infiltration from the peripheral circulation into the eye is likely due to IFNλ-driven activation of LCN-2.

[0119] To further validate the observation that IFNλ-induced increased LCN-2 levels in transmigrated neutrophils may enhance outer retinal degeneration, we used bone marrow-derived WT neutrophils, LCN-2 - / - Bone marrow-derived neutrophils from mice, WT neutrophils treated with IFNλ, neutrophils treated with conditioned medium from primary cultures of RPE cells overexpressing IFNλ, or neutrophils treated with recombinant LCN-2 were injected into NOD-SCID mice.

[0120] After 7 days, optical coherence tomography (OCT) analysis showed that mice injected with either IFNλ-treated WT neutrophils or recombinant LCN-2 exhibited alterations in the RPE and photoreceptor (inner and outer segment) layers (Figure 4(iii)-(v)). Quantitative analysis by spider plots revealed a decrease in the thickness of these layers in the experimental groups (Figure 4(ix)). No significant changes were observed in mice treated with vehicle and / or WT neutrophils (Figure 4(i), (ii) and (ix)). In addition, LCN-2 - / - Neutrophils and LCN-2 cells treated with IFNλ - / - Neutrophils did not show any degenerative changes (Figure 4(vi)-(ix)), suggesting a pathogenic role of LCN-2 in retinal degeneration.

[0121] Hematoxylin-eosin staining of retinal sections from NOD-SCID mice injected with either IFNλ-treated WT neutrophils or recombinant LCN-2 showed degenerative changes in the outer nuclear layer (ONL) along with the photoreceptor layer (disruption of the inner segment and outer segment [IS / OS] junction) and the RPE-Bruch's membrane-choriocapillaris complex compared with vehicle control or WT neutrophil-injected mice. Furthermore, measurement of retinal layer thickness from these sections by spider plot showed severe loss or thinning of the IS / OS and RPE layers in mice injected with either IFNλ-treated WT neutrophils or recombinant LCN-2 compared with vehicle or WT neutrophil-treated groups. In addition, immunofluorescence studies confirmed increased loss of photoreceptors and RPE cells in NOD-SCID mice injected with either IFNλ-treated WT neutrophils or recombinant LCN-2 versus controls, as evidenced by decreased staining for rhodopsin (labeling rod photoreceptors) and RPE 65 (retinal pigment epithelium-specific 65 kDa protein) in the retinas of these mice.

[0122] The NOD-SCID mouse data provide evidence that IFNλ mediates LCN-2 activation in neutrophils, thereby inducing their transmigration to the retina and enhancing retinal degeneration. Thus, administration of one or more inhibitors of IFNλ may treat or prevent retinal degeneration and rescue the AMD-like phenotype.

[0123] [Example 6] This example demonstrates that LCN-2 / Dab2 association regulates neutrophil infiltration.

[0124] The above observations prompted further investigation into the possible molecular mechanisms by which neutrophils infiltrate the retina and thereby contribute to the pathogenesis of AMD. LCN-2 has previously been shown to regulate neutrophil chemotaxis and cell migration of cancer cells (Shao et al., J. Invest. Dermatol., 136, 1418-1428 (2016), and Du et al., Biochim. Biophys. Acta, 1853: 2240-2250 (2015)). To determine whether IFNλ-mediated LCN-2 activation in neutrophils is responsible for the increased adhesion and transmigration, we performed a human proteome high-throughput array to identify LCN-2 binding partners that may play a specific role in cell adhesion and migration. We determined that LCN-2 interacts with Dab2. This was confirmed by pull-down assays, which showed increased association between LCN-2 and Dab2 in IFNλ-exposed neutrophils compared with untreated or control conditioned medium-treated neutrophils.

[0125] It has been previously reported that Dab2 binds to integrin β1 and regulates its internalization, thereby regulating cell migration. Dab2 is also known to be a negative regulator of cell adhesion, especially during inflammatory processes. Furthermore, expression of extracellular integrin β1 drives cell adhesion to the endothelial cell surface of various tissues, thereby facilitating transmigration into the tissue. We hypothesized that such an increase in the association between LCN-2 and Dab2 could regulate extracellular integrin β1 levels by regulating the Dab2 / integrin β1 axis, thereby promoting neutrophil adhesion and transmigration into the retina.

