Methods for treating age-related macular degeneration

Abstract

Methods for treating age-related macular degeneration (AMD) or reducing the risk of developing it in subjects are disclosed. These methods may include selecting subjects who have AMD or are at risk of developing it, and administering to the subjects an effective amount of a drug that inhibits HERV-K. Drugs that inhibit HERV-K include, but are not limited to, antibodies, inhibitory RNA molecules, antiretroviral agents, HERV-K-targeting CRISPR / Cas13 systems, and angiogenins. HERV-K-targeting CRISPR / Cas13 systems are also provided. These methods may also include selecting subjects who have AMD or are at risk of developing it, and administering to the subjects an effective amount of a drug that increases ANG activity.

Description

[Technical Field]

[0001] Cross-references to related applications This asserts the benefit pursuant to U.S. Provisional Application No. 63 / 453,006, filed on 17 March 2023, which is incorporated herein by reference in its entirety.

[0002] Areas of disclosure The present invention relates to the inhibition of human endogenous retrovirus K (HERV-K) and / or the increase of angiogenin (ANG) activity for the treatment of retinal diseases, specifically age-related macular degeneration (AMD).

[0003] Sequence List The sequence listing was submitted as an XML file (88,776 bytes) in the form of a file named "Sequence.xml" created on March 14, 2024, and is incorporated herein by reference. [Background technology]

[0004] background Age-related macular degeneration (AMD) is the leading cause of blindness worldwide (as outlined in Lim et al., The Lancet 379 (9827): 1728-1738, 2012 and Mitchell et al., The Lancet 392 (10153): 1147-1159, 2018). As many countries experience aging populations, more than 20% of the world's population may have AMD. Progressive AMD, including neovascular age-related macular degeneration (exudative) and geographic atrophy (late-onset and atrophic), is associated with substantial progressive visual impairment. Major risk factors include smoking, nutritional factors, cardiovascular disease, and genetically identifiable traits, including genes that regulate complement, lipid, angiogenesis, and extracellular matrix pathways. More than 50 susceptibility loci have been identified, including the CFH and ARMS2 genes.

[0005] Accurate diagnosis of AMD involves both clinical and visual examinations, including retinal photography, angiography, and optical coherence tomography. Dietary supplementation with antioxidants has been shown to slow disease progression but not eliminate it. Current treatment for neovascular age-related macular degeneration includes intravitreal injections of anti-VEGF agents. Evidence suggests that three commonly used anti-VEGF therapies—ranibizumab, aflibercept, and bevacizumab—have similar efficacy. However, there remains a need for additional medications that can be used to treat AMD and reduce the risk of developing AMD. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lim et al., The Lancet 379 (9827): 1728-1738, 2012 [Non-Patent Document 2] Mitchell et al., The Lancet 392 (10153): 1147-1159, 2018 [Overview of the project] [Means for solving the problem]

[0007] Summary of Disclosure Methods for treating age-related macular degeneration (AMD) or reducing the risk of developing it in subjects are disclosed. In some embodiments, these methods include selecting subjects who have or are at risk of developing AMD; and administering to the subjects an effective amount of a HERV-K inhibitor, e.g., administration to the eyes. HERV-K inhibitors can reduce a) the amount of human endogenous retrovirus (HERV)-K; b) the production of mRNA encoding the HERV-K protein; c) the translation of HERV-K mRNA; d) the amount of HERV-K protein in the subject; e) the inhibitory activity of HERV-K mRNA and / or protein, or any combination of a) to e). Exemplary agents, but not limited to, include antibodies, inhibitory RNA molecules, inhibitory peptides, antiretroviral agents, and CRISPR / Cas13 systems targeting HERV-K. In some embodiments, the HERV-K inhibitor is angiogenin, or an agent that increases angiogenin expression and / or activity (e.g., a viral vector expressing ANG). In some embodiments, angiogenin expression and / or activity are increased by at least 50%, at least 100%, at least 200%, at least 500%, or more in the eyes, e.g., in RPE cells of the eye.

[0008] CRISPR / Cas13 systems and ribonucleoprotein (RNP) complexes for targeting HERV-K are also provided, as well as isolated cells containing them.

[0009] In a further embodiment, a method is provided for treating age-related macular degeneration (AMD) in a subject or reducing the risk of developing it, comprising: selecting a subject who has AMD or is at risk of developing it; and administering to the subject an effective amount of a drug that increases angiogenin (ANG) activity.

[0010] The aforementioned and other features of this disclosure will become more apparent from the following detailed description of several embodiments, which follows references to the attached drawings. [Brief explanation of the drawing]

[0011] [Figure 1A] Figure 1A is a series of digital images showing induced pluripotent stem (iPSC)-derived retinal pigment epithelium (iRPE) cells treated with complement-incompetent human serum (CIHS, left image) or complement-competent human serum (CCHS, right image). CCHS induces the formation of sub-RPE deposition (drusen), as indicated by the changes in APOE immunostaining from the apical membrane (lateral view) to the basilar membrane of the iRPE. TW = Transwell membrane, on which iRPE cells grow. [Figure 1B] Figure 1B is a graph showing the transepithelial resistance (TER) of the iRPE monolayer after CIHS (gray bars) and CCHS (white bars) treatments. [Figure 2] Figure 2 is a schematic diagram of the genome structure of an endogenous retrovirus: a retrovirus containing two long-terminal repeat sequences (5' and 3' LTRs) flanking the internal coding sequences of three basic retroviral genes (Gag, Pol, and Env). GAG = glycosaminoglycan, POL = polymerase, ENV = envelope. [Figure 3A]Figures 3A and 3B are graphs showing RNA-seq analysis of CC-HS-treated iRPE transcriptome-related genomic loci exhibiting differentially elevated HERV-K mRNA. A) Bioinformatics analysis of RNA-seq data identified differentially expressed HERV-K in human serum-treated iPSC-RPE. 5% CCHS was added to both apical and basal media of iRPE grown on Transwell for 48 hours, and HERV-K RNA levels from LTRs and ORFs containing HERV-K loci (approximately 80), shown as adjusted depth values ​​(signal reads), were normalized to transcript length. B) Adjusted depth values ​​of the top 16 differentially expressed HERV-K loci in the human genome in CCHS-treated samples were compared to a control in complement-incompetent human serum (5% CIHS). Each graph shows the precise location of the endogenous retroviral locus. Chromosome number is the first number, followed by the position in base pairs. The location's genome ID is shown in parentheses. [Figure 3B] Same as above. [Figure 4A] Figures 4A and 4B are graphs showing mRNA of HERV-K components in iRPE that were specifically elevated with persistently low levels of CCHS. A) Representative image of RNA seq data, as seen by Integrative Genomics Viewer, showing aligned reads of differentially expressed HERV-K loci. Only HERV-K reads, and not reads for HERV-P, were increased by CCHS. B) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) quantification of HERV-K components GAG, ENV, and POL in 0.1% CCHS-treated iRPE shows a specific increase in HERV-K ENV and GAG, and not HERV R, in cultured iRPE. CCHS (5%) was added to fully differentiated monolayers of iRPE and left for 2, 4, and 6 days. Data are calculated as double change (CCHS / CHIS). Mean ± SEM. N=3, p<0.05 is considered statistically significant. The symbols indicate individual iRPE donors. The stars indicate statistically significant data. [Figure 4B] Same as above. [Figure 5] Figure 5 shows a digital image of iPRE cells. Sustained low levels of complement-competent human serum (CCHS) increase HERV-K ENV levels in iRPE immunofluorescence detection, showing increased HERV-K ENV protein levels in CCHS (0.1%)-treated iRPE cells 4 and 6 days after treatment. Confocal laser microscopy image of IF signal (HERV-K, phalloidin, and DAPI are shown), scale bar 20 μM. [Figure 6] Figure 6 shows digital images of the localization of HERV-K expression in iRPE cells using RNAscope. Increased HERV-K (ENV) mRNA was detected in both CCHS-treated iRPE cells and the eyes of AMD patients. Top left: The detection procedure is validated by including PPIB (peptidyl prolyl isomerase B) mRNA probe as a positive control. Under the same conditions, HERV-K was specifically detected in CCHS-treated iRPE cells 48 hours after exposure (bottom left), in addition to cells treated with CIHS (center left). Cross-sections of eyes obtained from a human AMD patient (90 years old, right panel) were subjected to HERV-K RNA detection using RNASCOPE®. The data show that HERV-K RNA is present in the RPE and choroid of human patients but not in age-matched control eyes. [Figure 7]Figures 7A-7B are graphs showing the reduction in CCHS-induced secretion of pro-inflammatory cytokines upon treatment with antiviral drugs or TLR inhibitors. iRPEs grown on Transwell were pre-treated overnight with an antiviral drug (tenofovir, 20 μM), a TLR3 / dsRNA complex inhibitor (10 μM), or the TLR4 inhibitor TAK-242 (5 μM), followed by incubation with CIHS (5%) or CCHS (5%) for 48 hours. Apical and basal media were collected and analyzed by Multiplex Luminex assay to detect IL-6, IL-8, IL-18, IL-1β, and IFNβ. Levels of IL-18, IL-1β, and IFNβ were below the detection range. The CCHS-induced increases in (A) IL-8 and (B) IL-6 were reduced by these inhibitors in two biological replicates. The results indicate that antiviral therapy as a preventive measure for atrophic AMD, and combination therapy with anti-VEGF for exudative AMD, can be used in pill form and topical eye drops. [Figure 8A] Figures 8A and 8B show lipid-staining iRPE treated with CCHS and / or TLR inhibitors. TLR inhibitors reduce CCHS-induced lipid accumulation. A) iRPE was pretreated with a TLR3 or TLR4 inhibitor and CCHS. iRPE was stained with BODIPY® fluorescent dye (stains lipids accumulating under RPE) (10 μM) or DAPI (stains DNA), and images were captured using a laser-scanning confocal microscope (40x). B) Quantification of fluorescence signals. Data are representative of two biological replicates. [Figure 8B] Same as above. [Figure 9]Figures 9A-9C show the preparation of lentiviral constructs expressing HERV-K ENV. A) Schematic diagram illustrates the HERV-K ENV expression cassette of the lentiviral construct. B) Detection of HERV-K ENV protein in IPSCs. AMDCD cells were transduced with the HERV-K ENV lentiviral construct (MOI 1). After 48 hours, total protein lysates were analyzed for HERV-K ENV by Western blotting using GAPDH as a loading control. The data show an increase in HERV-K ENV protein in overexpressing cells. C) Live imaging of the mcherry fluorescence signal shows that the amount of transduced lentivirus is equivalent to the level of protein expression in fully differentiated iRPE 14 days after transduction. iRPE was transduced for 16 hours with lentivirus (MOI - multiplicity of infection - 0.5 or 3) in 5% RPE medium supplemented with 5 ug / ml polybron. The culture medium was replaced with fresh medium, and incubation continued for another 14 days. The medium was refreshed every other day. [Figure 10A]Figures 10A–10C show that HERV-K ENV is sufficient to induce RPE degeneration similar to the AMD phenotype. HERV-K-ENV expressed via lentiviral transduction reduces TER and induces lipid accumulation in iRPE. A) Transepithelial resistance (TER), measured using EVOM, was reduced by HERV-K ENV overexpression. B) Images of immunostaining of HERV-K ENV (upper panel) and staining with Alexa 488 conjugate BODIPY® fluorescent dye (lower panel) were acquired under a laser scanning confocal microscope (LSM980) using a 40x objective lens. C) Quantification of HERV-KENV (upper graph) and BODIPY® fluorescent dye (lower graph) in iRPE overexpressing HERV-K. Four images were captured for each sample. Signals were quantified using Image J according to an established method and normalized to the number of cells shown. The data are the mean ± SEM signal count per cell from three biological replicates. ****P<0.0001, ***P<0.005, **p<0.05. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 11A]Figures 11A-11C are graphs showing that CCHS progressively reduces mRNA levels and induces angiogenin (ANG) secretion in iRPE cells. A) CCHS (5%) was added to a fully differentiated monolayer of iRPE cells and left for 48 hours. Total RNA was subjected to RNA-seq analysis. The data show that CCHS specifically reduced ANG mRNA levels, while it did not reduce those of Dicer cells. Data are calculated as a twofold change (CCHS / CHIS). Mean ± SEM. N=3, p<0.05 is considered statistically significant. The symbols indicate iRPE donor cells. B) ANG mRNA levels progressively decreased with CCHS treatment. CCHS (0.1%) was added to both apical and basal media of iRPE cells and refreshed daily for 2, 4, and 6 days. Total RNA was analyzed for ANG and Dicer expression by qRT-PCR using CIHS-treated cells as a control. N=3, p<0.05. C) CCHS (0.1%) reduced ANG secretion into iRPE medium. CCHS or CIHS was added to iRPE medium as described in B). Apical and basal media were collected separately at days 2, 4, and 6. The amount of ANG was quantified by the QUANTIKINE® ELISA kit. Data are mean ± SEM of secretory activity. N=3, p<0.05 is considered statistically significant. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 12] Figure 12 is a digital image showing that CCHS progressively reduces intracellular angiogenin (ANG) in iRPE. CCHS (0.1%) reduced intracellular ANG in iRPE. IPRE was treated with CCHS or CIHS (5%) for 48 hours. Total protein lysates were analyzed for expressed ANG by Western blotting (top). In a parallel experiment, cells were fixed in 4% PFA for 20 minutes and stained with an antibody against human ANG. ANG (bottom) expression was visualized by immunofluorescence (ZO-1) on cells and by DAPI on the nuclei. Scale bar: 20 μM. [Figure 13A] Figures 13A-13B are graphs showing that CCHS reduces HERV-K-specific ANG target-transfer RNA fragments (tRFs) in iRPE. A) CCHS specifically reduced tRFs that regulate HERV-K levels in iRPE. CCHS (CIHS as control) was added to fully differentiated iRPE and left for 3, 24, and 48 hours. Total RNA was extracted and subjected to adapter-mediated tRF RT-qPCR to detect either tRFs that specifically bind to HERV-K (3”tiR088(LysCTT(n)), 3006B--LysTTT, tRF-3 3002A pro-AGG, or tRF-3 3016 / 18 / 22B LysCTT(n), as shown in (B), or tRFs that do not bind (3009B Leu TAA). [Figure 13B] Same as above. [Figure 14] Figures 14A–14B show a gene therapy strategy (A) for reducing HERV-K levels in CC-HS treated iRPE cells. HERV-K is generated from multiple loci in the genome, and these loci have slight sequence variations. Therefore, to develop gene therapy for HERV-K, RNA generated from most of these different loci of HERV-K is targeted. Cas13 is a nuclease that can target RNA using complementary guide sequences. Using bioinformatics analysis, three guide sequences (SEQ ID NOs. 1–3) (B) were identified in HERV-K used to target HERV-K expression in RPE cells. These sequences were designed against the GAG ​​region of HERV-K mRNA, as it is the RNA start. Degradation of the RNA start leads to degradation of the rest of the RNA. [Figure 15A]The sequences showed complementarity across multiple HERV-K loci and also demonstrated good consensus for CAS13Rx-based targeting. The genomic distributions of the three guides used are shown in the table. Sequence IDs 1–3 are shown in bold and underlined. Consensus sequences for gRNA1 (sequence ID 18), gRNA2 (sequence IDs 34 and 51), and gRNA3 (sequence ID 59) are shown. Various alignments to the genome are shown for gRNA1 (sequence IDs 19–33), gRNA2 (sequence IDs 35–50 and 52–58), and gRNA3 (sequence IDs 60–75). [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 16] Figures 16A–16B show the induction of Cas13Rx expression using doxycycline. (A) is a Western blot showing HA-tagged Cas13Rx overexpressed using a lentiviral construct. High expression of HA-tagged Cas13Rx is observed at an infection multiplicity (MOI) of 1.0. (B) is a digital image showing HA-tagged-Cas13Rx enzyme expression in iRPE cells with a lentiviral vector at an MOI of 1.0. [Figure 17A-B]Reverse complement-induced lipid accumulation by Cas13Rx-mediated HERV K knockdown. (A) iRPE cells were transduced with a lentiviral expressing HA-tagged CAS13Rx under the control of a doxycycline-inducible promoter to overexpress Cas13Rx. Seven days of doxycycline treatment induced CAS13Rx expression (Figures 16A-16B). At this stage, iRPE cells were transduced with another lentiviral construct expressing one of three guide RNAs alone or together, and a control guide RNA. After a further seven days, allowing high levels of guide RNA expression, the cells were treated with CC-HS. Four days after CC-HS or CI-HS treatment, HERV-K levels were measured in cells and monolayer TER, and BODIPY® levels were measured in cells. (B) shows TER graphs for CIHS (gray bars) and CCHS (black bars) treated samples transduced with either scrambled guide RNA (NEG) or guide RNA (Cas13Rx) or guide RNA #2 (Figure 15). Only guide RNA #2 is able to rescue TER downregulated by CCHS treatment. (C) Upper panel: Digital images of CIHS or CCHS treated iRPE transduced with either scrambled control (NEG) or guide RNA #2. Increased HERV-K expression induced by CCHS (comparing CIHS NEG samples and CCHS NEG samples) is downregulated by guide RNA 2 for HERV-K. Lower panel: Digital images of CIHS or CCHS treated iRPE transduced with either scrambled control (NEG) or guide RNA #2. Increased lipid deposition in CCHS induced by CCHS (measured as a BODIPY® signal) is downregulated by guide RNA 2 for HERV-K. (D) The quantification of HERV-K levels seen in the upper panel of Figure 17C indicates that guide RNA #2 can downregulate HERV-K levels in CCHS-treated iRPE cells.(E) The quantification of BODIPY® levels seen in the lower panel of Figure 17C indicates that guide RNA #2 can downregulate BODIPY® in CCHS-treated iRPE cells. [Figure 17C] Same as above. [Figure 17D-E] Same as above. [Figure 18] Figure 15 shows reverse complement-induced lipid accumulation of Cas13Rx-mediated HERV K knockdown by all three selected guides. Digital images of CCHS-treated AMD patient 1-iRPE cells transduced with scrambled control (NEG) or with any of the three guide RNAs #1, 2, or 3 individually or in combination. CCHS induced increased lipid deposition (measured as a BODIPY® signal). The BODIPY® signal is downregulated by all three guides to HERV-K when transduced in CCHS-treated cells. The graph on the left shows quantification of digital imaging data. [Figure 19] Figure 15 shows reverse complement-induced lipid accumulation of Cas13Rx-mediated HERV K knockdown by selected guides (examples shown for two guides). Digital images of CCHS-treated AMD patient 2-iRPE cells transduced with either a scrambled control (NEG) or one of the two guide RNAs #1, 2. CCHS induced increased lipid deposition (measured as a BODIPY® signal). The BODIPY® signal is downregulated by the guide for HERV-K when transduced in CCHS-treated cells. CIH treatment is a negative control for the study. The graph on the left shows quantification of digital imaging data. [Figure 20] Schematic diagram of a method for testing the role of exogenously expressed angiogenins (ANG) in protecting against CCHS-induced iRPE injury. [Figure 21] A bar graph showing ANG overexpression caused by lentivirus. [Figure 22] Digital image showing that ANG prevented CCHS-induced HERVK increase. [Figure 23]Digital image showing that ANG reduced CCHS-induced lipid accumulation. [Figure 24] A bar graph showing that ANG overexpression prevents CCHS-induced TER reduction in iRPE. [Figure 25A-B] (A) The schematic diagram illustrates the domain structure of the transgene in the expression vector, where HERV-K ENV is under the RPE-specific human VMD2 promoter (also known as the best1 promoter). (B) qPCR data from genotyping of 6 HERV-K ENV-established mice. (C) qRT-PCR identified two established mouse strains—established strain 1 and established strain 6—showing significantly higher expression of HERV-K ENV. These established mouse strains (1 & 6) were backcrossed with C57BL / 6J to obtain a pure C57BL / 6 background. In induced pluripotent stem cell-derived RPEs (iPSC-RPEs), complement-competent human serum (CC-HS) increased HERV-K ENV, mimicking pathogenic changes during early AMD, consistent with increased lipid accumulation, cytokine release, and loss of epithelial cell morphology (Sharma R, et.al., Nat Commun. 2021 Dec 15;12(1):7293. doi: 10.1038 / s41467-021-27488-x.PMID: 34911940, 36550275). [Figure 25C] Same as above. [Figure 26A-B]HERV-K ENV overexpression induces lipid accumulation in transgenic RPE. (A-B) Immunostaining images show higher HERV-K ENV expression in flat mounts of RPE isolated from the eyes of 1-2 month old transgenic mice (A), and quantification of the HERV-K ENV signal is shown in (B). (C-D) Immunostaining images show higher HERV-K ENV expression in flat mounts of RPE isolated from the eyes of 7-8 month old transgenic mice (C), and corresponding quantification data is shown in (D). (E-F) Immunostaining images show higher lipid droplet staining with BODIPY® in 1-2 month old HERV-K ENV overexpressing mice (E), and corresponding quantification data shows significantly higher lipid droplet signaling in homozygous HERV-K ENV mice (F). (G~H) Immunostaining images show higher lipid droplet staining with BODIPY® in 7-8 month old HERV-K ENV overexpressing mice (G), and corresponding quantified data show significantly higher lipid droplet levels in homozygous HERV-K ENV mice (H). Four images per sample were captured under laser scanning according to an established method and normalized to the size of the imaging area being displayed. Data are the ratio of raw counts obtained from transgenic RPE compared to wild-type RPE for n>3 biological replicates. **P<0.005; *p<0.05. [Figure 26C-D] Same as above. [Figure 26E-F] Same as above. [Figure 26G-H] Same as above. [Figure 27] Lipid droplets co-localize with the HERV-K ENV protein in the RPE of transgenic mice. (A) The front view of the image shows the localization of HERV-K ENV (magenta) and lipid droplets (stained with BODIPY®, green) in 7-8 month old transgenic mice compared to wild-type mice. (B) The 3D rendering of panel A shows the subcellular localization of HERV-K ENV and lipid droplets. [Figure 28]Appearance of hyperfluorescence spots in eyes overexpressing HERV-K ENV. (A) Autofluorescence (AF) images of the eyes of 7-8 month old wild-type + / + (WT, left) and heterozygous HERV-K ENV tg / + (center) or homozygous HERV-K ENV tg / tg transgenic mice (right) show increased autofluorescence deposition (arrows) in transgenic mice compared to WT and heterozygotes. (B) Light microscopy images of H&E-stained 5 μm tissue sections of WT, heterozygous and homozygous HERV-K ENV mice. Labeling of retinal anatomical layers including ganglion cell layer (GCL); internal plexiform layer (IPL); internal granular layer (INL); external plexiform layer (OPL); external granular layer (ONL); internal / external segment layer (IS / OS) and nearby RPE and choroid. Blue arrows mark abnormal cells in the SRS within the misaligned external segment. [Figure 29] Appearance of subretinal and RPE intracellular vacuoles in eyes overexpressing HERV-K ENV. (A-D) Transmission electron (TEM) microscopy images of the photoreceptor-RPE-choroid complex in 7-8 month wild-type + / + (A) and heterozygous HERV-K ENV tg / + (B) or homozygous HERV-K ENV tg / tg transgenic mice (C, D) show lipid aggregation (white arrows, B), abnormal subretinal cells (horizontal black arrows, C), vacuoles (white arrows, C), and disrupted tight junctions (black arrowheads, D) in the eyes of transgenic mice. [Figure 30A]Combinations of antiviral drugs showed a greater effect in blocking the lipid accumulation effect induced by CCHS. A) Flowchart of the experimental process and assay. Mature iPSC-RPEs grown in 96 cell cultures were treated in three replicates with single or combination drugs for 24 hours before the addition of complement-competent human serum (CC-HS) (0.1%) and complement-incompetent human serum (CI-HS) (0.1%) as a control, and untreated cells were also included for baseline comparison. B) The figure outlines the application format of single and combination drug treatments, as well as a plate map of the applications. The drugs tested were metformin hydrochloride (3 mM); darunavir (50 μM); L-745,870 trihydrochloride (6 μM, a very potent and selective D4 dopamine receptor blocker); and tenofovir (10 μM, a nucleoside reverse transcriptase inhibitor (NRTI)). C) Combinations with antiviral drugs showed a higher rescue effect against CC-HS-induced lipid accumulation than single drugs alone. Mature iRPE cells grown in 96-cell cultures were treated in three replicates with single drugs or combinations 24 hours before the addition of CC-HS and CI-HS. Cells were fixed and labeled with Alexa 488 conjugate BODIPY® fluorescent dye (10 μM) and ZO-1 monoclonal antibody Alexa FLUOR® 594 (1:200), and then imaged using an ANDOR confocal scanning microscope (Oxford system, BC43). Four images were randomly captured for each sample. Images were exported from their respective channels. The BODIPY® signal was quantified using Image J according to an established method and normalized to the size of the imaging area being displayed. Raw counts of lipid deposition were obtained from 3 replicates per sample group, with 4 images per replicate. ****P<0.0001; ***p<0.0005. Data were analyzed and plotted using PRISM. D) Boundary images of ZO-1 labeled cells were subjected to REShAPE analysis to evaluate cell area, aspect ratio, and hexagonality. Data were analyzed and plotted using the R program. E-F) Combination with antiretroviral drugs inhibits CC-HS-induced IL-8 and IL-6 release.100 μL of medium was collected from each well of a 96-well cell culture plate and diluted 50-fold before performing a Luminex-based multiplex human assay. The amounts of IL-8 and IL-6 in the medium were analyzed using a Luminex-based multiplex human cytokine assay protocol. The cytokine concentration values ​​for three replicates of each sample are shown. [Figure 30B] Same as above. [Figure 30C] Same as above. [Figure 30D] Same as above. [Figure 30E-F] Same as above. [Figure 31] Application of AAV8-shRNA HERV-K ENV as a therapeutic agent for treating AMD in in vitro and in vivo studies. A) A schematic diagram illustrates the workflow of an in vitro experiment using iRPE to test the effect of AAV8-shRNA HERV-K ENV in rescuing cellular defects caused by anaphylatoxin (CC-HS). Either a naive iRPE or an iRPE overexpressing HERV-K ENV is transduced at a MOI of 10⁴–10⁵ with AAV HERV-K ENV or AAV control, four days before the addition of CC-HS (0.1%) or control CIHS (0.1%). The medium containing anaphylatoxin is refreshed daily for three days. Samples are collected on day four and the corresponding assay is performed to detect the rescue effect. B) A schematic diagram of the procedure for determining the effect of AAV-shRNA HERV-K ENV in reversing RPE degeneration caused by HERV-K ENV. [Modes for carrying out the invention]

[0012] array The nucleic acid and amino acid sequences listed in the attached sequence listing are shown using standard letter abbreviations for nucleotide bases and single-letter codes for amino acids, as defined in U.S. Patent Rule 1.822. Only one strand of each nucleic acid sequence is shown, but it is understood that complementary strands are included by any reference to the shown strand. Sequence IDs 1-3 are exemplary nucleic acid sequences of gRNA. Sequence ID 4 is an exemplary nucleic acid sequence of HERV-K. Sequence ID 5 is an exemplary nucleic acid sequence encoding Cas13Rx. Sequence ID 6 is an exemplary HERV-K Gag amino acid sequence (GENBANK® accession number AAL16779.1). Sequence ID 7 is an exemplary HERV-K Gag amino acid sequence (GENBANK® accession number AAL60056.1). Sequence ID 8 is an exemplary HERV-K Env amino acid sequence (GENBANK® accession number AAL16780.1). Sequence ID 9 is an exemplary Cas13Rx protein (without signal peptide) (GENBANK® accession number QMT62609.1). Sequence ID 10 is an exemplary nucleic acid sequence encoding Cas13Rx (GENBANK® accession number MN934322.1). Sequence ID 11 is an exemplary amino acid sequence of human ANG. Sequence IDs 12 and 13 are exemplary genomic DNA corresponding to the tRNA-CTT and tRNA-TTT of the tRNA fragments. Sequence ID 14 is an exemplary nucleic acid sequence encoding human ANG. Sequence IDs 15-16 are the nucleic acid sequences of the primers. Sequence ID 17 is the nucleic acid sequence of the reporter. Sequence ID 18 is the consensus nucleic acid sequence for gRNA1. Sequence IDs 19-33 are nucleic acid alignment sequences for gRNA1. Sequence IDs 34 and 51 are the consensus nucleic acid sequences for gRNA2. Sequence IDs 35-50 and 52-58 are nucleic acid alignment sequences for gRNA2. Sequence ID 59 is the consensus nucleic acid sequence for gRNA3. Sequence IDs 60-75 are nucleic acid alignment sequences for gRNA3.

[0013] Detailed description of several aspects Human endogenous retroviruses (HERVs) and elements containing long-terminal repeat-like sequences constitute up to 8% of the human genome. See U.S. Published Patent Application 2008 / 0019979A1. There are more than 200 distinct groups and subgroups of HERVs. HERVs lose their infectivity due to mutations, and generally, they are primarily non-infectious retroviral remnants. However, open reading frames (ORFs) have been observed for ERV3, HERV-E 4-1, and HERV-K.

[0014] Members of the HERV-K superfamily, characterized by the presence of primer binding sites for lysine-tRNA, are the most biologically active. Only HERV-K appears to possess a complete set of open reading frames typical of replication-competent mammalian retroviruses. The K family contains a central open reading frame (cORF) and is equivalent to the HIV-1 Rev protein. HERV-K was originally identified by its homology to mouse mammary tumor virus (MMTV) and is transcriptionally active in breast cancer tissue, as well as in several human cancer tissues, including tumor cell lines such as human breast cancer cell line T47D and teratoma carcinoma cell line GH. HERV-K env mRNA is frequently expressed in human breast cancer, and HERV-E mRNA is expressed in prostate and ovarian cancer. See U.S. Published Patent Application 2008 / 0019979A1 disclosing an antibody that specifically binds to the HERV-K protein, as well as U.S. Patents 10,723,787 and 10,981,976.

[0015] It is disclosed herein that HERV-K expression is associated with the AMD phenotype in human cells in the retinal pigment epithelium (RPE). Furthermore, inhibition of HERV-K transcription and translation using inhibitory RNA molecules, inhibitory peptides, antiretroviral agents, and CRISPR / Cas13 or Cas9 can reduce the pathological phenotype of human RPE cells. In addition, agents that increase ANG activity prevented CCHS-induced HERVK increase, reduced CCHS-induced lipid accumulation, and prevented CCHS-induced TER reduction in RPE cells.

[0016] Methods for treating age-related macular degeneration (AMD) or reducing the risk of developing it in subjects are disclosed. These methods include selecting subjects who have AMD or are at risk of developing it, and administering to the subjects an effective amount of a drug that inhibits HERV-K. CRISPR / Cas13 systems and RNPs that can be used in methods for treating AMD are also provided herein. Methods for treating subjects who have AMD or are at risk of developing it are also disclosed by administering to the subjects an effective amount of a drug that increases ANG activity.

[0017] I. Terminology Unless otherwise stated, technical terms are used according to their conventional usage. Definitions of many common terms in molecular biology can be found in Krebs et al. (eds.), Lewin's genes XII, published by Jones & Bartlett Learning, 2017. Where used herein, the singular forms “a,” “an,” and “the” refer to both singular and plural unless the context indicates otherwise. For example, the term “a protein” includes one or more proteins and can be considered equivalent to the phrase “at least one protein.” Where used herein, the term “comprises” means “includes.” Unless otherwise indicated, “about” means within 5 percent. It should be further understood that any base size or amino acid size, and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for descriptive purposes unless otherwise indicated. Many methods and materials similar to or equivalent to those described herein can be used, but particularly preferred methods and materials are described below. In case of any conflict, this specification, including the definitions of terms, shall prevail. Furthermore, materials, methods, and examples are merely illustrative and not intended to be limiting. To facilitate an overview of various embodiments, the following definitions of terms are provided.

[0018] Administration: Introduction of a composition (e.g., one containing an agent that increases or decreases the transcription or translation of HERV-K) to a subject via a selected route. Administration may be localized or systemic. For example, if the route is intravenous, the composition is administered by introducing the composition into the subject's vein. Similarly, if the route is intramuscular, the composition is administered by introducing the composition into the subject's muscle. If the selected route is oral, the composition is administered by taking the composition orally. Exemplary routes of administration useful in the methods disclosed herein include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intraosseous, and intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, transvaginal, and inhalation routes. Administration may also be localized, for example, to the eye of the subject. In specific embodiments, administration is topical to the eye, for example, via eye drops. In specific embodiments, administration is intraocular, for example, subretinal, directly to the retina, on the choroid or intravitreal injection.

[0019] As used herein, the term “co-administered” (or “co-administered”) means, for example, the administration of two or more drugs within approximately two hours of each other as part of a clinical treatment regimen. In other embodiments, “co-administered” means the administration of two or more drugs within one hour of each other. In other embodiments, “co-administered” means the administration of two or more drugs within 30 minutes of each other. In other embodiments, “co-administered” means the administration of two or more drugs within 15 minutes of each other. In other embodiments, “co-administered” means the administration of two or more drugs at the same time, either as part of a single formulation or as part of multiple formulations administered by the same or different routes. A single “dose” means the co-administration of drugs at the same time.

[0020] Age-related macular degeneration (AMD): AMD is caused by damage to the macula of the retina. The onset of AMD can be asymptomatic, but it gradually worsens over time, generally resulting in blurred vision or loss of central vision in one or both eyes. Recognizing faces, driving, reading, or performing other activities of daily living may be difficult, and hallucinations may also occur. AMD typically occurs in older adults, e.g., those around 50 years of age and older. Genetic factors and smoking may play a role. Diagnosis involves a complete eye examination, and the severity can range from early, middle, and late types, where the late type may further include "atrophic" and "exudative" forms.

[0021] Atrophic AMD (also known as atrophic AMD) develops over time, in which the macular tissue thins and is destroyed. Symptoms may include visual distortion, reduced central vision in one or both eyes, a need for bright light for reading or working at close range, increased difficulty adapting to low light levels, increased blurring of printed words, decreased color intensity or brightness, and difficulty recognizing faces. Atrophic AMD is diagnosed by examining the fundus for drusen, testing for central vision defects (e.g., using an Amsler grid to identify whether straight lines in a grid appear weak, broken, or distorted, indicating the presence of atrophic AMD); fluorescein or indocyanine green angiography (to examine for abnormal vascular or retinal changes); and / or optical coherence tomography (to examine for thinning, thickening, or swelling of the retina). Currently available treatments include rehabilitation to adapt to loss of central vision (low vision rehabilitation) and implantation of telephoto lenses.

[0022] Exudative AMD (also known as progressive neovascular AMD) follows atrophic AMD and involves abnormal vascular growth and fluid accumulation in the fundus, which can lead to macular elevation and cause vision loss or visual distortion. In addition to the symptoms of atrophic AMD, symptoms of exudative AMD may include a well-defined blurred spot or blind spot in the visual field, general blurring of vision, and sudden onset and rapid worsening of symptoms. Currently available treatments include drug therapies that direct the cessation of new vascular growth, such as bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), and aflibercept (EYLEA®); photodynamic therapy; photocoagulation; and low vision rehabilitation.

[0023] Drug: Any substance or combination of substances useful to achieve a target or outcome; for example, a substance or combination of substances useful to treat AMD. Drugs may include proteins, nucleic acid molecules (e.g., gRNA), compounds, small molecules, organic compounds, inorganic compounds, or other molecules of interest. Drugs may also include therapeutic agents (e.g., antiretroviral agents), diagnostic agents, or pharmaceutical agents.

[0024] Angiogenin (ANG): A protein also known as ribonuclease 5, which is a stimulant of new blood vessel formation in vivo. ANG hydrolyzes cellular RNA, which can lead to modulation of protein synthesis levels, interacts with DNA, and causes promoter-like increases in ribosomal (r)RNA expression. ANG enhances rRNA transcription by binding to CT-rich angiogenin-binding elements. ANG has catalytic activity similar to RNase A, which preferentially binds to the 3' side of pyrimidines and follows a transphosphorylation / hydrolysis mechanism. Unlike RNase A, which lacks base specificity, ANG typically cleaves the 3' side of cytidylic acid or uridylic acid residues when the pyrimidine is followed by adenine, but is not limited to all potential cleavage sites. ANG shows preferential cleavage of single-stranded RNA as a substrate and cleaves tRNA. Although ANG has been shown to bind to DNA in vivo, it does not cleave DNA. See Sheng and Zu, Acta Biochimica et Biophysica Sinica 48: 3990-410, 2016. ANG overexpression is related to tRNA. Glu tRNA Gly tRNA Lys tRNA Val tRNA His tRNA Asp , and tRNA SeCAngiogenin selectively cleaves subsets of tRNA containing tRNAs, generating tRNA halves and 16-30 nucleotide-long tRF-5s called tiRNAs (see Zu et al. RNA 294: P16930-16941, 2019). Angiogenin-mediated tRNA cleavage occurs at their anticodon loops, a process highly regulated by tRNA modification. TiRNAs have been shown to inhibit ribosome assembly and are associated with stem cell maintenance. Some tRNA fragments are complementary to the HERV-K coding sequence and may play a role in its degradation. Lys-tRNA CTT and Lys-tRNA-TTT are complementary to HERV-K. Increased ANG activity leads to tRNA fragments complementary to the HERV-K coding sequence, thereby reducing HERV-K activity and / or expression.

[0025] Animals: Living multicellular vertebrates, including, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

[0026] Antibody: A polypeptide containing at least a light-chain or heavy-chain immunoglobulin variable region that specifically recognizes and binds to an epitope (e.g., an antigen, e.g., a HERV-K protein (e.g., Gag, Pol, or Env, or a fragment thereof). This includes intact immunoglobulins, as well as their variants and parts known in the art, e.g., Fab' fragment, F(ab)'2 fragment, single-chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv"). The scFv protein is a fusion protein in which the light-chain variable region and the heavy-chain variable region of an immunoglobulin are linked by a linker, whereas in dsFv, the chain is mutated to introduce a disulfide bond to stabilize the chain association. This term also includes genetically modified forms, e.g., chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL);Kuby, J., Immunology, 3 rd See also Ed., WH Freeman & Co., New York, 1997.

[0027] Typically, an immunoglobulin has heavy and light chains. Each heavy and light chain contains a constant region and a variable region (the regions are also known as "domains"). In combination, the heavy and light chain variable regions specifically bind to an antigen. The light and heavy chain variable regions contain "framework" regions interrupted by three hypervariable regions, also called "complementary determining regions" or "CDRs". The extent of the framework regions and CDRs is defined (see Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference herein). The Kabat database is currently maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0028] CDRs are primarily responsible for binding to an antigen epitope. The CDRs of each chain are typically designated CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and typically also specified by the chain in which a particular CDR is located. Thus, V H CDR3 is located within the variable region of the heavy chain of the antibody in which it is found, while V L CDR1 is CDR1 from the variable region of the light chain of the antibody in which it is found.

[0029] A reference to "V H " or "VH" refers to the variable region of an immunoglobulin heavy chain, including those of Fv, scFv, dsFv, or Fab. A reference to "V L " or "VL" refers to the variable region of an immunoglobulin light chain, including those of Fv, scFv, dsFv, or Fab.

[0030] A "monoclonal antibody" is an antibody produced by a single clone of a B lymphocyte or by a cell, in which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells from the fusion of myeloma cells and immunosplenic cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0031] A “humanized” immunoglobulin is an immunoglobulin containing a human framework region and one or more CDRs from non-human (e.g., mouse, rat, or synthetic) immunoglobulins. The non-human immunoglobulin providing the CDRs is referred to as the “donor,” and the human immunoglobulin providing the framework is referred to as the “acceptor.” In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. A constant region is not required to be present, but if present, it must be substantially identical to the human immunoglobulin constant region, i.e., at least about 85–90%, e.g., about 95% or higher. Thus, perhaps with the exception of the CDRs, all parts of the humanized immunoglobulin are substantially identical to the corresponding parts of the native human immunoglobulin sequence. A “humanized antibody” is an antibody containing humanized light chain and humanized heavy chain immunoglobulins. The humanized antibody binds to the same antigen as the donor antibody providing the CDRs. The acceptor framework of the humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids obtained from the donor framework. Humanized or other monoclonal antibodies may have additional conserved amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering (see, for example, U.S. Patent No. 5,585,089).

[0032] Antiretroviral agents: Agents that specifically reduce or inhibit the expression or replication activity of retroviruses (or viral elements of endogenous retroviruses in the human genome) in cells or retroviruses from infected cells. Non-limiting examples of antiretroviral agents that are drugs and can be used in the manner disclosed include fusion inhibitors (e.g., enfvirtide), entry inhibitors (e.g., maraviroc), nucleoside and nucleotide reverse transcriptase inhibitors (e.g., lamivudine, zidovudine, abacavir, tenofovir, TAF, TDF, FTC), non-nucleoside reverse transcriptase inhibitors (NNRTIs) (e.g., delavirdine, efavirenz, nevirapine, and other diarylpyrimidine (DAPY) derivatives), protease inhibitors (e.g., indinavir, ritonavir, darunavir, atazanavir), and integrase inhibitors (e.g., elvitegravir, raltegravir, dolutegravir). Antiretroviral agents useful in this method are disclosed, for example, in Tyagi et al., Inhibition of human endogenous retrovirus-K by antiretroviral drugs, Retrovirology 14: 21 (13 pages), 2017, which is incorporated herein by reference.

[0033] Antiretroviral therapy (ART): A therapeutic treatment to inhibit retroviruses, which includes administering at least one antiretroviral agent (e.g., one, two, three, or four antiretroviral agents) to an individual (e.g., one infected with a retrovirus) during the course of treatment. An example regimen is a combination of TAF, FTC, and EVG. In some cases, ART includes highly active antiretroviral therapy (HAART).

[0034] Caspases (Cas): Enzymes that are cysteine-aspartate proteases, cysteine ​​aspartases, or cysteine-dependent aspartate-targeting proteases. Caspases are a family of protease enzymes that play an essential role in programmed cell death. They are named caspases due to their specific cysteine ​​protease activity, where cysteine ​​in the active site nucleophilically attacks and cleaves only the target protein after the aspartate residue.

[0035] cDNA (complementary DNA): A piece of DNA lacking internal non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.

[0036] Cobicistat (COBI): 1,3-thiazole-5-ylmethyl(2R,5R)-(5-{[(2S)-2-[(methyl{[2-(propan-2-yl)-1,3-thiazole-4-yl]methyl}carbamoyl)amino]]-4-(morpholin-4-yl)butanamide}-1,6-diphenylhexane-2-yl)carbamate. Cobicistat is a cytochrome P450 3A inhibitor that acts as a pharmacokinetic enhancer to increase the efficacy of HIV antiretroviral drugs. It is used to increase the bioavailability of other antiretroviral agents. Cobicistat is marketed as TYBOST® and is also known as GS-9350.