[0126] To explore the role of LCN-2, we cultured WT and LCN-2 mice with either recombinant IFNλ or conditioned medium from IFNλ-overexpressing RPE cells. - / - Bone marrow-derived neutrophils from wild-type mice were used. Flow cytometry studies revealed increased extracellular integrin β1 expression in IFNλ-exposed neutrophils from wild-type mice, accompanied by a decrease in integrin β1. In addition, co-immunoprecipitation data showed no change in the binding between Dab2 and integrin β1 upon IFNλ exposure versus controls.

[0127] These results support an altered Dab2-mediated internalization of integrin β1 in IFNλ-exposed neutrophils, due to an increased association between LCN-2 and Dab2 specifically in IFNλ-exposed cells.

[0128] IFNλ exposure LCN-2 - / - Since we observed neutrophil homing to the eyes of neutrophil-injected NOD-SCID mice, the expression of adhesion-related surface proteins appears to be downregulated in the absence of LCN-2. Based on these observations, we hypothesized that LCN-2 regulates the expression of extracellular adhesion molecules, which in turn regulate cell attachment and transmigration. However, putative overlapping pathways may be involved in regulating neutrophil infiltration upon exposure to IFNλ.

[0129] Enhanced neutrophil adhesion to fibrinogen-coated plates and transmigration of IFNλ-treated normal neutrophils across fibrinogen-coated transwell chambers were observed. In addition, LCN-2 - / - We found that extracellular expression of integrin β1 was increased in naive neutrophils from mice. This data supports the findings of IFNλ-treated LCN-2 - / - This is in sharp contrast to our previous observation that neutrophils have reduced surface expression of integrin β1.

[0130] Previous studies have shown that integrin β1 surface expression on neutrophils can be regulated by several independent signaling cascades during inflammatory processes. - / - It seems plausible that neutrophil integrin β1 is upregulated independently of the IFNλ / LCN-2 / Dab2 pathway. However, extracellular integrin β1 levels and its internalization were significantly increased after IFNλ treatment compared to IFNλ-exposed WT neutrophils, whereas these LCN-2 - / - However, the adhesion and transmigration properties were not significantly different between LCN-2 cells exposed to IFNλ. - / - There was a large decrease in neutrophils and in integrin β1-silenced normal neutrophils, but no change in cell viability.

[0131] These results suggest that LCN-2 regulates Dab2-mediated internalization of integrin β1, which is crucial for cell adhesion and migration of IFNλ-exposed neutrophils.

[0132] [Example 7] This example describes a method to identify IFNλ inhibitors, which can be used to inhibit neutrophil activation and treat or delay AMD.

[0133] Identification of DARPin-based inhibitors DARPins (acronym for designed ankyrin repeat proteins) are engineered antibody-mimetic proteins that typically exhibit highly specific and high affinity target protein binding, as described in Pluckthun et al., Annu. Rev. Pharmacol. Toxicol., 55: 489-511 (2015) and Binz et al., J. Mol. Biol., 332: 489-503 (2003)).

[0134] The DARPin consensus sequence is DxxGxTPLHLAAxxGHLEIVEVLLKZGADVNAx (SEQ ID NO: 7), where x=any amino acid except Cys, Pro or Gly, and Z=His, Asp or Tyr.

[0135] Reverse translation of the DARPIN consensus sequence into the 99 base sequence of the most likely codons results in gatnnnnnnggcnnnaccccgctgcatctggcggcgnnnnnnggccatctggaaattgtggaagtgctgctgaaataaggcgcggatgtgaacgcgnnn (SEQ ID NO:8), where n = any base (either A, T, G or C) that does not result in Cys, Pro or Gly.

[0136] Preparation of new libraries for all predicted sequence possibilities.

[0137] Binding affinity to immobilized target protein IFNL1 interferon lambda 1 (Homo sapiens) is assessed by ribosome display, where IFNL1 interferon lambda 1 [Homo sapiens (human)] with gene ID: 282618 has the following nucleotide sequence and translated amino acid sequence:

[0138] Nucleotide sequence (603nt): JPEG2025063043000002.jpg82154

[0139] Translated amino acid sequence (200 aa): JPEG2025063043000003.jpg28152

[0140] The screened DARPins targeting human IFNL1 are used as candidate drug(s) for neutralizing IFNL1 expression in the retina of human dry AMD patients.

[0141] RNA interference (RNAi) The human IFNL1 interferon lambda 1 nucleic acid sequence (SEQ ID NO: 9) was analyzed and the following shRNA target sequences were identified (Table 1).