[0037] Control: Reference standard. In some embodiments, the control is a negative control sample obtained from healthy patients (e.g., those without AMD), or subjects treated with a carrier, untargeted nucleic acid sequence, scrambled nucleic acid / amino acid sequence, or untreated cells derived from healthy patients. In other embodiments, the control is a positive control sample obtained from patients treated with an active agent. In yet another embodiment, the control is a historical control or standard reference value or range of values ​​(e.g., previously tested control samples, e.g., a group of patients with known prognosis or outcome, or a group of samples representing baseline or normal values).

[0038] Differences between the test sample and the control may be increases or, conversely, decreases. Differences may be qualitative or quantitative, e.g., statistically significant differences. In some cases, the difference is an increase or decrease compared to the control of at least about 5%, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500%.

[0039] CRISPR (Clustered Regularly Arranged Short Palindromic Sequence Repeats) / Cas (CRISPR-related Protein) editing systems are engineered nuclease systems based on bacterial systems used for genome manipulation. These are, in part, based on the adaptive immune responses of many bacteria and archaea. Such methods can be used to allow genetic material to be added, removed, or modified at specific locations, for example, in target DNA or RNA sequences (e.g., La nucleic acid sequences). Therefore, CRISPR / Cas systems can be used for nucleic acid targeting (e.g., DNA or RNA), for example, to detect target DNA or RNA, to modify target DNA or RNA at any desired location, or to cleave target DNA or RNA at any desired location. Thus, such methods can be used, for example, to modify the expression of HERV-K Gag, Pol, or Env by introducing mutations to silence expression, for example, by knocking out the La gene.

[0040] In one example, the method involves editing DNA, such as a genome, and using the Cas9 nuclease. The Cas9 nuclease cleaves DNA, creating double-strand breaks at sites defined by the 20-nucleotide complementary strand sequence contained within the crRNA transcript, thereby generating blunt ends. Thus, the CRISPR / Cas system can be manipulated to create double-strand breaks at desired targets in the cell's genome, utilizing the cell's endogenous mechanisms to repair damage induced by homologous recombination repair (HDR) or non-homologous end joining (NHEJ). In another example, the method involves editing RNA, such as HERV-K Gag, Pol, or Env RNA, and using the Cas13 nuclease. Examples of Cas13 nucleases (also known as CasM) include Cas13a, Cas13b, Cas13c, Cas13Rx, Cas13x, and Cas13y (see, for example, WO2019 / 040664, US11,293,011, and US10,392,616).

[0041] In some cases, CRISPR arrays contain at least a DR-spacer-DR-spacer. This feature was used to identify the Cas13Rx protein family. In bacteria, the array is transcribed as a single transcript (containing multiple crRNA units), which is then processed into individual crRNAs by the Cas13Rx protein and other RNases. CRISPR often associates with the cas gene encoding a CRISPR-related protein (e.g., the Cas13Rx protein). The CRISPR / Cas system can be used for RNA targeting, for example, to modify a target HEV-K RNA at any desired location.

[0042] Denatured variants: Polynucleotides encoding peptides that contain sequences that are denatured as a result of genetic coding. There are 20 native amino acids, most of which are defined by two or more codons. Thus, all denatured nucleotide sequences are included in this disclosure as long as the amino acid sequence of the polypeptide encoded by the nucleotide sequence is not altered.

[0043] Downregulation or knockdown: When used in reference to the expression of a molecule, e.g., target RNA, it refers to any process that results in a reduction in the production of target RNA, but in some cases does not result in the complete elimination of the target RNA product or target RNA function. In some cases, downregulation or knockdown does not result in the complete elimination of detectable target RNA expression or target RNA activity. In some cases, the target RNA is a coding HERV-K RNA. In some cases, the target RNA is a non-coding HERV-K RNA.

[0044] Downregulation or knockdown involves any detectable reduction of the target HERV-K RNA. In certain cases, the detectable target HERV-K RNA in cells or cell-free systems is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to the control (such an amount of target HERV-K RNA detected in the corresponding normal cells or sample) (e.g., a reduction of 40%–90%, 40%–80%, or 50%–95%). In one example, the control is the relative amount of expression in cells that do not contain Cas13 or guide RNA.

[0045] Emtricitabine; 2'-deoxy-5-fluoro-3'thiacitidine (FTC). FTC is a nucleoside reverse transcriptase inhibitor (NRTI) used in the treatment of HIV infection in adults and children, and is marketed under the brand name EMTRIVA® (emtricitabine), formerly COVIRACIL®. Emtricitabine is also marketed under the brand name TRUVADA® in a fixed-dose combination with tenofovir disoproxil fumarate (Viread). A fixed-dose triple combination of emtricitabine, tenofovir, and efavirenz (marketed by Sustiva, Bristol-Myers Squibb) has been approved by the U.S. Food and Drug Administration (FDA) under the name ATRIPLA®. Emtricitabine constitutes one-quarter of a four-drug ("Quad") combination known as STRIBILD®.

[0046] A regulatory sequence is a nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence to which it is ligated. A regulatory sequence is ligated to a nucleic acid sequence if it controls and regulates the transcription of the nucleic acid sequence, and, where appropriate, the translation of the nucleic acid sequence. Thus, a regulatory sequence may include appropriate promoters, enhancers, transcriptional terminators, the start codon (i.e., ATG) before a protein-coding gene, splicing signals for introns, maintenance of the correct gene reading frame to allow proper translation of mRNA, and stop codons. The term “regulatory sequence” includes at least a component whose presence can affect expression, and may also include additional components that make its presence advantageous, such as a leader sequence and a fusion partner sequence. A regulatory sequence may include a promoter.

[0047] A promoter is the smallest sequence sufficient to direct transcription. These promoter elements are also included, sufficient to make promoter-dependent gene expression controllable in a cell-type specific, tissue-specific, or inductively by an external signal or drug, and such elements may be located in the 5' or 3' region of the gene. Both constitutive and inductive promoters are included (see, e.g., Bitter et al., 1987, Methods in Enzymology 153, 516-544). For example, in bacterial cloning, inductive promoters such as the bacteriophage lambda pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) can be used. In one embodiment, in mammalian cell cloning, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., retroviral long-terminal repeat sequences; adenovirus late promoter; vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide transcription of nucleic acid sequences.

[0048] Heterogeneous: Biological components originate from different genetic sources that are not found together in nature. Components may be host cells, genes, or regulatory regions, such as promoters. Heterogeneous components are not found together in nature, but they can function together, for example, if a heterogeneous promoter is operably ligated to a gene.

[0049] Host cell: A cell into which a vector can be transmitted and into which its DNA is expressed. The cell may be prokaryotic or eukaryotic. The cell may be mammalian, for example, a human cell. This term also includes any offspring of the target host cell. It is understood that not all offspring may be identical to the parent cell, as mutations can occur during replication. However, when the term “host cell” is used, such offspring are included. In one example, the host cell is a retinal cell.

[0050] Human endogenous retroviruses (HERVs): Endogenous viral elements in the human genome that closely resemble and may originate from retroviruses, making up about 8% of the genome. ERVs are vertically inherited proviral sequences. ERVs lack most transposon function and are typically non-infectious, often defective genomic remnants in the retroviral replication cycle. They are distinguished as germline proviral retroelements due to their integration into the nuclear genome of the host cell and reverse transcription. There are more than 200 distinct groups and subgroups of HERVs. Most HERVs have lost their infectivity due to mutations, and generally, they are primarily non-infectious retroviral remnants. However, open reading frames (ORFs) have been observed for ERV3, HERV-E 4-1, and HERV-K.

[0051] Members of the HERV-K superfamily, characterized by the presence of primer binding sites for lysine-tRNA, are the most biologically active. HERV-K appears to possess a complete set of open reading frames typical of replication-competent mammalian retroviruses. The K family contains a central open reading frame (cORF) and is equivalent to that of the HIV-1 Rev protein. HERV-K is transcribed during embryogenesis, from the 8-cell stage up to stem cell induction. HERV-K is also transcriptionally active in breast cancer tissue, as well as in several human cancer tissues, including tumor cell lines such as human breast cancer cell line T47D and teratoma carcinoma cell line GH; see U.S. Published Patent Application 2008 / 0019979A1.

[0052] Inhibiting or treating a disease: Inhibiting a disease, for example, AMD, means preventing the complete onset of the disease. In some examples, inhibiting a disease means reducing the symptoms of a particular disease. "Treatment" refers to a therapeutic intervention that restores the signs or symptoms of a disease or a disease-related condition. Treatment can be measured using any objective or subjective parameters, such as success or signs of success in attenuation or recovery of an injury, pathology or condition, including remission, remission, reduction or making symptoms more tolerable to the patient, slowing the rate of degeneration or decline, not debilitating the final point of degeneration, or improving the physical condition of the subject. Treatment may be evaluated by objective or subjective parameters, including the results of a health examination or test, such as a vision test.

[0053] Inhibitory nucleic acid molecules: These include inhibitory RNA and DNA molecules, e.g., antisense oligonucleotides, siRNAs, microRNAs (miRNAs), shRNAs, or ribozymes. Any type of antisense compound that specifically targets and modulates the expression of the nucleic acids encoding Gag, Pol, or Env of HERV-K is intended for use. The antisense compound specifically hybridizes with the Gag, Pol, or Env nucleic acid molecule and modulates its expression. These compounds can be introduced as single-stranded, double-stranded, cyclic, branched, or hairpin compounds and may contain structural elements, e.g., internal or terminal bulges or loops. A double-stranded antisense compound may be two strands hybridized to form a double-stranded compound, or a single strand with sufficient self-complementarity to allow for complete or partial hybridization and formation of a double-stranded compound. In some cases, antisense oligonucleotides are single-stranded antisense compounds, which then hybridize to mRNA encoding the Gag, Pol, or Env protein, resulting in mRNA cleavage when the resulting double helix is ​​recognized by RNaseH. In some cases, miRNAs are single-stranded RNA molecules, e.g., about 21–23 nucleotides long, that are at least partially complementary to the mRNA molecule that regulates gene expression through the RNAi pathway. In further cases, shRNAs are RNA oligonucleotides that form a tight hairpin, which are cleaved into siRNAs. siRNA molecules are generally about 15–40 nucleotides long, e.g., 20–25 nucleotides long, and may have 0–5 nucleotide overhangs at the 3' or 5' ends, or they may have blunt ends. Generally, one strand of the siRNA is at least partially complementary to the nucleic acid molecule encoding Gag, Pol, or Env. Antisense compounds that specifically target the Gag, Pol, or Env gene can be prepared by designing compounds that are complementary to the target nucleotide sequence, e.g., the mRNA sequence.Antisense compounds do not need to be 100% complementary to the nucleic acid molecules encoding Gag, Pol, or Env in order to specifically hybridize to the target and specifically control its expression. For example, in the case of antisense compounds or double-stranded compounds, the antisense strand of the compound may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% complementary to the nucleic acid molecules encoding Gag, Pol, or Env. Methods for screening antisense compounds for specificity are publicly known (see, for example, the United States).

[0054] Inhibitory peptides: Blocking peptides competitively inhibit protein-protein interactions by mimicking one of their binding domains, or “binding epitopes.” The term “mimotope” refers to a peptide macromolecule that replicates, or “mimics,” an epitope structure that elicits the same specific antibody response as the intact macromolecule from which it originated. This term includes peptides that competitively disrupt protein-protein interactions by binding to one of their partner’s binding domains, including those that interfere with the interaction.

[0055] Isolated: “Isolated” biological components (e.g., nucleic acid molecules or proteins or organelles) are substantially separated or purified from other biological components, i.e., other chromosomes and extrachromosomal DNA and RNA, proteins, and organelles, within the cells of the organism in which the component naturally exists. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. This term also includes nucleic acids and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids and proteins.

[0056] Labeling: A detectable compound or composition that is directly or indirectly conjugated to another molecule to facilitate its detection. Specific, non-limiting examples of labeling include fluorescent tags, enzyme linking, and radioisotopes.

[0057] Mammals: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

[0058] Modulation: To change in a statistically significant manner. Modulation can be an increase or a decrease. Those skilled in the art can identify appropriate assays for determining a statistically significant increase or decrease in a parameter. These include, but are not limited to, Student's t-test or the paired ratio t-test.

[0059] Nucleic acid molecules: polymers composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and their naturally occurring synthetic analogs) linked via phosphodiester bonds, associated naturally occurring structural variants, and their naturally occurring synthetic analogs. Therefore, this term includes nucleotide polymers in which nucleotides and the links between them include naturally occurring synthetic analogs such as, for example, but not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acid (PNA). Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" typically refers to short polynucleotides, generally about 50 or fewer nucleotides. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it will be understood that this also includes RNA sequences (i.e., A, U, G, C) where "T" is replaced by "U".

[0060] Conventional notation used herein to describe nucleotide sequences is as follows: the left end of a single-stranded nucleotide sequence is the 5' end, and the direction of the left end of a double-stranded nucleotide sequence is referred to as the 5' direction. The direction of 5'-3' addition from the nucleotide to the nascent RNA transcript is referred to as the transcription direction. The DNA strand having the same sequence as the mRNA is called the "coding strand," the sequence on the DNA strand having the same sequence as the mRNA transcribed from that DNA and located 5' relative to the 5' end of the RNA transcript is called the "upstream sequence," and the sequence on the DNA strand having the same sequence as the RNA and located 3' relative to the 3' end of the encoding RNA transcript is called the "downstream sequence."

[0061] "cDNA" refers to DNA that is complementary to or identical to mRNA, in either single-stranded or double-stranded form.

[0062] "Coded" refers to the intrinsic properties of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which has either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties derived therefrom, and which serve as a template for the synthesis of other polymers and macromolecules in biological processes. Therefore, if the transcription and translation of mRNA produced by a gene produce a protein in a cell or other biological system, that gene codes for a protein. Both coding strands, whose nucleotide sequences are identical to the mRNA sequence and are typically provided in sequence listings and used as templates for transcription, may be said to code for a protein or other product of that gene or cDNA. Unless otherwise specified, "nucleotide sequences coding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and code for the same amino acid sequence. Protein and RNA coding nucleotide sequences may contain introns.

[0063] "Recombinant nucleic acids" refer to nucleic acids that have nucleotide sequences that do not naturally combine. This includes nucleic acid vectors containing amplified or assembled nucleic acids that can be used to transform suitable host cells. Host cells containing recombinant nucleic acids are referred to as "recombinant host cells." Genes are then expressed in recombinant host cells to produce, for example, "recombinant polypeptides." Recombinant nucleic acids can also play non-coding roles (e.g., promoters, origins of replication, ribosome binding sites, etc.).

[0064] If a polynucleotide whose first sequence specifically hybridizes with a polynucleotide whose second sequence is, then the first sequence is "antisense" with respect to the second sequence.

[0065] Terms used to describe the sequence relationships between two or more nucleotide or amino acid sequences include "reference sequence," "selected from," "comparison window," "identical," "percentage of sequence identity," "substantially identical," "complementary," and "substantially complementary."

[0066] For sequence comparison of nucleic acid sequences, typically one sequence acts as a reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, and if necessary, subsequence coordinates and sequence algorithm program parameters are specified. Default program parameters are used. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981; by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI); or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds 1995 supplement)).

[0067] Nucleoside analog reverse transcriptase inhibitors (NRTIs): An early class of antiretroviral drugs. To be incorporated into viral DNA, NRTIs must be activated in cells by the addition of phosphate groups to their deoxyribose moieties to form NRTI triphosphates. This phosphorylation step is carried out by cell kinase enzymes. Examples of NRTIs include zidovudine, didanosine, zalcitabine, stabudine, lamivudine, abacavir, and emtricitabine (also known as FTC).

[0068] Nucleotide analog reverse transcriptase inhibitors (NtRTIs): NtRTIs are nucleotide analogs of cytidine, guanosine, thymidine, and adenosine that are useful in treating HIV infection. For example, tenofovir (and its related prodrugs) is an NtRTI adenosine analog.

[0069] An open reading frame (ORF) is a sequence of nucleotide triplets (codons) that encode amino acids without any internal stop codons. These sequences are typically translatable into proteins.

[0070] Operatively linked: When a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, such as a sequence encoding a polypeptide, the promoter is operably linked to the coding sequence. Generally, operably linked DNA sequences are in close proximity and in the same reading frame if necessary to join two protein-coding regions.

[0071] Pharmacopoeia-acceptable carriers: Useful pharmacopoeia-acceptable carriers are conventional ones. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975) describes compositions and formulations suitable for the pharmacopoeia of the therapeutic agents disclosed herein.

[0072] Generally, the characteristics of the carrier depend on the specific method of administration used. For example, parenteral formulations typically include an injectable liquid as a vehicle, such as a pharmaceutically and physiologically acceptable liquid, e.g., water, physiological saline, equilibrium salt solution, aqueous dextrose, glycerol, etc. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the administered pharmaceutical composition may contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffers, e.g., sodium acetate or sorbitan monolaurate.

[0073] Drug enhancers: Substances that increase the bioavailability and bioefficacy of the active substance they are combined with, without possessing any activity of their own at the dose used. These drugs are also known as “bioenhancers.” Increased bioavailability means an increase in the level of the drug, for example, in the blood. Increased bioefficacy means an increase in the effectiveness of the drug, at least in part, due to the increase in bioavailability. COBIs are drug enhancers.

[0074] Polypeptide: Any chain of amino acids, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). With respect to polypeptides and proteins, the word "about" indicates an integer amount. For example, a polypeptide with "about" 29 amino acids in length is 28-30 amino acids long. Therefore, a polypeptide with "about" a specified number of residues can be one amino acid shorter or one amino acid longer than the specified number. A fusion polypeptide contains the amino acid sequences of a first polypeptide and a second different polypeptide (e.g., heterologous polypeptide) and can be synthesized as a single amino acid sequence. Recombinant polypeptides have an amino acid sequence that does not exist naturally or is composed of two otherwise separate segments of an amino acid sequence.

[0075] Promoter: A set of nucleic acid regulatory sequences that direct the transcription of nucleic acids. A promoter includes necessary nucleic acid sequences near the transcription start site, for example, the TATA element in the case of a polymerase type II promoter. A promoter may also include distal enhancer or repressor elements, which may be located several thousand base pairs from the transcription start site, as needed. A promoter may be a constitutively active promoter (i.e., a promoter that is constitutive in its active / "ON" state), an inductive promoter (i.e., a promoter whose state is active / "ON" or inactive / "OFF" is controlled by an external stimulus, such as the presence of a specific temperature, compound, or protein), a spatially restricted promoter (e.g., a tissue-specific promoter, a cell-type-specific promoter, etc.), or a temporally restricted promoter (i.e., a promoter is in an "ON" or "OFF" state during a specific stage of embryonic development or a specific stage of a biological process). RPE-specific promoters can be used to specifically drive expression in the RPE. Some examples of promoters include those for genes such as DCT (ID#1638), tyrosinase (ID#7299), VMD2 (ID#7439), and RPE65 (ID#6121). Choroid-specific promoters can also be used, such as carbonic anhydrase 4 (ID#762) and PLVAP (ID#83483). Examples of photoreceptor-specific promoters include rhodopsin (ID#6010), NR2E3 (ID#10002), and NRL (ID#4901).

[0076] Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. For example, a purified protein preparation is one in which the protein referred to is purer than the protein in its natural environment within the cell. For example, a protein preparation is purified so that the protein represents at least 50% of the total protein content of the preparation. Similarly, a purified nucleic acid molecule preparation is one in which the nucleic acid molecules, including complex mixtures, are purer than those found in the environment. A purified population of nucleic acids or proteins is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure, respectively, or free from other nucleic acids or proteins.

[0077] RNA editing is a type of genetic manipulation in which an RNA molecule (or ribonucleotide of RNA) is inserted, deleted, or replaced in the genome of an organism using a manipulated nuclease (e.g., Cas13Rx protein). This creates site-directed strand breaks at desired locations in the RNA. The induced breaks are repaired, resulting in targeted mutations or repairs. The CRISPR / Cas method disclosed herein, for example, using the Cas13Rx protein and HERV-K-specific gRNAs, can be used to edit the sequence of one or more target HERV-K RNAs.

[0078] Sequence Identity: The similarity between amino acid sequences is expressed in terms of similarity between sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in units of identity (or similarity or homology) percentage, with higher percentages indicating greater similarity between the two sequences. Homologs or variants of polypeptides, when aligned using standard methods, exhibit a relatively high degree of sequence identity.

[0079] Methods for aligning sequences for comparison are publicly known. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, 1970, J Mol Biol 48, 443-453; Higgins and Sharp, 1988, Gene 73, 237-244; Higgins and Sharp, 1989, CABIOS 5, 151-153; Corpet et al., 1988, Nucleic Acids Research 16, 10881-10890; and Pearson and Lipman, 1988, Proc Natl Acad Sci USA 85, 2444-2448. Altschul et al., 1994, Nature Genet 6, 119-129, present detailed considerations for sequence alignment methods and homology calculations.

[0080] The NCBI's basic local alignment search tool (BLAST) (Altschul et al., 1990, J Mol Biol 215, 403-410) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) on the internet, for use in combination with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Instructions on how to determine sequence identity using this program are available on the NCBI website.

[0081] Polypeptide homologs and variants are typically characterized by possessing at least 75%, e.g., at least 80%, of sequence identity, counted across full-length alignment with the polypeptide amino acid sequence using NCBI Blast 2.0, gapped blastp, set to default parameters. For amino acid sequence comparisons of more than approximately 30 amino acids, Blast 2 sequence function is used with the default BLOSUM62 matrix set to default parameters (gap elongation cost of 11 and gap cost per residue). When aligning shorter peptides (less than approximately 30 amino acids), alignment must be performed using Blast 2 sequence function with the PAM30 matrix set to default parameters (open gap 9, elongation gap 1 percentile). Proteins with even higher similarity to the reference sequence will exhibit increased identity percentages, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity when evaluated by this method. When fewer sequences than the whole are compared for sequence identity, homologs and variants typically have at least 80% sequence identity over a short window of 10–20 amino acids, and depending on their similarity to the reference sequence, they can have at least 85%, or even 90% or 95% sequence identity. Methods for determining sequence identity over such short windows are available on the NCBI website. Overall, these ranges of sequence identity are provided for guidance purposes only, and it is entirely possible to obtain very important homologs that lie outside the provided range.

[0082] Therefore, in some cases, a polypeptide or nucleic acid sequence variant is typically characterized by possessing at least about 75% sequence identity, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as counted across the full-length alignment with the amino acid or nucleotide sequence of interest. Sequences with even higher similarity to the reference sequence will exhibit an increasing percentage of identity, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, when evaluated in this manner. When fewer sequences than the whole are compared for sequence identity, homologs and variants typically have at least 80% sequence identity across a short window of 10–20 amino acids (or 30–60 nucleotides), and may have at least 85%, or at least 90% or 95%, sequence identity, depending on their similarity to the reference sequence. Methods for determining sequence identity across such short windows are available on the NCBI website.

[0083] As used herein, a reference to “at least 90% identity” (or similar language) means “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” of a given reference array.

[0084] Subjects: Living multicellular vertebrates in categories including humans and non-human mammals, e.g., non-human primates, rats, mice, dogs, cats, horses, cattle, and pigs. In specific embodiments, the subject is human. In additional embodiments, subjects having AMD are selected.

[0085] Tenofovir Prodrug: Tenofovir (9-R-[(2-phosphonomethoxy)propyl]adenine), an acyclic nucleotide analog of dAMP, is a potent in vitro and in vivo inhibitor of human immunodeficiency virus type 1 (HIV-1) replication. In cells, tenofovir is sequentially phosphorylated by AMP kinase and nucleoside diphosphate kinase to the active species, tenofovir diphosphate, which acts as a competitive inhibitor of HIV-1 reverse transcriptase, terminating the growth of the viral DNA strand. Tenofovir disoproxil fumarate (TDF), marketed as VIREAD®, is an oral prodrug of tenofovir, approved for marketing in many countries as a once-daily tablet (300 mg) in combination with other antiretroviral agents for the treatment of HIV-1 infection.

[0086] U.S. Patents 7,390,791 and 7,803,788, both incorporated herein by reference, disclose prodrugs of phosphonate nucleotide analogs that are also useful in therapy. 9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosphenyl]-methoxy]propyl]adenine 16 is an isopropylalaninylphenyl ester prodrug of tenofovir. Tenofovir alafenamide (TAF) is also known as GS-7340. TAF is marketed under the name VEMLIDY®. The hemi-fumarate form of TAF is also useful in the methods disclosed herein. TAF exhibits potent anti-HIV activity against HIV-1 in T cells, activated peripheral blood mononuclear lymphocytes (PBMCs), and macrophages, with activity 500-1000-fold enhanced compared to tenofovir. TAF also possesses an enhanced ability to deliver parent tenofovir to PBMCs and other lymphoid tissues in vivo and increase their accumulation. TAF can be prepared as described in U.S. Patent No. 7,390,791, incorporated herein by reference.

[0087] FTC / TAF / EVG / COBI (containing 150 mg of EVG, 150 mg of COBI, 200 mg of FTC, and 10 mg of TAF), also known as GENVOYA®, is approved for the treatment of pre-existing HIV infections in subjects. EVG, FTC, and TAF have been shown to inhibit viral replication. Cobicistat enhances the efficacy of the combination, for example, by inhibiting enzymes in the liver and intestinal wall that metabolize EVG. The use of FTC / TAF / EVG / COBI for the treatment of pre-existing HIV infections is disclosed, for example, in U.S. Patent Publication US2015 / 0105350, entitled “Combination Therapy Containing Tenofovir Alafenamide Hemifumarate and Cobicistat for Use in the Treatment of Viral Infections,” which is incorporated herein by reference. The FTC / TAF / EVG / COBI combination drug is manufactured and marketed by Gilead Sciences.

[0088] Therapeutic dose: The amount of composition or cells necessary to achieve the desired effect in the subject being treated. For example, this may be the amount required to inhibit vision loss and / or retinal degeneration. When administered to a subject, the dosage that achieves the target tissue concentration for which the in vitro effect has been demonstrated is generally used.

[0089] The therapeutically effective dose may vary depending on one or more of the subject and disease state being treated, the subject's weight and age, the severity of AMD, and the mode of administration, which can be readily determined by those skilled in the art. Beneficial therapeutic effects may include the usability of diagnostic determination; recovery of AMD symptoms, improvement of vision, or delay of vision loss; and reduction or prevention of the onset of AMD symptoms. In one embodiment, “effective dose” is an amount sufficient to reduce the symptoms of AMD by, for example, at least 10%, at least 20%, at least 50%, at least 70%, or at least 90% (compared to no administration of the therapeutic agent), or to delay their onset or progression. The term also applies to doses that enable the expression of Cas13 and / or gRNA and enable the targeting (e.g., detection or modification) of target HERV-K RNA.

[0090] Transfer (t)RNA: Adapter molecules composed of RNA, typically 76-90 nucleotides long, that transport amino acids to ribosomes. Complementation of the 3-nucleotide codon in mRNA by the anticodon in tRNA results in protein synthesis. The human genome contains 497 nuclear genes encoding cytoplasmic tRNA molecules. These cytoplasmic tRNA genes can be grouped into 49 families according to their anticodon characteristics. Shorter molecules are produced after cleavage of mature tRNA. When cleaved by ANG, these molecules have a characteristically unusual cyclic phosphate at their 3' end and a hydroxyl group at their 5' end, playing a role in RNA interference, specifically in the repression of retroviruses and retrotransposons that use tRNA as a primer for replication. These molecules are called tiRNA or tRF (see below) (see Schorn and Martienssen, Trends Cell Biol. 28(10): 793-906, 2018).

[0091] tRNA-derived stress-inducing RNA (tiRNA) or tRNA fragment (tRF): A class of small non-coding RNAs produced through the specific cleavage of tRNA by endonucleases. Based on their enzymatic cleavage sites, tRNA-derived fragments are classified into two main types: tRNA halves (also known as tiRNA) and small tRNA fragments. TiRNA, as an important subtype of tRNA-derived fragment, is produced by angiogenins; see Tao et al., J Cell Physiol. 235:683-690, 2020.

[0092] Unit dosage form: A physically distinct unit, such as a capsule, tablet, or solution, which is suitable as a unit dosing for human patients, and each unit contains a predetermined amount of one or more active ingredients calculated to produce a therapeutic effect in combination with at least one pharmaceutically acceptable diluent or carrier, or a combination thereof. The unit dosing formulation contains a daily dose or an appropriate fraction of the active ingredient. tRNA-derived fragments (or tRFs) are short molecules that appear after cleavage of mature tRNA or precursor transcripts.

[0093] Upregulation and Knock-in: When used in reference to the expression of molecules, e.g., targets, “upregulation” refers to any process that results in an increase in the production of the RNA of interest. For example, upregulation increases detectable RNA expression or RNA activity. “Knock-in” is an increase in expression resulting from the introduction of a nucleic acid molecule encoding the protein of interest, e.g., ANG, tRNA, or tRNA fragment.

[0094] Upregulation includes any detectable increase in RNA. In certain cases, detectable RNA in cells or cell-free systems increases by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to the control (such amount of ANG RNA detected in the corresponding untreated cells or sample) (e.g., a decrease of 40%–90%, 40%–80%, or 50%–95%).

[0095] A vector is a nucleic acid molecule that is introduced into a host cell to produce a transformed host cell. A vector may contain a nucleic acid sequence, such as an origin of replication, that enables it to replicate in the host cell. A vector may also contain one or more select marker genes and other genetic elements known in the art. Examples of vectors include plasmid vectors, which contain plasmids for expression in Gram-negative and Gram-positive bacterial cells. Exemplary vectors include those for expression in E. coli and Salmonella. Examples of vectors include viral vectors, such as, but are not limited to, retrovirus, lentivirus, adeno-associated virus (AAV), orthopox, tripox, fowlpox, capripox, butapox, adenovirus, herpesvirus, alphavirus, baculovirus, Sindbisvirus, vaccinia virus, and poliovirus vectors. Examples of vectors include vectors for expression in yeast cells or mammalian cells. In some embodiments, the vector is a lentivirus (e.g., a lentivirus vector with integration defects) or an adeno-associated virus (AAV) vector.

[0096] Certain vectors can direct the expression of genes to which they are operably ligated. Such vectors are referred to herein as “expression vectors.” Common expression vectors are often in the form of plasmids. Recombinant expression vectors may contain nucleic acids provided herein in a form suitable for nucleic acid expression in host cells (e.g., guide RNA (RNA sequence or one that can be expressed from an RNA sequence), nucleic acid encoding the Cas13Rx protein), meaning that a recombinant expression vector contains one or more regulatory elements, which may be selected based on the host cell used for expression and are operably ligated to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably ligated” is intended to mean that the nucleotide sequence of interest is ligated to the regulatory element in a manner that enables the expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system, or in the host cell if the vector is introduced into the host cell). It will be recognized by those skilled in the art that the design of an expression vector may depend on factors such as the selection of the host cell to be transformed, the desired level of expression, etc. The vector can be introduced into a host cell to generate transcripts, proteins, or peptides encoded by the nucleic acids described herein, such as fusion proteins or peptides (e.g., clustered, regularly arranged short palindromic sequence repeats (CRISPR) transcripts, proteins, enzymes, their mutant forms, their fusion proteins, etc.).

[0097] Viruses are microscopic infectious organisms that reproduce within living cells. Viruses essentially consist of a core of single nucleic acid surrounded by a protein coat and possess the ability to replicate only within living cells. "Viral replication" is the generation of additional viruses through the emergence of the life cycle of at least one virus.

[0098] A "retrovirus" is an RNA virus, meaning its viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse-transcribed into a DNA intermediate, which is very efficiently integrated into the chromosomal DNA of the infected cell. This integrated DNA intermediate is called a provirus. The wild-type retrovirus genome encodes polymerase, glycosaminoglycans, and envelope proteins.

[0099] The term "lentivirus" is used in its traditional sense to describe a genus of viruses that contain reverse transcriptase. Lentiviruses include "immunodeficiency viruses," which include human immunodeficiency virus (HIV) types 1 and 2 (HIV-I and HIV-II), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV).

[0100] II. HERV-K inhibitors Methods for treating AMD in subjects and methods for reducing the risk of developing AMD are disclosed herein. In some embodiments, the methods can reduce retinal degeneration by, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%, at least 95%, at least 100%, at least 200%, or even more than 500%, and / or halt vision loss or improve vision. These methods use one or more agents that reduce or inhibit HERV-K activity by, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%, at least 95%, or 100%. In some embodiments, 100% inhibition of HERV-K activity is not required for the disclosed methods for treating AMD. Agents that treat or reduce the risk of AMD in a subject may reduce one or more of the following: a) the amount of human endogenous retrovirus (HERV)-K, b) the production of mRNA encoding the HERV-K protein, c) the translation of the mRNA; d) the amount of HERV-K protein in the subject; e) the inhibitory activity of HERV-K mRNA and / or protein, or any combination of a, b, c, d, and e. Exemplary agents are disclosed below and can be used in any combination. In some embodiments, agents that inhibit HERV-K reduce the amount of HERV-K locally in the subject, for example, in the subject's retina. In other embodiments, the agent is administered systemically. Preferred agents are discussed in more detail below. In addition, the treatments listed below can be used in any combination. In some embodiments, antiretroviral agents are administered in combination with the CRISPR / Cas13 system. In some embodiments, the treatment reduces the amount of protein activity and / or viral mRNA.

[0101] Inhibitory peptides are useful. Inhibitory peptides are also disclosed, for example, in Hoffman et al., J. Virol. 94(23) e01682-20, 2000, which is incorporated herein by reference. Inhibitory peptides are also available for HIV (see Egerer et al., Mol. Ther. 19(7):1236-1244, 2011) and other retroviruses (see Kotler et al., PNAS 85:4185-4189, 1988).

[0102] In one embodiment, the drug that inhibits KERV-K is ANG, for example, a vector that expresses ANG.

[0103] A. Antibodies and their antigen-binding fragments In some embodiments, the drug is an antibody or its antigen-binding fragment that specifically binds to the HERV-K protein, for example, a monoclonal antibody that specifically binds to Gag, Pol, or Env of HERV-K, but is not limited to these.

[0104] An exemplary complete nucleic acid sequence for HERV-K is: [ka] [ka] [ka] That is the case. An example HERV-K Gag amino acid sequence (GenBank AAL16779.1) is: [ka] That is the case.

[0105] An example of the HERV-K Pol amino acid sequence (GenBank AAL60056.1) is: [ka] That is the case.

[0106] An example HERV-K Env amino acid sequence (GenBank AAL16780.1) is: [ka] That is the case.

[0107] Antibodies that specifically bind to the Gag, Pol, or Env of HERV-K are available; see U.S. Published Patent Application 2008 / 0019979A1 disclosing antibodies that specifically bind to the HERV-K protein. Additional antibodies that bind to the HERV-K envelope protein are disclosed, for example, in U.S. Patents 10,723,787 and 10,981,976, both of which are incorporated herein by reference. In some embodiments, an antibody or its antigen-binding fragment that specifically binds to the Gag, Pol, or Env of HERV-K is used in the manner disclosed. In some embodiments, the antibody or antigen-binding fragment is delivered locally to the eye. In other embodiments, the antibody or antigen-binding fragment is delivered systemically to the subject. In further embodiments, a nucleic acid molecule encoding the antibody or antigen-binding fragment is delivered locally to the eye or systemically to the subject.

[0108] Antibodies against the HERV-K envelope protein are commercially available from MyBioSource under catalog numbers MBS602670 and MBS603725, and from United States Biological under catalog numbers E2286-13-100ug and E2286-14-100ug. Antibodies against the HERV-K envelope protein are also commercially available from LSBio under catalog number LS-C65286-100, and from antibodies-online under catalog number ABIN472658.

[0109] Antibodies that specifically bind to Gag, Pol, or Env and substantially reduce or inhibit their activity (e.g., reduction by at least 20%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100%) are useful in the methods disclosed herein. Examples of antibodies include monoclonal antibodies, human antibodies, humanized antibodies, dehumanized antibodies, and immunoglobulin (Ig) fusion proteins. Fully human antibodies and humanized antibodies can also be produced using methods known to those skilled in the art.

[0110] Polyclonal antagonist antibodies can be prepared, for example, by immunizing a suitable subject (e.g., a human or veterinary subject) with the HERV-K Gag, Pol, or Env protein. The titer of the anti-HERV-K Gag, Pol, or Env antibody in the immunized subject can be monitored over time using immobilized HERV-K Gag, Pol, or Env, or its epitope, for example, by enzyme-linked immunosorbent assay (ELISA). In one example, an antibody molecule that specifically binds to HERV-K Gag, Pol, or Env can be isolated from a mammal (e.g., from serum) and further purified by isolating the IgG antibody, for example, using protein A chromatography. In some embodiments, the antibody can also be selected using a functional assay to detect, for example, inhibition of HERV-K function.

[0111] Antibody-producing cells can be obtained from a target, such as an immunized target, and used to prepare monoclonal antibodies (Kohler and Milstein Nature 256:495 49, 1995; Brown et al., J. Immunol. 127:539 46, 1981; Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77 96, 1985; Gefter, ML et al. (1977) Somatic Cell Genet. 3:231 36; Kenneth, RH in Monoclonal Antibodies: A New Dimension In Biological Analyses. Plenum Publishing Corp., New York, NY (1980); Kozbor et al. Immunol. Today 4:72, 1983; Lerner, EA (1981) Yale J. Biol. See Med. 54:387 402; Yeh et al., Proc. Natl. Acad. Sci. 76:2927 31, 1976). In one example, an immortalized cell line (typically myeloma) is fused to mammalian lymphocytes (typically spleen cells) immunized with HERV-K Gag, Pol, or Env, and the resulting hybridoma cell culture supernatant is screened to identify hybridomas that specifically bind to the target polypeptide and produce monoclonal antibodies that inhibit the polypeptide's function.

[0112] In one embodiment, to generate hybridomas, the immortalized cell line (e.g., myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, mouse hybridomas can be produced by fusing lymphocytes derived from mice immunized with HERV-K Gag, Pol, or Env, or their epitopes, with an immortalized mouse cell line. In one example, a mouse myeloma cell line sensitive to a culture medium containing hypoxanthine, aminopterin, and thymidine ("HAT medium") is used. Any of several myeloma cell lines available from the American Type Culture Collection (ATCC), Rockville, MD, including, for example, P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653, or Sp2 / O-Ag14 myeloma lines, can be used as a fusion partner. HAT-sensitive mouse myeloma cells can be fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells obtained from fusion are then selected using HAT medium, which kills unfused (and unproductively fused) myeloma cells. Hybridoma cells that produce the desired monoclonal antibody can be detected, for example, by screening the hybridoma culture supernatant for the produced antibody that binds to HERV-K Gag, Pol, or Env, using an immunological assay (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)).

[0113] As an alternative to the preparation of monoclonal antibody secretory hybridomas, monoclonal antibodies that specifically bind to HERV-K Gag, Pol, or Env can be identified and isolated by screening recombinant combinatorial immunoglobulin libraries (e.g., antibody phage display libraries) using HERV-K Gag, Pol, or Env, or their epitopes, to isolate immunoglobulin library members that specifically bind to polypeptides. Library members with specific activity, such as activity to bind to HERV-K Gag, Pol, or Env, or activity to inhibit HERV-K in in vitro assays, can be selected. Kits for constructing and screening phage display libraries are commercially available (e.g., Pharmacia and Stratagene, to some extent). Examples of methods and reagents particularly suitable for use in the preparation and screening of antibody display libraries can be found, for example, in U.S. Patent No. 5,223,409; PCT Publication No. WO90 / 02809; PCT Publication No. WO91 / 17271; PCT Publication No. WO92 / 18619; PCT Publication No. WO92 / 20791; PCT Publication No. WO92 / 15679; PCT Publication No. WO92 / 01047; PCT Publication No. WO93 / 01288; PCT Publication No. WO92 / 09690; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978 7982, 1991; Hoogenboom et al., Nucleic Acids Res. 19:4133 4137, 1991.

[0114] In one example, the sequence of the specificity-determining region of each CDR is determined. Residues outside the SDR (specificity-determining region, e.g., non-ligand-containing region) are substituted. For example, in any of the CDR sequences, up to one, two, or three amino acids may be substituted. Chimeric antibodies can be generated that contain a framework region from one antibody and CDRs from different antibodies. For example, humanized antibodies can be generated. The antibody or antibody fragment may be a CDR derived from a donor monoclonal antibody that binds to HERV-K Gag, Pol, or Env, or its epitope, as well as an immunoglobulin derived from a human acceptor immunoglobulin heavy chain and light chain framework, and a humanized immunoglobulin having a heavy chain and light chain variable region framework.

[0115] Humanized monoclonal antibodies can be produced by transferring CDRs (which specifically bind to HERV-K Gag, Pol, or Env) derived from the heavy and light chain variable regions of donor mouse immunoglobulins into human variable domains, and then substituting human residues in the framework region where affinity preservation is required. The use of antibody components derived from humanized monoclonal antibodies prevents potential problems associated with the immunogenicity of the constant region of donor antibodies. Techniques for generating humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci. USA 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993. The antibody may be of any isotype, but in some embodiments, the antibody is IgG, including, but not limited to, IgG1, IgG2, IgG3, and IgG4.

[0116] In one embodiment, the sequence of the humanized immunoglobulin heavy chain variable region framework may be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Therefore, the sequence of the humanized immunoglobulin heavy chain variable region framework may be at least about 75%, at least about 85%, at least about 99%, or at least about 95% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Mutations can be produced in the human framework region and the humanized antibody framework region (see, for example, U.S. Patent No. 5,585,089).

[0117] Exemplary human antibodies are LEN and 21 / 28 CL. Sequences of many human heavy and light chain frameworks are publicly known. Generally, antibodies, e.g., human antibodies or humanized antibodies, are at least 10 7 M -1 For example, at least 10 8 M -1 , at least 5 × 10 8 M -1 or at least 10 9 M -1 It binds specifically to HERV-K Gag, Pol, or Env, and / or their epitopes, with an affinity constant of . 8 M -1 , at least 5 × 10 8 M -1 or at least 10 9 M -1 The antibody specifically binds to HERV-K Gag, Pol, or Env, or their epitopes, with an affinity constant of [value missing]. The antibody may be a fully human antibody.

[0118] Antibodies, such as mouse monoclonal antibodies, chimeric antibodies, and humanized antibodies, include not only the full-length molecule but also fragments thereof, such as Fab, F(ab')2, and Fv, which contain heavy and light chain variable regions and can bind to specific epitope determinants. These antibody fragments retain some ability to selectively bind to their antigens or receptors. Examples of these fragments include: (1) Fab, a fragment containing a monovalent antigen-binding fragment of an antibody molecule, can be produced by digesting the whole antibody with the enzyme papain to obtain an intact light chain and a portion of one heavy chain; (2) Fab', fragments of the antibody molecule, can be obtained by treating the whole antibody with pepsin and then reducing it to obtain intact light chain and heavy chain portions; two Fab' fragments are obtained for each antibody molecule; (3)(Fab')2, an antibody fragment that can be obtained by treating the entire antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, a genetically engineered fragment containing a variable region of the light chain and a variable region of the heavy chain expressed as two strands; and (5) A single-chain antibody (e.g., scFv) is defined as a genetically engineered molecule containing a variable light chain region and a variable heavy chain region linked by a suitable polypeptide linker, as a genetically fused single-chain molecule.