[0142] [Table 1]

[0143] Each of these sequences is cloned and packaged into the AAV2 and / or AAV8 delivery systems.

[0144] [Example 8] This example demonstrates that βA1-crystallin is required for lysosomal function in RPE cells.

[0145] Cryba1 has two gene products: βA3- and βA1-crystallin. To study the functions of βA3- and βA1-crystallin, βA3-crystallin knockout and βA1-crystallin knockout were created. As shown in Figure 5A, the wild-type sequence of Cryba1 shows dual ATG start sites for βA3- and βA1-crystallin. In the βA1-knockdown mutant, CCACC was inserted just before the first start site to reinforce the Kozak consensus sequence, thereby making the first start codon stronger (Figure 5B). In the βA3-knockout mutant, an A to G change inactivates the first start site (Figure 5C). A silent mutation (ACC to ACG) was also introduced to prevent binding and re-cutting of the sequence by gRNA after homologous recombination repair (Figure 5C).

[0146] Knockdown of βA1-crystallin caused inflammation in RPE cells as indicated by increased levels of interferons (e.g., IFNα, IFNβ, and IFNλ), CXCL1, and TNFα protein (Figure 6). Elevated levels of these cytokines were not observed in RPE cells from wild-type or βA3-crystallin knockout mice (Figure 6).

[0147] In addition, knockdown of βA1-crystallin caused oxidative damage in RPE cells, as indicated by superoxide (SOD2) expression (Figure 7A-B). Compared to SOD2 expression in RPE cells from wild-type mice, a significant increase in SOD2 expression was observed in Cryba1 cKO and βA1-crystallin knockdown mice (Figure 7B). No increase in SOD2 expression was observed in RPE cells from βA3-crystallin knockout mice (Figure 7B).

[0148] These results demonstrate that loss of βA1-crystallin led to inflammation and oxidative damage in RPE cells and support the idea that administration of βA1-crystallin mitigates the alterations in lysosomal function in RPE cells that are observed in disorders including AMD, diabetic retinopathy and diabetic macular edema, as well as neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0149] βA1-crystallin can be administered to RPE cells by any suitable means, including in a vector (e.g., an AAV vector). An exemplary vector (scCAG_humCryba1) is shown in FIG. 8, and its corresponding nucleic acid sequence is shown in FIG. 9.

[0150] [Example 9] This example demonstrates that βA1-crystallin can be used to treat diabetic retinopathy (DR).

[0151] DR is one of the leading causes of blindness with no currently available treatment, and is estimated to affect 400 million people in 2020. Decades of DR research have led to a more precise definition of the disease as a neurodegenerative disorder that precedes and coexists with vascular changes. One important gap in the understanding of the disease is that the contribution of a key player that may contribute to DR pathology during the various stages of DR progression, namely the retinal glial cell population, has not been considered in pathological retinal modeling.

[0152] Recently, inhibitors against protein tyrosine phosphatase 1B (PTP1B), a ubiquitously expressed phosphatase and a known negative regulator of the leptin and insulin signaling pathways, are in clinical trials for the treatment of type 2 diabetes. Elevated PTP1B phosphatase activity is involved in several disease processes, including DR. Most of these small molecules are pharmacological inhibitors and therefore suffer from side effects and also lack the specificity absolutely essential to target PTP1B. Therefore, there is a strong need for new inhibitors of PTP1B.

[0153] The present inventors have discovered a protein (biological inhibitor) that directly binds to PTP1B and inhibits its abnormal activity in DR, and have demonstrated the efficacy of the drug in a specific rat model of the disease (Nuc1).

[0154] The Nuc1 rat model is characterized by a spontaneous mutation in the Cryba1 gene, which encodes the βA3 / A1-crystallin protein. Heterozygotes exhibit nuclear cataracts, while homozygotes exhibit microphthalmia. The Nuc1 rat model exhibits symptoms of persistent fetal vasculature (PFV) disease of the eye. The Nuc1 rat model of DR is unique because it is the only animal model that exhibits microaneurysms, a hallmark of DR in human patients, as the disease progresses (see Figures 11 and 12).