[0119] Methods for producing these fragments are disclosed, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988. In some examples, the variable region includes a variable region of the light chain and a variable region of the heavy chain, expressed as individual polypeptides. Fv antibodies are typically about 25 kDa and contain a complete antigen-binding site with three CDRs for each heavy chain and each light chain. To produce these antibodies, V H and VL It can be expressed in host cells from two individual nucleic acid constructs. H and V L When expressed in non-proximal locations, the Fv antibody chains are typically held together by non-covalent interactions. However, these chains tend to dissociate upon dilution, and therefore methods have been developed to crosslink the chains through glutaraldehyde, intermolecular disulfides, or peptide linkers. For example, Fv can be disulfide-stabilized Fv (dsFv), where the heavy-chain and light-chain variable regions are chemically linked by disulfide bonds.

[0120] In an additional example, the Fv fragment is linked by a peptide linker. H and V L These single-chain antigen-binding proteins (scFv) are linked by oligonucleotides. H and V L It is prepared by constructing a structural gene containing a DNA sequence encoding the domain. The structural gene is inserted into an expression vector, which is then introduced into a host cell such as E. coli. The recombinant host cell synthesizes a single polypeptide chain having a linker peptide that cross-links the two V domains. Methods for generating scFv can be found, for example, in Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science 242:423, 1988; U.S. Patent No. 4,946,778; Pack et al., Bio / Technology 11:1271, 1993; and Sandhu, as mentioned above.

[0121] Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression of the DNA encoding the fragment in E. coli. Antibody fragments can also be obtained by conventional methods of pepsin or papain digestion of the whole antibody. For example, an antibody fragment can be produced by enzymatically cleaving the antibody with pepsin to provide a 5S fragment represented as F(ab')2. This fragment can be further cleaved using a thiol reducing agent and, optionally, a blocking group for the sulfhydryl group resulting from the cleavage of disulfide links to produce a 3.5S Fab' monovalent fragment. Alternatively, enzymatic cleavage using pepsin directly produces two monovalent fragments, Fab' and Fc (see U.S. Patents 4,036,945 and 4,331,647, and the references contained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

[0122] Other methods for cleaving antibodies, such as separation of the heavy chain to form a monovalent light-heavy chain fragment, further cleavage of the fragment, or other enzymatic, chemical, or genetic techniques, may also be used, insofar as the fragment binds to the antigen recognized by the intact antibody. Any of the antigen-binding fragments described herein may be useful.

[0123] Conservative variants of antibodies can be generated. Such conservative variants used in antibody fragments, for example, dsFv fragments or scFv fragments, exhibit correct folding, as well as V H and V LIt retains essential amino acid residues necessary for stabilization between regions and preserves the charge characteristics of the residues to conserve the low pI and low toxicity of the molecule. Amino acid substitutions (e.g., up to 1, up to 2, up to 3, up to 4, or up to 5 amino acid substitutions) H and V L These substitutions can be performed in a region to increase yield. In some embodiments, these substitutions are performed in the framework region and not in the CDR. A table of conserved amino acid substitutions is provided above. The amino acid sequence of the antibody of interest may be outlined by identifying one or more amino acids in the HERV-K Gag, Pol, or Env sequence, identifying conserved substitutions, and generating conserved variants using molecular techniques.

[0124] Effector molecules, such as a detectable or therapeutic moiety, can be linked to antibodies that specifically bind to HERV-K Gag, Pol, or Env using any number of means. Both covalent and non-covalent attachment means can be used. The procedure for attaching the effector molecule to the antibody varies depending on the chemical structure of the effector. Polypeptides typically contain various functional groups, such as carboxylic acid (COOH), free amine (-NH2), or sulfhydryl (-SH) groups, which are available for reaction with suitable functional groups on the antibody that result in the binding of the effector molecule. Alternatively, the antibody may be derivatized to expose or attach additional reactive functional groups. Derivatization may involve the attachment of any number of linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to conjugate the antibody to the effector molecule. The linker can form covalent bonds to both the antibody and the effector molecule. Suitable linkers include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody and effector molecules are polypeptides, the linker may be conjugated to the constituent amino acids through their side chain groups (e.g., through disulfide linkage to cysteine) or to the amino and carboxyl groups of the alpha carbons of the terminal amino acids.

[0125] The nucleic acid sequence encoding the antibody can be synthesized, for example, by cloning a suitable sequence, or by the phosphotriester method described in Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method described in Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramidite method described in Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; or, for example, by using an automated synthesizer as described in Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984, Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, They can be prepared by direct chemical synthesis, such as the solid-phase phosphoramidite triester method described in 1981, and the solid-phase support method described in U.S. Patent No. 4,458,066. Chemical synthesis produces single-stranded oligonucleotides, which can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerization using a DNA polymerase that uses a single strand as a template. Longer sequences may be obtained by ligation of shorter sequences produced by chemical synthesis.

[0126] Exemplary nucleic acid coding sequences encoding antibodies that specifically bind to HERV-K Gag, Pol, or Env are prepared by cloning techniques. Sufficient instructions to guide those skilled in the art, including examples of appropriate cloning and sequencing techniques and numerous cloning exercises, can be found in Sambrook et al., above, Berger and Kimmel (eds.), above, and Ausubel, above. Product information from manufacturers of biological reagents and laboratory equipment also provides useful information. Such manufacturers include SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (San Diego, CA), and Applied Biosystems (Foster City, CA), as well as other commercial sources.

[0127] Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), and autologous persistent sequence replication systems (3SR). A wide variety of cloning methods, host cell types, and in vitro amplification methodologies are known.

[0128] In one example, a useful antibody is prepared by inserting cDNA encoding a variable region derived from an antibody that specifically binds to HERV-K Gag, Pol, or Env into a vector containing cDNA encoding an effector molecule (EM). The insertion is performed so that the variable region and EM are read in frame and a single consecutive polypeptide is generated. Thus, the encoded polypeptide contains a functional Fv region and a functional EM region. In one embodiment, cDNA encoding a detectable marker (e.g., an enzyme) is ligated to scFv such that the marker is located at the carboxyl terminus of scFv. In another example, the detectable marker is located at the amino terminus of scFv. In a further example, cDNA encoding a detectable marker is ligated to the heavy chain variable region of an antibody that specifically binds to HERV-K Gag, Pol, or Env such that the marker is located at the carboxyl terminus of the heavy chain variable region. The heavy chain variable region can then be ligated to the light chain variable region of an antibody that specifically binds to HERV-K Gag, Pol, or Env using disulfide bonds. In yet another example, a cDNA encoding a marker is ligated to the light chain variable region of an antibody that binds to HERV-K Gag, Pol, or Env, such that the marker is located at the carboxyl terminus of the light chain variable region. The light chain variable region can then be ligated to the heavy chain variable region of an antibody that specifically binds to HERV-K Gag, Pol, or Env using disulfide bonds.

[0129] Once nucleic acids encoding an antibody or a functional fragment thereof are isolated and cloned, the protein can be expressed in recombinantly engineered cells, such as bacterial cells, plant cells, yeast cells, insect cells, and mammalian cells. One or more DNA sequences encoding an antibody or a functional fragment thereof can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. This term also includes any offspring of the target host cell. It should be understood that not all offspring may be identical to the parent cell, as mutations may occur during replication.

[0130] A polynucleotide sequence encoding an antibody or a functional fragment thereof (e.g., scFV) can be ligated to an expression regulatory sequence. The expression regulatory sequence ligated to the coding sequence is ligated so that the expression of the coding sequence is achieved under conditions compatible with the expression regulatory sequence. Expression regulatory sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, pre-start codons (i.e., ATGs) of protein-coding genes, splicing signals for introns, maintenance of the correct reading frame of the gene to enable proper translation of mRNA, and stop codons. RNA encoding the disclosed antibody may also be useful.

[0131] Polynucleotide sequences encoding antibodies or their functional fragments may be manipulated to allow insertion or incorporation of sequences and inserted into expression vectors, including plasmids, viruses, or other vehicles that can be expressed in either prokaryotes or eukaryotes. Hosts may include microorganisms, yeasts, insects, and mammals. Methods for expressing DNA sequences having eukaryotic sequences or prokaryotic viral sequences can be used. Biologically functional viral and plasmid DNA vectors that can be expressed and replicated in a host can be used.

[0132] Host cells may be transformed using recombinant DNA. If the host is a prokaryote, such as E. coli, competent cells capable of DNA uptake can be prepared from cells collected after the exponential growth phase and subsequently treated by the CaCl2 method. Alternatively, MgCl2 can be used. Transformation may also be carried out, if desired, after the formation of host cell protoplasts or by electroporation.

[0133] When the host is a eukaryote, methods of DNA transfection such as calcium phosphate coprecipitation, mechanical procedures such as microinjection, electroporation, insertion of a liposome-encapsulated plasmid, or viral vectors may be used. Eukaryotic cells can also be co-transformed with an antibody or a functional fragment thereof, and a second exogenous DNA molecule encoding a selectable phenotype, such as a polynucleotide sequence encoding the herpesthymidine kinase gene. Another method is to transiently infect or transform eukaryotic cells with eukaryotic viral vectors, such as simian virus 40 (SV40) or bovine papillomavirus, to express the protein (see, e.g., Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). Expression systems, such as plasmids and vectors, can be used to generate proteins in cells including higher eukaryotic cells, such as COS, CHO, HeLa, and myeloma cell lines.

[0134] The isolation and purification of recombinantly expressed polypeptides can be carried out, for example, using preparative chromatography and immunological separation. Once expressed, recombinant antibodies can be purified, for example, using ammonium sulfate precipitation, affinity columns, column chromatography, etc. (see R. Scopes, Protein Purification, Springer-Verlag, NY, 1982 in general). Substantially pure compositions with at least about 90–95% homogeneity are disclosed herein, and 98–99% or higher homogeneity can be used for pharmaceutical purposes. Once partially or homogeneously purified as desired, the polypeptides should not be substantially endotoxin-free when used therapeutically.

[0135] Methods for the expression and / or refolding into a suitable active form of single-chain antibodies, including single-chain antibodies derived from bacteria such as E. coli, are applicable to the antibodies disclosed herein. See Buchner et al., Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991; Huse et al., Science 246:1275, 1989 and Ward et al., Nature 341:544, 1989.

[0136] In many cases, functional heterologous proteins from E. coli or other bacteria require isolation from inclusion bodies and solubilization and subsequent refolding using a strong denaturant. During the solubilization step, a reducing agent must be present to separate the disulfide bonds. An exemplary buffer with a reducing agent is 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, and 0.3 M DTE (dithioerythritol). Reoxidation of disulfide bonds can occur in the presence of reduced and oxidized low molecular weight thiol reagents, as described above in Saxena et al., Biochemistry 9: 5015-5021, 1970, Buchner et al.

[0137] Restoration is typically achieved by denaturing the protein in a refolding buffer and diluting the reduced protein (e.g., 100-fold). An example buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

[0138] As a modification to the two-chain antibody purification protocol, the heavy and light chain regions are solubilized and reduced separately and then combined in the refolding solution. Exemplary yields are obtained when these two proteins are mixed in a molar ratio that does not exceed a 5-fold molar excess of one protein relative to the other. After the redox shuffling is complete, it is desirable to add excess oxidized glutathione or other oxidative low molecular weight compounds to the refolding solution.

[0139] In addition to recombinant methods, the antibodies and their functional fragments disclosed herein can also be constructed, in whole or in part, using standard peptide synthesis. Solid-phase synthesis of polypeptides shorter than approximately 50 amino acids can be achieved by attaching the C-terminal amino acid of the sequence to an insoluble support, followed by the sequential addition of the remaining amino acids in the sequence. Techniques for solid-phase synthesis are described in Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963 and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984. Longer proteins may be synthesized by condensation of the amino and carboxyl terms of shorter fragments. A method for forming peptide bonds by activating the carboxyl terminus (for example, by using the coupling reagent N,N'-dicyclohexylcarbodimide) can be used. A pharmaceutical composition for the delivery of nucleic acid molecules encoding antibodies is disclosed below.

[0140] B. Inhibitory nucleic acid molecules Drugs that inhibit HERV-K may be inhibitory nucleic acid molecules. Inhibitory nucleic acids that reduce the expression and / or activity of HERV-K Gag, Pol, or Env can also be used in the manner disclosed herein. In some examples, such inhibitory nucleic acid molecules reduce the expression or activity of HERV-K Gag, Pol, or Env by at least 20%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100%. One embodiment is RNA interference (RNAi), such as, but not limited to, small inhibitory RNA (siRNA) or small hairpin RNA, which can be used to interfere with or inhibit the expression of a target. RNAs that specifically target HERV-K Gag, Pol, or Env are commercially available, for example, from Santa Cruz Biotechnology, Inc., ThermoFisher Scientific, and Sigma Aldrich. PCT Publication WO2017 / 059122 discloses the use of polynucleotide sequences in the LTR of the HERV-K genome, where the polynucleotide sequences contain eight or more consecutive pyrimidine bases to reduce HERV-K transcription. There are 10 inhibitory mRNAs disclosed as useful (SEQ ID NOs. 1-10 in PCT Publication WO2017 / 059122). Exemplary commercially available RNAi sequences specific to HERV-K Gag, Pol, or Env that can be used in the manner disclosed include those that target any portion of the HERV-K open reading frame.

[0141] Generally, siRNA is produced by the cleavage of relatively long double-stranded RNA molecules by dicers or DCL enzymes (Zamore, Science, 296:1265-1269, 2002; Bernstein et al., Nature, 409:363-366, 2001). In animals and plants, siRNA is assembled into RISC, which guides the sequence-specific ribonucleic acid degradation activity of RISC, thereby resulting in the cleavage of mRNA or other RNA target molecules in the cytoplasm. In the nucleus, siRNA also guides heterochromatin-associated histone and DNA methylation, resulting in transcriptional silencing of individual genes or large chromatin domains.

[0142] This disclosure provides RNA suitable for interfering with or inhibiting the expression of HERV-K Gag, Pol, or Env. The RNA can also be used to inhibit the LTR, promoter, or enhancer of HERV-K. Other parts that affect the binding of transcription factors to the LTR are also useful. In some embodiments, the inhibitory RNA includes a double-stranded RNA of about 19 to about 40 nucleotides having a sequence substantially identical to a portion of the mRNA or transcript of the target gene, e.g., HERV-K Gag, Pol, or Env, or any regulatory sequence, e.g., an LTR, whose expression interference or inhibition is desired.

[0143] For the purposes of this disclosure, RNA sequences that are "substantially identical" to a specific portion of the mRNA or transcript of a target gene whose expression interference or inhibition is desired differ from the specific portion of the mRNA or transcript of the target gene by about 30% or less, and in some embodiments, by about 10% or less or 5% or less. In certain embodiments, the RNA sequence is exactly identical to a specific portion of the mRNA or transcript of the target gene (e.g., HERV-K Gag, Pol, or Env transcript). Target sequences are provided in Figures 15A–15B.

[0144] Therefore, the siRNAs disclosed herein comprise double-stranded RNA having a 3' or 5' overhang having approximately 15 to approximately 40 nucleotides in length and 0 to 5 nucleotides in length in each strand, where the sequence of the double-stranded RNA is substantially identical to a portion of the mRNA or transcript of the nucleic acid encoding HERV-K Gag, Pol, or Env (see above). In certain examples, the double-stranded RNA contains approximately 19 to approximately 25 nucleotides, e.g., 20, 21, or 22 nucleotides, substantially identical to the nucleic acid encoding HERV-K Gag, Pol, or Env. In additional examples, the double-stranded RNA contains approximately 19 to approximately 25 nucleotides, 100% identical to the nucleic acid encoding HERV-K Gag, Pol, or Env. It should be noted that in this context, "approximately" refers only to integer amounts. In one example, "approximately" 20 nucleotides refers to nucleotides with a length of 19 to 21 nucleotides.

[0145] With respect to overhangs on double-stranded RNA, the length of the overhang is independent between the two strands in that the length of one overhang does not depend on the length of the overhang on the other strand. In specific examples, the length of the 3' or 5' overhang is 0 nucleotides on at least one strand, and in some cases, it is 0 nucleotides on both strands (hence blunt dsRNA). In other examples, the length of the 3' or 5' overhang is 1 to 5 nucleotides on at least one strand. More specifically, in some examples, the length of the 3' or 5' overhang is 2 nucleotides on at least one strand, or 2 nucleotides on both strands. In certain examples, a dsRNA molecule has a 3' overhang of 2 nucleotides on both strands.

[0146] Therefore, in a particular embodiment, the double-stranded RNA contains 20, 21, or 22 nucleotides, and the length of the 3' overhang is 2 nucleotides in both strands. In the embodiments of RNA provided herein, the double-stranded RNA contains about 40–60% adenine + uracil (AU) and about 60–40% guanine + cytosine (GC). More specifically, in a particular example, the double-stranded RNA contains about 50% AU and about 50% GC.

[0147] RNA further comprising, for example, at least one modified ribonucleotide in the sense strand of a double-stranded RNA is also disclosed herein. In certain examples, the modified ribonucleotide is located in the 3' overhang of at least one strand, or more specifically, in the 3' overhang of the sense strand. Examples of modified ribonucleotides are intended to include ribonucleotides comprising detectable labels (e.g., fluorophores, e.g., rhodamine or FITC), thiophosphate nucleotide analogs, deoxynucleotides (considered modified because the base molecule is ribonucleic acid), 2'-fluorouracil, 2'-aminouracil, 2'-aminocytidine, 4-thiouracil, 5-bromouracil, 5-iodouracil, 5-(3-aminoallyl)-uracil, inosine, or 2'O-Me-nucleotide analogs.

[0148] Nucleotide analogs may also involve modifications to the sugar moiety of the nucleotide. For example, the 2'OH group may be replaced by H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, or COOR, where R is substituted or unsubstituted C. l These include ~C6 alkyl, alkenyl, alkynyl, and aryl compounds. Other possible modifications are described in U.S. Patents 5,858,988 and 6,291,438. Locked nucleic acids (LNAs), often referred to as isolated RNA, are modified RNA nucleotides. The ribose portion of an LNA nucleotide is modified by an extra crosslink connecting the 2' oxygen and 4' carbon atoms.

[0149] Antisense and ribozyme molecules for HERV-K Gag, Pol, or Env are useful in the methods disclosed herein. An antisense nucleic acid is a DNA or RNA molecule that is complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American 262:40, 1990). In cells, antisense nucleic acids hybridize with the corresponding mRNA to form a double-stranded molecule. Antisense nucleic acids interfere with mRNA translation because cells do not translate double-stranded mRNA. Antisense oligomers of about 15 nucleotides can be used because they are readily synthesized and less likely to cause problems than larger molecules when introduced into target cells producing HERV-K Gag, Pol, or Env (see, e.g., Marcus-Sakura, Anal. Biochem. 172:289, 1988).

[0150] Antisense oligonucleotides can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides long. Antisense nucleic acids can be constructed using chemosynthesis and enzymatic ligation reactions. For example, antisense nucleic acid molecules can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the double helix formed between the antisense and sense nucleic acids. Phosphothioate derivatives and acridine-substituted nucleotides can be used, for example. Examples of modified nucleotides that can be used to produce antisense nucleic acids include, among others, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridin (thiouridin-e), 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, and inosine.

[0151] The use of oligonucleotides to halt transcription is a triple-strand strategy in which the oligonucleotide wraps around the double-helix DNA, forming a triple-stranded helix. Therefore, these triple-stranded compounds can be designed to recognize specific sites on selected genes (Maher, et al., Antisense Res. and Dev. 1(3):227, 1991; Helene, C., Anticancer Drug Design 6(6):569), 1991). This type of inhibitory oligonucleotide is also useful in the methods disclosed herein.

[0152] Ribozymes, which are RNA molecules capable of specifically cleaving other single-stranded RNAs in a manner similar to DNA restriction endonucleases, are also useful. By modifying the nucleotide sequences encoding these RNAs, it is possible to recognize specific nucleotide sequences within RNA molecules and manipulate molecules that cleave them (Cech, J. Amer. Med. Assn. 260:3030, 1988). Because they are sequence-specific, only mRNA with a particular sequence is inactivated.

[0153] There are two basic types of ribozymes: the tetrahymena type (Hasselhoff, Nature 334:585, 1988) and the "hammerhead" type. Tetrahymena ribozymes recognize sequences that are 4 nucleotides long, while "hammerhead" ribozymes recognize sequences that are 11 to 18 nucleotides long. As the recognition sequence length increases, the sequence is more likely to be exclusively present in the target mRNA species. Therefore, hammerhead ribozymes are preferred over tetrahymena ribozymes for inactivating specific mRNA species, and 18-nucleotide recognition sequences are preferred over shorter recognition sequences.

[0154] siRNA and other inhibitory nucleic acid molecules can be administered as therapeutic agents using various delivery systems. Such systems include, for example, encapsulation in liposomes, microparticles, microcapsules, and nanoparticles; recombinant cells capable of expressing therapeutic molecules (see, e.g., Wu et al., J. Biol. Chem. 262, 4429, 1987); and the construction of therapeutic nucleic acids as part of retroviruses or other vectors. Nucleic acid molecule delivery is discussed below.

[0155] C.CRISPR / Cas13 As disclosed in PCT Publication WO2019 / 040664, incorporated herein by reference, the Cas13 protein (and coding sequence) and guide molecule (e.g., gRNA and coding sequence) can be used in a CRISPR / Cas system to target one or more RNA molecules, e.g., HERV-K. A CRISPR / Cas system for RNA targeting comprises two common components: (1) the Cas13 protein or its coding sequence (whose expression may be driven by a promoter), and (2) a guide nucleic acid molecule, e.g., RNA (gRNA) (whose expression may also be driven by a promoter), which is specific to the target RNA. When introduced into cells, for example, (1) as Cas13 mRNA and gRNA, (2) as part of a single vector or plasmid, or split into multiple vectors or plasmids, (3) as separate Cas13 proteins and guide molecules, or (4) as an RNP complex of the Cas13 protein and guide molecule, the guide molecule guides Cas13 to the target RNA (e.g., HERV-K). In some embodiments, Cas13 is Cas13a, Cas13b, Cas13c, Cas13Rx, Cas13x, or Cas13y. In certain non-limiting examples, Cas13 is Cas13Rx. Exemplary Cas13 sequences are found in PCT Publication WO2019 / 040664, US11,293,011, and US10,392,616, and are also provided below. In some examples, CRISPR / Cas system administration for HERV-K RNA targeting is directed at the eye.

[0156] In some embodiments, Cas13 is Cas13Rx. An exemplary nucleic acid sequence encoding Cas13Rx is: [ka] [ka] (See Addgene, available on the internet (.addgene.org / 138149 / sequences / 2771-5668)). Another exemplary nucleic acid sequence encoding Cas13Rx is: [ka] [ka] That is the case. An exemplary Cas13Rx protein sequence is: [ka] That is the case.

[0157] If the Cas13Rx protein has a native HEPN domain or is fused to a suitable effector domain with RNase activity, RNA can be cleaved. If the Cas13Rx protein has a mature HEPN domain, the guide array can process mature gRNA, but the target RNA is not cleaved. Using this system, RNA sequences can be easily targeted and, for example, edited or detected by the effector domain as needed. Therefore, as an example, the Cas13Rx protein is represented by Sequence IDs 1, 2, 3, 4, 5, 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, in PCT Publication WO2019 / 040664, which is incorporated herein by reference. 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, 1 07, 108, 109, 110, 111, 112, 113, 138, 147, 19, 153, 155, 158, 160, 162, 164, 166, 168, 170, 175, 177, 179, 181, 183, 185, 187, 189, 194, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 229, 231, 233, 235, 237, 2 It has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to 39, 241, 243, 245, 247, 249, 253, 278, 279, 280, 281, 282, 283, 284, 285, 292, 293, 294, 295, or 296.

[0158] In one example, the Cas13 protein is expressed and purified in recombinant cells. The resulting purified Cas13 protein, along with a suitable guide molecule specific to the target RNA, is then introduced into the retina where one or more RNAs can be targeted. In some examples, the Cas13 protein and the guide nucleic acid molecule are introduced into the retina as separate components. In other examples, the purified Cas13 protein is complexed with a HERV-K specific guide nucleic acid (e.g., gRNA), and this ribonucleoprotein (RNP) complex is introduced into retinal cells (e.g., using transfection or injection).

[0159] When the Cas13 protein and HERV-K guide nucleic acid molecule are present in retinal cells, one or more HERV-K RNAs can be targeted. In some embodiments, Cas13 is Cas13a, Cas13b, Cas13c, Cas13Rx, Cas13x, or Cas13y. In other embodiments, Cas13 is Cas13Rx.

[0160] In one example, the Cas13 protein is expressed from a nucleic acid molecule in retinal cells containing the target HERV-K. In some such examples, the Cas13 protein is expressed from a vector, such as a viral vector (e.g., adenovirus vector, lentiviral vector, or baculovirus vector) or plasmid, which is introduced into retinal cells. This results in the production of the Cas13 protein in retinal cells. In addition, these nucleic acid molecules may be co-expressed in retinal cells with a HERV-K-specific guide nucleic acid molecule (e.g., gRNA).

[0161] In one example, multiple plasmids or vectors are used for RNA targeting. The nucleic acid molecule encoding Cas13 may be provided in, for example, one vector or plasmid, and the guide nucleic acid molecule (e.g., gRNA) may be provided in another plasmid or vector. Multiple plasmids or viral vectors can be mixed and introduced into retinal cells, either simultaneously or separately.

[0162] In some cases, multiple nucleic acid molecules are expressed from a single vector or plasmid. For example, a single vector may contain a nucleic acid molecule encoding Cas13, while another vector may contain a guide molecule.

[0163] In some cases, multiple different guide molecules (e.g., gRNAs) specific to each HERV-K (e.g., Gag, Pol, and / or Env targets) are present on a single array and / or vector. In one example, the method involves delivering multiple gRNAs (e.g., at least two, at least three, at least four, or at least five different gRNAs) that are part of an array (which may be part of a vector, e.g., a viral vector or plasmid). Once introduced into retinal cells, the array is processed into individual mature gRNAs by a Cas13 protein, e.g., a Cas13Rx protein. The nucleic acid molecules expressed from the vector may be under the control of a promoter and may contain a selection marker (e.g., antibiotic resistance) as needed.

[0164] In one example, RNA-targeting methods result in editing the sequence of the target RNA. For instance, by using a Cas13Rx protein with an immature HEPN domain (e.g., Sequence IDs 1, 3, 42, 62, 70, 82, 83, or 92 of PCT Publication WO2019 / 040664) and a gRNA containing at least one spacer sequence specific to the target RNA, the target RNA can be cleaved or truncated at a precise location. In some cases, such methods are used to reduce the expression of the target HERV-K RNA, which reduces the translation of Gag, Pol, and / or Env. Combinations of gRNAs for these targets are also useful.

[0165] For example, targeting a specific HERV-K RNA (e.g., Gag, Pol, and / or Env) can reduce the expression of the RNA-encoded protein. For instance, by using a Cas13Rx fusion protein with a mature HEPN domain and a translation repression domain (e.g., Pumilio or FBF PUF protein, deadenylase, CAF1, Argonaut protein, etc.) and a guide RNA containing at least one spacer sequence specific to the target HERV-K RNA, the expression of a specific HERV-K RNA, e.g., Gag, Pol, and / or Env, can be reduced. In some cases, Cas13Rx can be fused to a ribonuclease (e.g., the PIN endonuclease domain, NYN domain, SMR domain from SOT1, or the RNase domain from a Staphylococcal nuclease) or a domain that affects RNA stability (e.g., tristetraproline, or domains from UPF1, EXOSC5, and STAU1).

[0166] In another example, RNA aptamer sequences can be appended to or inserted into gRNA molecules, such as MS2, PP7, Qβ, and other aptamers. Proteins that specifically bind to these aptamers, such as the MS2 phage coat protein, can be fused to translation repression domains, ribonucleases, or domains that affect RNA stability. Since the Cas13 and gRNA complex guides the aptamer protein-effector domain to the target RNA, this aptamer-effector domain fusion can be used to target the target RNA. In one example, the target RNA is a HERV-K RNA encoding, for example, Gag, Pol, or Env. For example, a guide RNA containing the Cas13 protein and at least one spacer sequence specific to HERV-K RNA can be used. In some examples, HERV-K translation is reduced using such a method.

[0167] In some examples, the gRNAs that hybridize with one or more target HERV-K RNA molecules contain one or more direct repeat (DR) sequences, one or more spacer sequences, or one or more sequences containing a DR-spacer-DR-spacer. In some examples, one or more DR sequences are sequence numbers 129, 130, 131, 132, 133, 134, 135, 136, 137, 148, 150, 151, 152, 154, 156, 157, 159, 161, 163, 165, 167, 169, 176, 178, 180, 182, 184, 186, 188, 190, 191, 192, 193, 199, 201, 203, 201, 203, 201, 203, 201, 203, 201, 203, 201, 201, 203, 201, 191, 191, 192, 193, 199, 201, 203, 201, 19201, 203, 201, 191, 191, 191, 191, 191, 191, 191, 191, 191, 191, 191 It has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to 05, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, or 254. In one example, the gRNA contains additional sequences, such as aptamer sequences.

[0168] In some cases, multiple gRNAs are processed from a single array transcript, where each gRNA may differ, for example, to target different RNAs (e.g., two or more of HERV-K Gag, Pol, and Env) or to target multiple regions of a single RNA (e.g., HERV-K Gag, Pol, or Env).

[0169] The guide sequence may also contain one or more direct repeats (DRs). The DRs are the constant portion of the guide and contain strong secondary structures that facilitate the interaction between the Cas13 protein and the guide molecule.

[0170] Methods for targeting one or more target HERV-K RNA molecules are provided. Targeting an RNA molecule may include, for example, cleaving or desecrating one or more target HERV-K RNA molecules encoding Gag, Pol, and / or Env; inactivating or downmodulating one or more target HERV-K RNA molecules; inactivating or repressing the translation of one or more target HERV-K RNA molecules; visualizing, labeling, or detecting one or more target HERV-K RNA molecules; ligating one or more target HERV-K RNA molecules; editing one or more target HERV-K RNA molecules; tracking one or more target HERV-K RNA molecules; and masking one or more target HERV-K RNA molecules. In some examples, modifying one or more target HERV-K RNA molecules may include one or more RNA base substitutions, RNA base deletions, RNA base insertions, or cleavage of the target HERV-K RNA.

[0171] gRNA molecules can contain naturally occurring or non-naturally occurring nucleotides or ribonucleotides (e.g., LNA, or other chemically modified nucleotides or ribonucleotides, to protect the guide RNA from degradation). In some examples, the guide sequence is RNA. The guide nucleic acid can contain modified bases or chemical modifications (see, for example, Latorre et al., Angewandte Chemie 55:3548-50, 2016). The guide sequence directs the Cas13 protein to the target HERV-K RNA, thereby targeting the HERV-K RNA (e.g., modifying or detecting the RNA).

[0172] The ability of a guide sequence to direct the sequence-specific binding of the CRISPR complex to the target HERV-K RNA can be evaluated by any suitable assay. For example, sufficient components of the CRISPR system to form a CRISPR complex, including the guide sequence to be tested, may be provided to host cells having the corresponding target HERV-K RNA molecule, for example, by transfection with a vector encoding the components of the CRISPR sequence, followed by evaluation of preferential cleavage within the target sequence. Similarly, cleavage of the target HERV-K RNA sequence may be evaluated in vitro by providing the target HERV-K RNA, which is a component of the CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and by comparing the rates of binding or cleavage at the target HERV-K RNA between the reactions of the test and control guide sequences. Other assays are possible.

[0173] Also provided are vectors, e.g., viral vectors or plasmids (e.g., retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or herpes simplex viruses) containing a guide nucleic acid molecule that targets HERV-K. In some examples, the guide nucleic acid molecule is operably ligated to a promoter or expression regulatory element (specific examples of this are provided elsewhere in this application). As described elsewhere in this specification, such vectors may include other elements, e.g., a selection marker, e.g., an antibiotic, e.g., puromycin, hygromycin, or a detectable marker, e.g., a gene encoding GFP or other fluorophores. The vector may be an adenovirus vector. The vector may be a lentivirus vector. The vector may be a baculovirus vector. In some embodiments, the lentivirus vector, adenovirus vector, or baculovirus vector operably ligated to a nucleic acid molecule encoding Cas13, e.g., Cas13Rx, includes an inducible promoter (e.g., a doxycycline-inducible promoter), a constitutive promoter (e.g., CMV), or a tissue-specific promoter (e.g., RPE-specific).

[0174] The guide molecule may contain one or more regions referred to as spacers. The spacers hybridize with the target HERV-K RNA and have sufficient complementarity with the target HERV-K RNA sequence to direct the sequence-specific binding of the Cas13 protein, e.g., Cas13d, to the target HERV-K RNA. Therefore, the spacers are variable portions of the guide sequence. In some examples, the spacers have 100% complementarity with the target HERV-K RNA (or the region of the HERV-K RNA being targeted), but the spacers may have less than 100% complementarity with the target RNA, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% complementarity with respect to the target RNA. In some embodiments, the gRNA specifically hybridizes to a) a nucleic acid molecule encoding RNA or gRNA encoding the HERV-K glycosaminoglycan (Gag) protein; b) a nucleic acid molecule encoding RNA or gRNA encoding the HERV-K polymerase (Pol) protein; or c) a nucleic acid molecule encoding RNA or gRNA encoding the HERV-K envelope (Env) protein. Exemplary guide molecules are provided as Sequence IDs 1-3 of this disclosure. Delivery of the nucleic acid molecule is described below.

[0175] Host cells transduced with the disclosed vectors are also provided. These host cells may be in vitro. In some embodiments, the cells may be retinal pigment epithelial cells, photoreceptor cells, or choroidal cells.

[0176] D. Antiretroviral agents One or more antiviral agents can inhibit HERV-K and thus are disclosed herein to be useful for treating AMD. In some embodiments, an effective amount of a reverse transcriptase inhibitor, such as lamivudine, zidovudine, abacavir, tenofovir, tenofovir prodrug, emtricitabine (FTC), or a pharmaceutically acceptable salt thereof, is administered to a subject having or at risk of developing AMD. In some embodiments, an effective amount of a reverse transcriptase inhibitor is administered to the subject. The reverse transcriptase inhibitor can be abacavir or zidovudine. The reverse transcriptase inhibitor can be efavirenz, etravirine or nevirapine. In a non-limiting example, the reverse transcriptase inhibitor is nevirapine. In other embodiments, an effective amount of an integrase inhibitor is administered to the subject. The integrase inhibitor can be raltegravir. In a further embodiment, an effective amount of a protease inhibitor can be administered to the subject. The protease inhibitor can be darunavir.

[0177] The disclosed methods can use an effective amount of an NRTI, NtRTI, integrase inhibitor, and / or protease inhibitor. Combinations of these agents are also useful. Suitable agents are also disclosed in Tyagi et al, supra, 2017, which is incorporated herein by reference.

[0178] Effective NtRTIs for treating AMD or reducing the risk of developing AMD include tenofovir, tenofovir prodrugs such as tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF), adefovir; 2',3'-dideoxy-3'-fluoroadenosine; 2',3'-dideoxy-3'-fluoroguanosine; 3'deoxy-3'-fluoro-5-O-[2-(L-valyloxy)-propionyl]guanosine, and pharmaceutically acceptable salts, esters, ester salts, nitrile oxides, and other prodrugs of any of the active agents. Effective NRTIs for treating AMD or helping to reduce the risk of developing AMD include FTC, lamivudine, zalcitabine, zidovudine, azidothymidine, didanosine, stabudine, and abacavir. Other antiretroviral agents, such as non-nucleoside reverse transcriptase inhibitors, protease inhibitors, fusion inhibitors, and combinations thereof, are also useful. Representative non-nucleoside reverse transcriptase inhibitors effective herein include, by example, delavirdin, efavirenz, nevirapine, and other diarylpyrimidine (DAPY) derivatives. Representative protease inhibitors effective herein include, by example, amprenavir, tipranavir, indinavir, saquinavir, lopinavir, ritonavir, fosamprenavir calcium, ritonavir, atazanavir sulfate, nelfinavir mesylate, darunavir, and combinations thereof. Examples of entry inhibitors effective herein as optional active ingredients in the inventive composition include, by example, enfvirtide, Schering C (Schering Plough), S-1360 (Shionogi), and BMS806 (Bristol Myers Squibb). Examples of derivatives and salts useful in the disclosed methods include salts such as alkali metal salts; esters such as acetates, butyrates, octinoates, palmitates, chlorobenzoates, benzoates, C1-C6 benzoates, succinates, and mesylates; salts of such esters; and nitrile oxides.

[0179] Any pharmaceutically acceptable salt, ester, ester salt, nitroxide, and prodrug of the active agent are also useful in the disclosed methods. In some embodiments, both an NRTI and / or an NtRTI are administered to the subject. The NRTI and / or NtRTI can be administered as a prodrug. As used herein, the term "prodrug" includes a compound that results in an active NRTI or NtRTI as a result of a spontaneous reaction, enzyme catalysis, metabolic clearance, or a combination thereof under physiological conditions when administered to a primate host. An exemplary NtRTI prodrug currently approved by the FDA for use in HAART is tenofovir disoproxil fumarate (TDF), which details TAF and the succinate of tenofovir in U.S. Patent No. 5,935,946. In some embodiments, the method includes the administration of tenofovir, TAF, and / or TDF. In further embodiments, an effective amount of a protease inhibitor, such as amprenavir, tipranavir, indinavir, saquinavir, lopinavir, ritonavir, fosamprenavir calcium, ritonavir, atazanavir sulfate, nelfinavir mesylate, and darunavir is administered. In a specific non-limiting example, an effective amount of darunavir is administered to the subject. In another specific non-limiting example, an effective amount of a) tenofovir, TAF, and / or TDF is administered to the subject and b) a protease inhibitor is administered to the subject. In another specific non-limiting example, an effective amount of a) tenofovir, TAF, and / or TDF is administered to the subject and b) darunavir is administered to the subject.

[0180] Other pharmaceutically active NRTIs, NtRTIs, or analogues of integrase inhibitors are also suitable for use. Pharmaceutically acceptable carriers or diluents include drugs that are compatible with the administration of other components and do not harm subjects who have AMD or are at risk of AMD. In some embodiments, the method comprises the administration of an effective dose of a) tenofovir, TAF and / or TDF, and b) FTC. In other embodiments, the method comprises the administration of an effective dose of a) tenofovir, TAF and / or TDF, and b) darunavir.

[0181] In some embodiments, NRTIs, NtRTIs, and, if necessary, integrase inhibitors are administered concurrently to the subject, for example, as a single formulation. This combination may be co-administered with or without an enhancer, for example, cobicistat (COBI), but is not limited to these. In some embodiments, the method includes administration of an effective dose of a) tenofovir, TAF and / or TDF, b) FTC, and c) COBI.

[0182] In some embodiments, the method utilizes a combination of at least one NRTI, at least one NtRTI, and optionally an integrase inhibitor. The integrase inhibitor may be elvitegravir (EVG). In some embodiments, the method includes the administration of an effective dose of a) tenofovir, TAF and / or TDF, b) FTC; c) COBI; and d) EVG. In further embodiments, an effective dose of a protease inhibitor, for example, darunavir, is administered to the subject.

[0183] In some embodiments, these can be formulated into dosage forms suitable for delivery by routes of administration including intraocular (e.g., into the eye), oral, rectal, topical, vaginal, or parenteral administration. Compositions and methods of formulation are provided, for example, in Remington's Science and Practice of Pharmacology, 20th Edition, Chapters 37-47, pages 681-929, where parenteral injection includes subcutaneous, intramuscular, intravenous, and intradermal injection. In some embodiments, administration is oral. Administration may be administered by injection into the eye as a suspension, delayed-release implant, or eye drops.

[0184] In some embodiments, the disclosed method involves co-administering to a subject, for example, a person having or at risk of developing AMD, a combination of a pharmaceutically effective amount of a nucleoside reverse transcriptase inhibitor, e.g., FTC, an effective amount of a nucleotide reverse transcriptase inhibitor, e.g., tenofovir, an effective amount of tenofovir prodrug, e.g., tenofovir salts, e.g., TDF, TAF, or another salt form of tenofovir. If necessary, the subject may also be administered an effective amount of an integrase inhibitor, e.g., EVG. If necessary, the subject may also be administered an effective amount of a protease inhibitor, e.g., darunavir. These combinations may be co-administered with or without a drug enhancer, e.g., COBI, an effective amount. In some embodiments, the co-administration is orally. In more embodiments, the co-administration is simultaneous.

[0185] In some embodiments, an effective amount of a nucleoside reverse transcriptase inhibitor, e.g., FTC, but not limited to, an effective amount of a nucleotide reverse transcriptase inhibitor, e.g., tenofovir or tenofovir prodrug, e.g., TDF or TAF, and optionally an effective amount of an integrase inhibitor, e.g., EVG, can be formulated into a single composition, e.g., a unit dose. Optionally, an effective amount of an enhancer, e.g., COBI, can be included in this same composition. In some embodiments, the composition can be formulated for oral administration. Thus, these active agents can be combined into a single unit dose and administered to subjects who have AMD or are at risk of developing it.

[0186] The doses of individual active components are administered in effective amounts to produce therapeutic concentrations of the active composition in the retina. Establishing effective concentrations for a given active agent in target cells, e.g., the retina, is recognized to involve factors concerning the agent, such as the route of administration, pharmacokinetics, absorption rate based on the route of administration, the effect of food on oral absorption, in vivo distribution, metabolic pathways, excretion pathways, the race, sex, and age of the subject, incidental side effects of single doses, side effects of long-term administration, and synergistic effects with co-administered active agents. Information on these factors to consider when administering the drug is available from the U.S. Food and Drug Administration (fda.gov / oashi / aids / virals.html). In some embodiments, administration according to this method utilizes the maximum recommended tolerable dose level for a given combination of active agents related to the HAART treatment protocol as a starting point.

[0187] In some embodiments, the method involves oral administration of TAF / TDF / tenofovir / salt to subjects with AMD or at risk of developing AMD. In more embodiments, the method involves oral co-administration of FTC, TAF / TDF / tenofovir / salt to subjects with AMD or at risk of developing AMD. In more embodiments, FTC, TAF / TDF / tenofovir / salt, and EVG are administered to subjects with AMD or at risk of developing AMD. In other embodiments, the method involves oral co-administration of FTC, TAF / TDF / tenofovir / salt, optionally EVG, and optionally COBI to subjects. In further embodiments, FTC, TAF / TDF / tenofovir / salt, EVG, and COBI are administered to subjects. In specific non-limiting examples, TAF is used in any of the above combinations. In other non-limiting examples, TDF is used in any of the above combinations.