[0155] In a human proteome high-throughput array (CDI Laboratories, Inc.), βA3 / A1-crystallin was determined to interact with PTP1B, an enzyme that is a link between metabolism and inflammation. In a pull-down assay, βA3 / A1-crystallin bound to PTP1B. βA3 / A1-crystallin, two closely related polypeptides produced by two different translation start sites with important regulatory functions in astrocytes, regulate PTP1B activity. βA3 / A1-crystallin was determined to be an uncompetitive inhibitor of PTP1B (see FIG. 13). Co-immunoprecipitation assays demonstrated that PTP1B was a binding partner of βA3 / A1-crystallin protein in wild-type astrocytes, but this binding was lost in βA3 / A1-crystallin mutant Nuc1 cells.

[0156] The inventors hypothesized that high glucose levels in diabetes induce glial abnormalities, particularly in astrocytes, that affect retinal homeostasis.

[0157] Loss of βA3 / A1-crystallin protein in the Nuc1 rat model indicated elevated PTP1B activity in astrocytes, which was significantly upregulated when astrocytes from Nuc1 rats were treated with high glucose (HG) (see FIG. 14A). Elevated levels of PTP1B were also observed in the vitreous humor of human DR patients (see FIG. 14B). Lower levels of crystallins compared to PTP1B were observed in samples from the vitreous humor of human patients with progressive DR (PDR), but not in normal samples (controls) (FIGS. 14C-14D).

[0158] A βA1-crystallin construct for gene therapy was prepared according to Figures 15A-15C. When βA1-crystallin was overexpressed in astrocytes from Nuc1 rats following administration of the βA1-crystallin vector, PTP1B activity was rescued to normal wild-type levels (see Figure 16).

[0159] These results support homology modeling data that binding of the open conformer of βA1 to PTP1B blocks substrate access to the active site, thereby inhibiting the tyrosine phosphatase activity of PTP1B, which is required to maintain homeostasis of PTP1B activity in astrocytes. In addition, administration of βA1-crystallin vector rescued the DR phenotype in Nuc1 rats (see Figure 17).

[0160] These observations prompted us to use the seahorse XF platform to delineate the extracellular flux of astrocytes by selectively blocking the glycolysis and OxPhos (oxidative phosphorylation) pathways with 2-deoxyglucose and rotenone / antimycin, respectively. Compared to wild-type cells exposed to high glucose, we observed an increase in extracellular acidification rate (ECAR - an indirect measure of glycolysis) and a decrease in mitochondrial respiration (oxygen consumption rate or OCR) in Nuc1 astrocytes treated with high glucose (see Figures 18A-18B). Overexpression of βA1-crystallin in Nuc1 astrocytes reduced ECAR and increased OCR to wild-type levels in Nuc1 astrocytes (see Figures 18A-18B). Furthermore, inhibiting the expression of PTPB1 by shRNA reduced ECAR and increased OCR in Nuc1 astrocytes.

[0161] In accordance with the increase in ECAR levels in high glucose-treated Nuc1 astrocytes, we also observed a significant increase in lactate levels in these cells, indicative of enhanced glycolytic flux (see FIG. 19A). This was rescued upon overexpression of βA1-crystallin (see FIG. 19A) or PTP1B knockdown. Taken together, the data confirm that the interaction of PTP1B with βA1-crystallin is essential to maintain normal glycolysis and oxidative phosphorylation in astrocytes.

[0162] It is known that OxPhos is key to the normal neuroprotective function exerted by astrocytes, and that reduced OxPhos results in mitochondria not producing enough ATP, leading to the generation of excess mitochondrial ROS (mROS). Nuc1 astrocytes exposed to high glucose showed a significant increase in mROS compared to WT cells (see FIG. 19B), which could be rescued by either βA1-crystallin overexpression (see FIG. 19B) or PTP1B knockdown.

[0163] In multifactorial diseases like DR, a group of patients with high glucose levels in diabetes may operate an abnormal stoichiometry between PTP1B and βA1-crystallin in astrocyte abnormalities that may link metabolic changes in the retina to immune dysregulation, possibly leading to the breakdown of the blood-retinal barrier (BRB) with the consequent infiltration of immune cells and leading to disease severity. Such can be efficiently blocked using the open conformer of βA1-crystallin, which shows an inhibitory effect on the tyrosine phosphatase activity of PTP1B.

[0164] Using a gene therapy approach to overexpress βA1-crystallin in the Nuc1 rat model to rescue the DR-like phenotype and electroretinogram (ERG), it was observed that gene therapy for βA1-crystallin could successfully improve retinal function. Thus, βA1-crystallin is the first of its kind inhibitor that can be used as a treatment for DR.