[0188] U.S. Published Patent Application No. 2015 / 0105350, incorporated herein by reference, discloses the use of FTC, TAF and other tenofovir prodrugs, EVG, and COBI for the treatment of HIV infection. As disclosed in this published patent application, oral doses of TAF may range from about 0.0001 to about 100 mg / kg body weight per day, for example, about 0.01 to about 10 mg / kg body weight per day, about 0.01 to about 5 mg / kg body weight per day, about 0.5 to about 50 mg / kg body weight per day, about 1 to about 30 mg / kg body weight per day, about 1.5 to about 10 mg / kg body weight per day, or about 0.05 to about 0.5 mg / kg body weight per day. As a non-limiting example, the daily dose for an adult human weighing approximately 70 kg is in the range of approximately 0.1 mg to approximately 1000 mg, or approximately 1 mg to approximately 1000 mg, or approximately 5 mg to approximately 500 mg, or approximately 1 mg to approximately 150 mg, or approximately 5 mg to approximately 150 mg, or approximately 5 mg to approximately 100 mg, or approximately 10 mg, and may be taken in the form of a single dose or multiple doses. In one embodiment, the oral dose of TAF may be in the form of a combination of drugs (e.g., TAF / FTC / EVG / COBI).

[0189] When COBI or a pharmaceutically acceptable salt thereof is combined with certain specific solid carrier particles (e.g., silica derivatives), the resulting combination has improved physical properties. For example, the resulting combination has lower hygroscopicity compared to COBI alone. In addition, the resulting combination is a free-flowing powder with a high loading value for COBI, acceptable physical and chemical stability, rapid drug release, and excellent compressibility. Therefore, the resulting combination can be easily processed into a solid dosage form (e.g., tablets). Thus, COBI can be used with any suitable solid carrier, provided that the resulting combination has physical properties that allow it to be formulated more easily than the parent compound. For example, suitable solid carriers include kaolin, bentonite, hectorite, colloidal aluminum magnesium silicate, silicon dioxide, magnesium trisilicate, aluminum hydroxide, magnesium hydroxide, magnesium oxide, and talc. In one embodiment, the solid carrier may include calcium silicate or magnesium aluminometasilicate. COBI can be coated into the pores and on the surface of the solid carrier. Useful and suitable silica derivatives are disclosed in PCT Publication WO03 / 037379.

[0190] Illustrative oral dosages useful in the disclosed method are (1) COBI: 10-500 mg, 50-500 mg, 75-300 mg, 100-200 mg, or 150 mg; (2) TAF: 1-60 mg, 3-40 mg, 5-30 mg, 8-20 mg, or 10 mg; (3) FTC: 10-500 mg, 50-500 mg, 75-300 mg, 150-250 mg, or 200 mg; and (4) EVG: 10-500 mg, 50-500 mg, 75-300 mg, 100-200 mg, or 150 mg. Tenofovir may be used in amounts less than 300 mg, less than 200 mg, and less than 100 mg. COBI can be used in doses of 50-500 mg, 100-400 mg, 100-300 mg, and 150 mg.

[0191] Tenofovir (or TDF or TAF, or another salt form) and COBI or a pharmaceutically acceptable salt thereof can be co-administered orally. Tenofovir (or TDF or TAF, or another salt form), COBI, FTC, and EVG can be co-administered. Tenofovir (or TDF or TAF) and COBI can be co-administered in a single pharmaceutical composition. Tenofovir (or TDF or TAF, or another salt form), COBI, FTC, and EVG can be co-administered in a single pharmaceutical composition. In cases where a pharmaceutically acceptable salt or complex of the drug is administered, the amount administered may be adjusted relative to the weight of the components added to form the salt or complex.

[0192] The method may include co-administration of 200 mg of FTC and 150 mg of EVG. The method may include co-administration of 150 mg of COBI, 100 mg or less of tenofovir, 150 mg of EVG, and 200 mg of FTC. The method may include co-administration of 150 mg of COBI, 200 mg or less of tenofovir, 150 mg of EVG, and 200 mg of FTC. The method may include co-administration of 150 mg of COBI, less than 300 mg of tenofovir, 150 mg of EVG, and 200 mg of FTC. The method may include co-administration of 150 mg of COBI, 50 mg of tenofovir, 150 mg of EVG, and 200 mg of FTC. In some specific, non-limiting examples, the method may include co-administration of 150 mg of EVG, 150 mg of COB, 200 mg of FTC, and 10 mg of TAF. These compositions can be administered orally. For additional dosing information, see U.S. Published Patent Application No. 2015 / 0105350, incorporated herein by reference. In some embodiments, GENVOYA® is administered to the subject. In further embodiments, administration is orally.

[0193] One or more additional drugs in an effective dose may be administered to the subject. These drugs may include, but are not limited to, an effective dose of L-745,870 trihydrochloride and an effective dose of metformin or metformin hydrochloride.

[0194] The active agent may be administered to the subject in any conventional manner. Examples of localized administration include intraocular, intraorbital, subconjunctival, subtenon's capsule, subretinal, or transscleral routes. In some embodiments, a considerably smaller amount of the component (compared to a systemic approach) may be more effective when administered locally (e.g., intravitreal) than when administered systemically (e.g., orally). In one embodiment, the system disclosed herein is delivered by intravitreal injection. Intravitreal injection carries a relatively low risk of retinal detachment. The components described herein may be administered using any method for administering drugs to the eye. In some embodiments, administration is from an internal reservoir (e.g., from an implant placed in an intraocular or extraocular location (see U.S. Patents No. 5,443,505 and 5,766,242)) or from an external reservoir (e.g., from an intravenous bag). The components can be administered by continuous release over a specific period of time from a sustained-release drug delivery device immobilized on the inner wall of the eye or via targeted transscleral controlled release to the choroid (see, for example, PCT / US00 / 00207, PCT / US02 / 14279, Ambati et al., Invest. Opthalmol. Vis. Sci. 41:1181-1185, 2000, and Ambati et al., Invest. Opthalmol. Vis. Sci. 41:1186-1191, 2000). Various devices suitable for localized administration of the components into the eye are known. See, for example, U.S. Patents 6,251,090, 6,299,895, 6,416,777, 6,413,540, and PCT Publication PCT / US00 / 28187. An additional method for intraocular administration is provided below, which can be used in conjunction with antiretroviral agents.

[0195] While active agents can be administered as compounds in their pure form, they can also be administered as one or more pharmaceutical compositions. Salts, carriers, or diluents must be acceptable in the sense that they are compatible with other components and not harmful to the recipient. Examples of carriers or diluents for oral administration include corn starch, lactose, magnesium stearate, talc, microcrystalline cellulose, stearic acid, povidone, crospovidone, dibasic calcium phosphate, sodium starch glycolate, hydroxypropyl cellulose (e.g., low-substituted hydroxypropyl cellulose), hydroxypropyl methylcellulose (e.g., hydroxypropyl methylcellulose 2910), and sodium lauryl sulfate. Pharmaceutical compositions can be prepared by any suitable method, such as methods well known in the art of pharmaceutical preparation, e.g., those described in Gennaro et al., Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., 1990), particularly Part 8: Pharmaceutical Preparations and their Manufacture. In some embodiments, the active agent is formulated for oral administration. In some embodiments, the active agent is formulated for localized administration to the eye.

[0196] Such a method includes the step of associating an active agent with a carrier or diluent and, optionally, one or more auxiliary components. Such auxiliary components include conventional components in the art, such as fillers, binders, excipients, disintegrants, lubricants, colorants, flavoring agents, sweeteners, preservatives (e.g., antimicrobial preservatives), suspending agents, thickeners, emulsifiers, and / or wetting agents. Pharmaceutical compositions useful in the methods disclosed herein can provide a controlled delayed release or sustained release of an active agent over a period of time. Controlled delayed release or sustained release of a drug can maintain the drug in the human bloodstream for a longer period than conventional formulations.

[0197] Pharmaceutical compositions include, but are not limited to, coated tablets, pellets, solutions (e.g., eye drops), powders, capsules, and dispersions in a medium insoluble in physiological fluids, or the release of the therapeutic compound follows the decomposition of the pharmaceutical composition due to mechanical, chemical, or enzymatic action. For oral administration, fine powders or granules may contain diluents, dispersants, and / or surfactants, which may be present, for example, in water or syrup, in capsules or sachets in a dry state, or in a non-aqueous solution or suspension that may contain a suspending agent, or in tablets that may contain binders and lubricants. When administered in the form of a liquid solution or suspension, the formulation may contain one or more active ingredients and purified water. Optional components in a liquid solution or suspension include suitable sweeteners, flavoring agents, preservatives (e.g., antimicrobial preservatives), buffers, solvents, and mixtures thereof. Components of the formulation may perform two or more functions. For example, a suitable buffer may also act as a flavoring agent and a sweetener. Suitable sweeteners include, for example, sodium saccharin, sucrose, and mannitol. A mixture of two or more sweeteners may be used. The sweeteners or mixtures thereof are typically present in an amount of about 0.001% to about 70% by weight of the total composition. Suitable flavoring agents may be present in the pharmaceutical composition to provide a flavor that makes it easier for humans to take the pharmaceutical composition orally. The flavoring agents or mixtures thereof are typically present in an amount of about 0.0001% to about 5% by weight of the total composition.

[0198] Preservatives can also be present in the composition. Suitable preservatives include, for example, methyl paraben, propyl paraben, sodium benzoate, and benzalkonium chloride. Mixtures of two or more preservatives may be used. The preservative or mixture thereof is typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Buffering agents can also be present in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. Mixtures of two or more buffering agents may be used. The buffering agent can be present in an amount of about 0.001% to about 4% by weight of the total composition.

[0199] If a liquid suspension is desired, a solvent can be used. Suitable solvents for liquid solutions or suspensions include, for example, sorbitol, glycerin, propylene glycol, and water. Mixtures of two or more solvents may be used. The solvent or solvent system can be present in an amount of about 1% to about 90% by weight of the total composition. These types of formulations are disclosed, for example, in U.S. Patent Application Publication No. 2015 / 0105350, which is incorporated herein by reference.

[0200] In a further aspect, the active agents (e.g., TAF / TDF / tenofovir / salt, FTC, EVG, darunavir, and optionally, COBI) are co-administered to the subject at a particular time point. In some aspects, the method utilizes dosages of multiple agents. In some aspects, each dosage can include the oral co-administration of FTC, TAF / TDF / tenofovir / salt, and EVG. In other aspects, each dosage can include the oral co-administration of FTC, TAF / TDF, EVG, and COBI. In a specific non-limiting example, TAF is included in the dosage.

[0201] Agents that increase E.ANG activity It is also disclosed herein that one or more agents that increase ANG activity, for example, by generating tRNA fragments that are inhibitory to HERV-K and thereby increasing ANG's ability to reduce HERV-K expression, may be useful in treating AMD. ANG itself, or nucleic acid molecules encoding ANG, can be used to treat AMD in a subject, or to reduce the risk of AMD. Increased ANG activity generates tRNA fragments that are complementary to the HERV-K coding sequence.

[0202] While not bound by theory, downregulation of ANG expression is associated with an increase in HERV-K mRNA. ANG, a ribonuclease (which cleaves double-stranded RNA), uses a tRNA fragment complementary to HERV-K. This tRNA fragment forms a double strand with HERV-K, allowing ANG to degrade this double-stranded RNA. Increased ANG expression (with or without the tRNA fragment) downregulates HERV-K expression, which can be used to treat AMD or reduce the risk of developing AMD. In some embodiments, drugs that increase ANG activity are useful for treating AMD. These drugs can increase ANG activity by, for example, about 1 to 10 times, for example, about 2 to 10 times, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. Methods are not limited to, but may include administration of ANG, nucleic acid molecules encoding ANG, tRNA fragments, or nucleic acid molecules encoding tRNA fragments. Combinations of these drugs are also useful.

[0203] 1. ANG protein An exemplary protein sequence for ANG is disclosed in GENBANK® accession number AAA51678.1, October 30, 1994, incorporated herein by reference.

[0204] An example amino acid sequence for ANG is also shown below: [ka] [ka] The amino acid sequence for the angiogenin precursor protein is provided by reference to GENBANK®, accession number NP_001091046, August 16, 2022. An exemplary nucleic acid sequence encoding ANG is provided by reference to GENBANK, accession number NM_001145.4, August 15, 2022. [ka]

[0205] Any fragment, variant, or precursor protein of ANG having ANG activity, and the nucleic acid molecule encoding such a protein, are useful in the methods disclosed herein, provided that they function to inhibit HERV-K. In some embodiments, a variant of ANG useful in the methods disclosed herein comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11 (or the nucleic acid encoding it). A variant of ANG may contain up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conserved substitution in SEQ ID NO: 11. The protein may be naturally occurring or recombinant. In some embodiments, a variant of ANG useful in the disclosed method contains, is essentially, or consists of an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11 (or the nucleic acid encoding it). In other embodiments, ANG contains, is essentially, or consists of SEQ ID NO: 11 (or the nucleic acid encoding it). In more embodiments, a fragment of ANG is useful in the disclosed method.

[0206] The use of ANG and its variants is disclosed, for example, in U.S. Published Patent Application No. 2013 / 0136727A1. ANG, its variants, and fragments can be prepared using recombinant methods, such as expression in host cells. Exemplary nucleic acid molecules can be prepared by cloning techniques (see below). Examples of appropriate cloning and sequencing techniques, as well as instructions sufficient to guide those skilled in the art through numerous cloning exercises, are publicly known (e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4)). th See ed, Cold Spring Harbor, New York, 2012 and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013).

[0207] Potential hosts include microorganisms, yeasts, insects, and mammals. DNA sequences containing eukaryotic or viral sequences can be expressed in prokaryotes or eukaryotes. Non-limiting examples of suitable host cells include bacterial cells, archaeal cells, insect cells, fungal (e.g., yeast) cells, plant cells, and animal cells (e.g., mammalian cells, e.g., human). Useful exemplary cells include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, HEK293 cells, Neurospora, and immortalized mammalian myeloma and lymphoid cell lines. Techniques for propagating mammalian cells in culture are well known (e.g., Helgason and Miller (Eds.), 2012, Basic Cell Culture Protocols (Methods in Molecular Biology), 4). thSee Ed., Humana Press. Examples of commonly used mammalian host cell lines include VERO and HeLa cells, CHO cells, and WI38, BHK, and COS cell lines, but cell lines such as those designed to provide higher expression, desired glycosylation patterns, or other features may be used. In some embodiments, the host cell may be HEK293 cells or their derivatives, e.g., GnTI - / - Examples include cells (ATCC® number CRL-3022) or HEK-293F cells.

[0208] Transformation of host cells with recombinant DNA can be carried out by conventional techniques. In some embodiments, when the host is a prokaryote, for example, E. coli (but not limited to E. coli), competent cells capable of DNA uptake can be prepared from cells collected after the exponential growth phase and subsequently treated by the CaCl2 method. Alternatively, heat shock, MgCl2, or RbCl can be used. Transformation can also be carried out, if desired, after the formation of host cell protoplasts or by electroporation.

[0209] When the host is a eukaryote, methods of DNA transfection such as calcium phosphate coprecipitation, conventional mechanical procedures such as microinjection, electroporation, insertion of plasmids encapsulated in liposomes, or viral vectors can be used. Eukaryotic cells can also be co-transformed with the disclosed antigen and a second exogenous DNA molecule encoding a selectable phenotype, such as a polynucleotide sequence encoding the herpesthymidine kinase gene. Another method is to transiently infect or transform eukaryotic cells with eukaryotic viral vectors, such as simian virus 40 (SV40) or bovine papillomavirus, to express the protein (see, e.g., Viral Expression Vectors, Springer press, Muzyczka ed., 2011). Suitable expression systems, such as plasmids and vectors, that are useful for generating proteins in cells include higher eukaryotic cell lines, such as COS, CHO, HeLa, and myeloma cell lines. ANG can also be produced using chemosynthesis.

[0210] 2. Nucleic acid molecules and tRNAs encoding ANG In some embodiments, nucleic acid molecules encoding ANG, precursors, variants, or fragments are also useful in the disclosed methods. By introducing nucleic acids encoding ANG, precursors, variants, or fragments, the amount of ANG is increased, and thus the activity of ANG is also increased.

[0211] In more embodiments, tRNAs complementary to HERV-K or its fragments may also be useful in the disclosed manner and may be encoded by DNA molecules (see gene ID: 100189107). Two tRNA fragments complementary to HERV-K (encoded by DNA) are LYS CTT (GCCCCACGTTGGGCGCCA, SEQ ID NO: 12) and LYS TTT (GTCCCTGTTCGGGCGCCA, SEQ ID NO: 13), see, for example, Tao et al., above, 2020. SEQ ID NOs: 12 and 13 are sequences derived from the genomic DNA of tRNA-CTT (gene ID: 100189107, May 13, 2022, National Library of Medicine, incorporated herein by reference) and tRNA-TTT (gene ID: 7206, May 13, 2022, National Library of Medicine, incorporated herein by reference). These tRFs or tiRNAs are complementary to HERV-K. A 3-nucleotide CCA may be added to the end of the tRNA and may not be complementary to HERV-K. All of these are useful in the methods disclosed herein. In some embodiments, the tRNA fragment is 16–22 nucleotides long, e.g., 17–21 nucleotides long, e.g., 16, 17, 18, 19, 20, 21, or 22 nucleotides long. In one embodiment, the tRNA fragment is 18 nucleotides long. In more embodiments, the tRNA is encoded by SEQ ID NO: 12 or SEQ ID NO: 13. In further embodiments, the tRNA contains a CCA.

[0212] Expressing one or both of these tRNA fragments can increase ANG activity and downregulate HERV-K expression. While tRFs are generated by ANG, AGO proteins may also be involved in targeting the HERV-K coding sequence using these tRFs. Therefore, increasing ANG activity can, in some embodiments, block HERV-K translation to GAG, POL, and ENV proteins, inducing the degradation of the entire mRNA due to a double-stranded loop between the tRFs and the HERV-K encoding RNA. See Shorn et al., Trends Cell Biol. 28(10): 793-806. doi:10.1016 / j.tcb.2018.05.006 (October 2018).

[0213] Expression of one or both of these tRNA fragments increases ANG activity. Therefore, in some embodiments, nucleic acid molecules encoding one or both of these tRNAs are useful in the disclosed methods. These tRNAs can be used in the disclosed methods with or without nucleic acid molecules encoding ANG or exogenous ANG.

[0214] Nucleic acid molecules can be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), and autologous persistent sequence replication systems (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to those skilled in the art. RNA molecules are also useful.

[0215] Polynucleotides encoding ANG (or its precursor, variant, or fragment) or tRNA may include recombinant DNA that is incorporated into a vector (e.g., an expression vector), a self-replicating plasmid or virus, or the genomic DNA of a prokaryotic or eukaryote, or that exists as a separate molecule independent of other sequences (e.g., cDNA). A nucleotide can be a ribonucleotide, a deoxyribonucleotide, or a modified form of either nucleotide. This term includes single and bimodal forms of DNA.

[0216] Polynucleotides encoding ANG, its precursors, variants, or fragments, or tRNA fragments, are useful in the disclosed manner and include DNA, cDNA, tRNA sequences themselves, and RNA sequences encoding ANG, its precursors, variants, or fragments. Silent mutations in coding sequences arise from the degeneracy (i.e., redundancy) of the genetic code, thereby allowing two or more codons to encode the same amino acid residue. Therefore, for example, leucine may be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine may be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine may be encoded by AAT or AAC; aspartic acid may be encoded by GAT or GAC; cysteine ​​may be encoded by TGT or TGC; alanine may be encoded by GCT, GCC, GCA, or GCG; glutamine may be encoded by CAA or CAG; tyrosine may be encoded by TAT or TAC; isoleucine may be encoded by ATT, ATC, or ATA. Tables showing standard genetic codes can be found in various sources (e.g., L. Stryer, 1988, Biochemistry, 3 rd Edition, WH 5 Freeman and Co., NY). Degenerate variants are also useful in the methods disclosed herein.

[0217] Additional nucleic acid molecules encoding ANG, its precursors, variants, or fragments, or tRNA fragments, can be readily generated using the amino acid sequences and genetic codes provided herein. Nucleic acid sequences encoding ANG, its precursors, variants, or fragments, or tRNA fragments, can be synthesized by any preferred method, for example, including cloning of a suitable sequence, or by the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; for example, using an automated synthesizer described in Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984, Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, It can be prepared by direct chemical synthesis, such as the solid-phase phosphoramidite triester method described in 1981; and the solid-phase support method of U.S. Patent No. 4,458,066. Chemical synthesis produces single-stranded (ss) oligonucleotides, which can be converted to double-stranded (ds) DNA by hybridization with a complementary sequence or by polymerization by DNA polymerase using a single strand as a template. Exemplary nucleic acids containing sequences encoding ANG, its precursors, variants, or fragments, or sequences encoding tRNA fragments, can be prepared by cloning.

[0218] Nucleic acid molecules encoding ANG, its precursors, variants, or fragments, or tRNA fragments, can be cloned or amplified by in vitro methods, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), auto-persistent sequence replication systems (3SR), and Qβ replicase amplification systems (QB). For example, polynucleotides encoding ANG, its precursors, variants, or fragments, or tRNA fragments, can be isolated by polymerase chain reaction of cDNA using primers based on the molecule's DNA sequence. A wide variety of cloning and in vitro amplification methodologies can be used. The PCR method is described, for example, in U.S. Patent No. 4,683,195; Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). Polynucleotides can also be isolated by screening a genomic or cDNA library using probes selected from sequences of desired polynucleotides under stringent hybridization conditions.