[0165] All publications, including publications, patent applications, and patents, cited in this specification are herein incorporated by reference to the same extent as if each individual publication was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0166] In the context of describing the invention (particularly in the context of the claims which follow), use of the terms "a" and "an" and "the" and "at least one" and similar referents should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item (A or B) selected from the listed items or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise indicated herein. The recitation of ranges of values ​​herein is intended to be used merely as a shorthand method of individually indicating each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the invention and does not impose limitations on the scope of the invention unless otherwise stated in the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0167] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments will become apparent to those of ordinary skill in the art upon reading the above description. The inventors expect that such variations will be employed by those of ordinary skill in the art as appropriate, and the inventors intend that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. A method of inhibiting neutrophil activation in retinal pigment epithelial (RPE) cells, comprising administering an inhibitor of interferon (IFN) lambda to a subject in need thereof.

2. 2. The method of claim 1, wherein the inhibitor is selected from the group consisting of a neutralizing anti-IFN lambda antibody, an antisense oligonucleotide, a small interfering ribonucleic acid (siRNA), a small hairpin RNA (shRNA), a non-antibody binding polypeptide, or a small chemical compound.

3. The method of claim 2, wherein the antibody is selected from the group consisting of an immunoglobulin, an antibody fragment, and an antibody analog.

4. The method of claim 3, wherein the antibody is an antibody fragment selected from the group consisting of Fab, Fab', F(ab')2, F(ab'), F(ab), Fv and scFv.

5. 4. The method of claim 3, wherein the antibody is an antibody analog selected from the group consisting of an affibody, a repebody, an affilin, a DARPin, a tetranectin, a microbody, a peptide aptamer, and an avimer.

6. The method of claim 2, wherein the shRNA targets one or more of the nucleotide sequences consisting of the group consisting of SEQ ID NOs: 11-17.

7. The method of claim 6, wherein the shRNA is contained in a vector.

8. The method of claim 7, wherein the vector is an adeno-associated (AAV) vector.

9. The method of claim 8, wherein the AAV vector is AAV2 or AAV8.

10. The method of claim 1 , wherein the inhibitor is administered by subretinal injection, intravitreal injection, or topical administration.

11. A method for restoring lysosomal function of RPE cells, comprising the step of administering to a subject in need thereof a polypeptide comprising βA1-crystallin or a nucleic acid encoding said polypeptide.

12. The method of claim 11, wherein the nucleic acid further comprises an hVMD2 promoter.

13. The method of claim 11 , wherein the nucleic acid is contained in a vector.

14. The method of claim 13, wherein the vector is an adeno-associated virus (AAV) vector.

15. The method of claim 14, wherein the AAV vector is AAV2 or AAV8.

16. The method of claim 11, wherein the polypeptide or a nucleic acid encoding the polypeptide is administered by subretinal injection, intravitreal injection or topical administration.

17. Vacuolar type H in RPE cells + The present invention relates to a method for rejuvenating V-ATPase (V-ATPase) activity in a subject in need thereof by regulating the assembly and disassembly of V-ATPase.

18. 10. The method of claim 1, wherein the subject has age-related macular degeneration or is at risk of developing AMD.

19. 19. The method of claim 18, wherein the AMD is dry AMD, and optionally the dry AMD includes geographic atrophy.

20. 19. The method of claim 18, wherein the AMD is wet AMD.

21. 2. The method of claim 1, wherein the subject has or is at risk of developing a neurodegenerative disease.

22. 22. The method of claim 21, wherein the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

23. 2. The method of claim 1, wherein the subject has diabetic retinopathy (DR) or is at risk of developing DR.

24. A method of treating diabetic retinopathy in a subject comprising administering to the subject a polypeptide comprising βA1-crystallin or a nucleic acid encoding the polypeptide.

25. 25. The method of claim 24, wherein the nucleic acid further comprises an hVMD2 promoter.

26. The method of claim 24, wherein the nucleic acid is contained in a vector.

27. 27. The method of claim 26, wherein the vector is an adeno-associated virus (AAV) vector.

28. The method of claim 27, wherein the AAV vector is AAV2 or AAV8.

29. 25. The method of claim 24, wherein the polypeptide or a nucleic acid encoding the polypeptide is administered by subretinal injection, intravitreal injection or topical administration.

30. The method of claim 1, wherein the subject is a human.

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