[0219] Typically, a polynucleotide sequence encoding ANG, its precursor, variant, or fragment, or a tRNA fragment, is operably ligated to a transcriptional regulatory sequence, such as a promoter and a polyadenylation signal. Any promoter can be used, which is a polynucleotide sequence recognized by the host cell's transcriptional mechanism (or the introduced synthetic mechanism) involved in the initiation of transcription. The polyadenylation signal is a polynucleotide sequence that directs the addition of a series of nucleotides at the ends of the mRNA transcript for proper processing and translation of the transcript and for the transport of the transcript from the nucleus to the cytoplasm.

[0220] Exemplary promoters include viral promoters, such as the cytomegalovirus pre-early gene promoter ("CMV"), herpes simplex virus thymidine kinase ("tk"), SV40 early transcription unit, polyomas, retroviruses, papillomaviruses, hepatitis B virus, and human and simian immunodeficiency viruses. Other promoters include mammalian genes, such as immunoglobulin heavy chains, immunoglobulin light chains, T cell receptors, and HLA DQα and DQ Promoter isolates from β, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II, HLA-DRα, β-actin, muscle creatine kinase, prealbumin (trans tyretin), elastase I, metallothionein, collagenase, albumin, fetoprotein, β-globin, c-fos, c-HA-ras, neuronal adhesion molecules (NCAM), α1-antitrypsin, H2B (TH2B) histone, type I collagen, glucose regulatory proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid (SAA), troponin I (TNI), platelet-derived growth factor, and dystrophin, as well as promoters specific to retinal pigment epithelial cells, such as RPE65, BEST1, DCT, TYR, or TYRP1 promoters (Nicoletti et al., Invests Ophthalmol. Vis. Sci. 1998). See Mar;39(3):637-44;Esumi et al., Hum. Mol. Gen. 18: 128-141, 2009;Takeda et al., Biochem. Biophys. Res. Comm. 300: 908-914, 2003;Murisier et al., Dev. Biol. 303: 838-847, 2007;and Beermann, Cell. Mol. Biol. (Noisy-le-grand) 45(7):961-8, 1999.

[0221] Promoters can be either inductive or constitutive. Inductive promoters are promoters that are inactive or exhibit low activity except in the presence of an inducer. Additional examples of promoters include, but are not limited to, MT II, ​​MMTV, collagenase, stromelysin, SV40, mouse MX gene, α-2-macroglobulin, MHC class I gene h-2kb, HSP70, proliferin, tetracycline-inducible, tumor necrosis factor, or thyroid-stimulating hormone gene promoters. An example of an inductive promoter is the interferon-inducible ISG54 promoter. In some embodiments, promoters are constitutive promoters that result in high levels of transcription upon introduction into host cells in the absence of additional factors. In more embodiments, the constitutive promoter is the human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus initial enhancer, cytomegalovirus (CMV), monkey virus 40, or herpes simplex virus thymidine kinase promoter (see Damdindorj et al., PLOS One 9(8): e106472, 2014). The promoter may be the human β-actin promoter, the human elongation factor-1α promoter, the β-actin promoter, the monkey virus 40 promoter, or the herpes simplex virus thymidine kinase promoter.

[0222] If necessary, the transcriptional regulatory sequence includes one or more enhancer elements, which are binding recognition sites for one or more transcription factors that increase transcription beyond the transcription observed for the minimal promoter alone, and are operably ligated to a polynucleotide encoding a nucleic acid molecule encoding an ANG, precursor, variant, or fragment, or a tRNA fragment. Introns that help stabilize mRNA and increase expression may also be included.

[0223] Polyadenylation signals may be present to ensure proper transcript termination and polyadenylation. Exemplary polyadenylation signals have been isolated from betaglobin, bovine growth hormone, SV40, and herpes simplex virus thymidine kinase genes.

[0224] Nucleic acid molecules encoding ANG, its precursors, variants, or fragments, or tRNA fragments, may be included in a viral vector, for example, to express a protomer to produce a corresponding protein, its variant, or fragment, or to encode a tRNA fragment in a host cell, or for administration to a subject disclosed herein. Typically, such a viral vector includes a nucleic acid molecule encoding ANG, its precursor, variant, or fragment, or tRNA fragments. In some examples, viral vectors encoding ANG, its precursor, variant, or fragment, or tRNA fragments, may be replication competent. For example, a viral vector may have mutations in the viral genome (e.g., insertions of nucleic acids encoding a protomer) that attenuate, but do not completely block, viral replication in a host cell.

[0225] Various viral vectors that can be used for nucleic acid-based therapies taught herein include adenoviruses or adeno-associated viruses (AAVs), herpesviruses, vaccines, or RNA viruses, such as retroviruses (including HVJ; see Kotani et al., Curr. Gene Ther. 4:183-194, 2004). In one embodiment, the retroviral vector is a mouse or tri-retrovirus, or a human or primate lentivirus derivative. Examples of retroviral vectors into which foreign genes may be inserted include, but are not limited to, Moloney's mouse leukemia virus (MoMLV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), and Rous sarcoma virus (RSV). In one embodiment, when the target is human, vectors such as gibbon leukemia virus (GaLV) can be used. Pseudotyped retroviral vectors containing heterologous envelope genes can be used. In one example, the viral vector is AAV.

[0226] Some retroviral vectors can incorporate multiple genes. These vectors can be transfected or incorporated with genes for selection markers so that transduced cells can be identified and produced. By inserting nucleic acids encoding ANG, its precursor, variant, or fragment, or tRNA fragments, along with another gene that can act as a viral envelope protein and also encode a ligand for a specific receptor on a target cell, the vector can now be target-specific. Retroviral vectors can be made target-specific by modifying the envelope protein, for example, by attaching sugars, glycolipids, or proteins. In a specific, non-limiting example, targeting is achieved by targeting the retroviral vector using antibodies.

[0227] Recombinant retroviruses are non-replicating by design and therefore require assistance to generate infectious vector particles. This assistance can be provided, for example, by using a helper cell system containing plasmids encoding all of the retrovirus's structural genes under the control of regulatory sequences within the long-terminal repeat (LTR). These plasmids lack nucleotide sequences that enable a packaging mechanism that recognizes the RNA transcript for inclusion. Examples of helper cell systems with a packaging signal deletion include, but are not limited to, ψ2, PA317, and PA12. These cell systems produce empty virions because the genome is not packaged. If a retroviral vector is introduced into such cells, where the packaging signal is intact but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virions can be generated.

[0228] Alternatively, NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol, and env using conventional transfection methods. These cells are then transfected with vector plasmids containing the target gene. The resulting cells release the retroviral vector into the culture medium.

[0229] Adenovirus vectors include replication-competent, replication-deficient, and weak forms. Defective viruses, such as adenovirus vectors or adeno-associated virus (AAV) vectors, that lack viral genes entirely or nearly entirely can be used. The use of defective viral vectors allows for administration to specific cells without the concern that the vector may infect other cells. A useful AAV vector is one that lacks replication. In some non-limiting cases, useful vectors are attenuated adenovirus vectors, e.g., those described by Stratford-Perricaudet et al. (J. Clin. Invest., 90:626-630 1992; La Salle et al., Science 259:988-990, 1993); or defective AAV vectors (Samulski et al., J. Virol., 61:3096-3101, 1987; Samulski et al., J. Virol., 63:3822-3828, 1989; Lebkowski et al., Mol. Cell. Biol., 8:3988-3996, 1988).

[0230] Recombinant AAV vectors can direct the expression and production of selected transgenic products in targeted cells. Therefore, recombinant vectors can contain at least all of the AAV sequences essential for inclusion and the physical structures necessary for infecting target cells.

[0231] AAV belongs to the family Parvoviridae and the genus Dependovirus. AAV is a small, non-enveloped virus that packages a linear single-stranded DNA genome. Both the sense and antisense strands of AAV DNA are packaged in the AAV capsid with equal frequency. In some embodiments, the AAV DNA comprises a nucleic acid containing a promoter operably ligated to a nucleic acid molecule encoding ANG, its precursor, variant, or fragment, or a tRNA fragment. Recombinant vectors, such as recombinant adenovirus vectors and recombinant adeno-associated virus (rAAV) vectors, containing the nucleic acid molecules disclosed herein are further provided. In some embodiments, the AAV is rAAV8 and / or AAV2. However, the AAV serotype may be any other suitable AAV serotype, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, or AAV12, or a hybrid of two or more AAV serotypes. An exemplary AAV8 vector is disclosed, for example, in PCT Publication WO2014 / 127196.

[0232] Gene therapy vectors utilizing AAV can infect both dividing and quiescent cells and can persist in an extrachromosomal state without being integrated into the host cell's genome. This disclosure intends to utilize rAAV for the methods disclosed herein.

[0233] Inclusion of AAV provides the ability to bind to and enter target cells, enter the nucleus, be expressed in the nucleus for extended periods, and have low toxicity. AAV can be used to transfect cells, and suitable vectors are known, see, for example, U.S. Published Patent Application No. 2014 / 0037585, incorporated herein by reference. Methods for generating rAAV suitable for gene therapy are known (see, for example, U.S. Published Patent Applications No. 2012 / 0100606; No. 2012 / 0135515; No. 2011 / 0229971; and No. 2013 / 0072548; and Ghosh et al., Gene Ther 13(4):321-329, 2006) and can be used in conjunction with the methods disclosed herein.

[0234] In some embodiments, the vector is an rAAV8 vector, an rAAV2 vector, or an rAAV9 vector. In a specific non-limiting example, the vector is an AAV8 vector. The AAV8 vector is disclosed, for example, in U.S. Patent No. 8,692,332, which is incorporated herein by reference. The locations and sequences of the capsid, rep 68 / 78, rep 40 / 52, VP1, VP2, and VP3 are disclosed in this U.S. Patent No. 8,692,332. The locations and hypervariable regions of AAV8 are also provided. In some embodiments, the vector is an AAV2 variant vector, for example, AAV7m8.

[0235] A useful vector in the method disclosed herein may contain a nucleic acid sequence encoding an intact AAV capsid, which may be derived from a single AAV serotype (e.g., AAV2, AAV6, AAV8, or AAV9). As disclosed in U.S. Patent No. 8,692,332, the useful vector may also be recombinant and therefore may contain a sequence encoding an artificial capsid containing one or more fragments of an AAV8 capsid fused to a heterologous AAV or non-AAV capsid protein (or fragment thereof). These artificial capsid proteins are selected from non-proximal portions of AAV2, AAV6, AAV8, or AAV9 capsids, or from capsids of other AAV serotypes. For example, the AAV vector may have a capsid protein containing one or more AAV8 capsid regions selected from VP2 and / or VP3, or from VP1, or a fragment thereof, selected from amino acids 1-184, 199-259; 274-446; 603-659; 670-706; and 724-738 of the AAV8 capsid presented as Sequence ID No. 2 in U.S. Patent No. 8,692,332. In another example, it may be desirable to change the start codon of the VP3 protein to GTG. Alternatively, AAV may contain one or more hypervariable regions of the AAV serotype 8 capsid protein, for example, aa185-198;aa260-273;aa447-477;aa495-602;aa660-669; and aa707-723 of the AAV8 capsid presented as SEQ ID NO: 2 in U.S. Patent No. 8,692,332.

[0236] Additional viral vectors that can be used to express ANG, precursors, variants, or fragments, or to encode tRNA fragments, include polyomas, i.e., SV40 (Madzak et al., 1992, J. Gen. Virol., 73:15331536), herpesviruses including HSV, EBV, and CMV (Margolskee, 1992, Curr. Top. Microbiol. Immunol., 158:67-90; Johnson et al., 1992, J. Virol., 66:29522965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199), Sindbisvirus (H. Herweijer et al., 1995, Human Gene Therapy 6:1161-1167; U.S. Patent Nos. 5,091,309 and 5,2217,879), alphavirus (S. Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al., 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377) and bird (Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J. Virol., 66:3391-3397), mouse (Miller, 1992, Curr. Top. Microbiol. Immunol., 158:1-24;Miller et al., 1985, Mol. Cell Biol., 5:431-437;Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737;Mann et al., 1985, J. Virol., 54:401-407), and of human origin (Page et al., 1990, J. Virol.Examples include retroviruses (64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739) and baculovirus (Autographa californica polynuclear polyhedron virus; AcMNPV) vectors.

[0237] Another targeted delivery system for polynucleotides encoding ANG, its precursors, variants, or fragments, or tRNA fragments, is colloidal dispersions. These systems are also useful in the methods disclosed. Colloidal dispersions include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. One colloidal dispersion is liposomes. Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. Large monolayer vesicles (LUVs), ranging in size from approximately 0.2 to 4 microns, have been shown to encapsulate a significant percentage of aqueous buffer containing large macromolecules. RNA, DNA, and intact virions can be encapsulated within the aqueous interior and delivered to cells in a biologically active form (Fraley et al., Trends Biochem. Sci. 6:77, 1981). In addition to mammalian cells, liposomes are used for the delivery of polynucleotides in plant, yeast, and bacterial cells. For liposomes to be an effective gene transfer vehicle, they must exhibit the following characteristics: (1) highly efficient encapsulation of target nucleic acids without impairing their biological activity; (2) preferential and substantial binding to target cells compared to non-target cells; (3) highly efficient delivery of the aqueous contents of the vesicle to the cytoplasm of target cells; and (4) accurate and effective expression of genetic information (Mannino et al., Biotechniques 6:682, 1988).

[0238] The composition of liposomes is typically a combination of phospholipids, especially those with high phase transition temperatures, often combined with steroids, particularly cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0239] Examples of lipids useful in liposome formation include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Diacylphosphatidylglycerol is particularly useful, where the lipid portion contains 14 to 18 carbon atoms, especially 16 to 18 carbon atoms, and is saturated. Examples of phospholipids include phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.

[0240] Liposome targeting can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, e.g., organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based on whether it is passive or active. Passive targeting utilizes the innate tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) in organs containing sinusoidal capillaries. Active targeting, on the other hand, involves modifying liposomes by coupling them to specific ligands, e.g., monoclonal antibodies, sugars, glycolipids, or proteins, or by altering the composition or size of liposomes to achieve targeting to organs and cell types other than those naturally occurring in localization sites.

[0241] Another targeted delivery system is the use of biodegradable and biocompatible polymer scaffolds for use in bone (see Jang et al., Expert Rev. Medical Devices 1:127-138, 2004). These scaffolds typically contain a mixture of one or more biodegradable polymers, e.g., saturated aliphatic polyesters, e.g., poly(lactic acid) (PLA), poly(glycolic acid), or poly(lactide-co-glycolide) (PLGA) copolymers, unsaturated linear polyesters, e.g., polypropylene fumarate (PPF), or microbially produced aliphatic polyesters, e.g., polyhydroxyalkanoates (PHA) (see Rezwan et al., Biomaterials 27:3413-3431, 2006; Laurencin et al., Clin. Orthopaed. Rel. Res. 447:221-236). By changing the proportions of various components, polymeric scaffolds with different mechanical properties can be obtained. An exemplary scaffold contains a PLA-to-PGA ratio of 75:25, but this ratio can vary depending on the specific application. Other exemplary scaffolds include surface bioerosion polymers, e.g., poly(anhydrous), e.g., trimellicylimidoglycine (TMA-gly) or pyromelitylimidoalanine (PMA-ala), or poly(phosphazene), e.g., high molecular weight poly(organic phosphazene) (P[PHOS]), and bioactive ceramics. The stepwise biodegradation of these scaffolds allows for the stepwise release of drugs or genes from the scaffold. Therefore, these polymeric carriers represent not only scaffolds but also drug or gene delivery systems. This system is applicable to the delivery of plasmid DNA and also to viral vectors, e.g., AAV or retroviral vectors, and transposon-based vectors.

[0242] The disclosed nucleic acid molecules may be contained in nanodispersion systems, see, for example, U.S. Patent No. 6,780,324; U.S. Patent Publication No. 2009 / 0175953. For example, the nanodispersion systems may include biologically active agents and dispersants (e.g., polymers, copolymers, or low molecular weight surfactants). Exemplary polymers or copolymers include polyvinylpyrrolidone (PVP), poly(D,L-lactic acid) (PLA), poly(D,L-lactide-co-glycolic acid) (PLGA), and poly(ethylene glycol). Exemplary low molecular weight surfactants include sodium dodecyl sulfate, hexadecylpyridinium chloride, polysorbate, sorbitan, poly(oxyethylene) alkyl ethers, poly(oxyethylene) alkyl esters, and combinations thereof. In one example, the nanodispersion system includes PVP and ODP or their variants (e.g., 80 / 20 w / w). In some examples, the nanodispersions are prepared using solvent evaporation; see, for example, Kanaze et al., Drug Dev. Indus. Pharm. 36:292-301, 2010; Kanaze et al., J. Appl. Polymer Sci. 102:460-471, 2006.

[0243] Dendrimers are synthetic three-dimensional macromolecules prepared stepwise from simple branched monomer units, and their properties and functionality can be easily controlled and modified. A dendrimer consists of an initiator core surrounded by layers of a selected polymer grafted onto the core, forming a branched macromolecular complex. Dendrimers are typically produced using polymers such as poly(amidoamine) or poly(L-lysine). Dendrimers can be synthesized from repeated additions of building blocks to a polyfunctional core (a branching approach to synthesis) or to a polyfunctional core (a convergent approach to synthesis), with each addition of a three-dimensional shell of building blocks resulting in the formation of higher-generation dendrimers. Polypropyleneimine dendrimers contain 100% protonated nitrogen and up to 64 terminal amino groups. Protonated groups are typically amine groups that can accept protons at neutral pH. For nucleic acid molecules, dendrimers may be formed from polyamidoamines and phosphorus containing compounds having a mixture of amine / amide or NP(O2)S as conjugate units. Dendrimers useful for the delivery of nucleic acid molecules are disclosed, for example, in PCT Publication 2003 / 033027, which is incorporated herein by reference.

[0244] The surface of the targeted delivery system may be modified in various ways. In the case of liposome-targeted delivery systems, lipid groups may be incorporated into the lipid bilayer of the liposome to maintain the targeted ligand in stable association with the liposome bilayer. Various linking groups can be used to attach the lipid chain to the targeted ligand.

[0245] In another embodiment, mRNA can be used to directly deliver nucleic acids encoding ANG, its precursors, variants, or fragments to cells. In yet another embodiment, tRNA fragments are delivered. In some embodiments, mRNA-based nucleic acid vaccines may offer a potent alternative to the previously mentioned approaches. mRNA delivery eliminates safety concerns regarding DNA integration into the host genome and can be directly translated in the cytoplasm of host cells. Furthermore, the simple cell-free in vitro synthesis of RNA avoids the manufacturing complexities associated with viral vectors. Two exemplary RNA forms that can be used to deliver nucleic acids are conventional non-amplifying mRNA (see, e.g., Petsch et al., "Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection," Nature biotechnology, 30(12):1210-6, 2012) and self-amplifying mRNA (see, e.g., Geall et al., "Nonviral delivery of self-amplifying RNA vaccines," PNAS, 109(36): 14604-14609, 2012; Magini et al., "Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge," PLoS One, 11(8):e0161193, 2016; and Brito et al., "Self-amplifying mRNA vaccines," Adv Genet., (See 89:179-233, 2015)

[0246] 3. CRISR / Cas9 This disclosure includes methods for site-directed modification of nucleic acid molecules in cells to introduce ANG, or other molecules that increase ANG activity, such as one or more tRNA fragments. These modifications may include, but are not limited to, site-directed insertions and nucleotide replacements ("knock-ins") that result in increased ANG activity. These modifications can be performed anywhere in the genome, particularly in genomic elements, including coding sequences, regulatory elements, and non-coding DNA sequences. However, in some embodiments, insertions are performed at safe harbor loci (see Pavani and Amendola, Front Genome Ed., https: / / doi.org / 10.3389 / fgeed.2020.609650, 20 January 2021).

[0247] Any number of such insertions can be made in any order or combination. Gene expression may be modified using such methods, for example, by increasing the expression of ANG or tRNA fragments. These modifications include "knock-ins" of nucleic acid molecules encoding ANG and one or more tRNA fragments. Techniques for performing such modifications by genome editing include, among others, the use of the CRISPR-Cas system, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). These modifications can be used to introduce (knock-in) nucleic acid molecules encoding ANG, precursors, variants, or fragments into safe harbor loci in the genome. These modifications can also be used to introduce (knock-in) nucleic acid molecules encoding tRNA fragments into safe harbor loci in the genome. Cas9-mediated integration of genes is disclosed, for example, in Li et al., G3 10(2):467-473. doi: 10.1534 / g3.119.400810 (2020) and Jacinto et al., J Cell Mol Med. 24:3766-3778, DOI: 10.1111 / jcmm.14916 (2020).

[0248] A typical CRISPR system consists of two components: CRISPR-associated nuclease 9 (Cas9) and one or more guide RNAs (gRNAs), each containing CRISPR RNA (crRNA) and transactivated CRISPR RNA (tracrRNA). Simple gene disruption can be produced by cleavage of the target site, followed by nucleic acid modification, e.g., deletion, and repair via the non-homologous end-joining pathway (NHEJ). Target recognition by crRNA occurs through complementary base pairing with the target DNA, which directs the cleavage of the foreign sequence using the Cas protein. In some embodiments, DNA recognition by guide RNA and the resulting cleavage by endonucleases require complementary base pairing with a protospacer-adjacent motif (PAM) (e.g., 5'-NGG-3') and protospacer region in the target (Jinek et. al., Science. 337:816-821, 2012). The PAM motif recognized by Cas9 varies for different Cas9 proteins. Any Cas9 protein can be used in the systems and methods disclosed herein, including mutant Cas9 proteins (e.g., those having R691A, D10A, H840A, or combinations of such substitutions). In other embodiments of the systems and methods disclosed herein, a promoter is operably linked to the nucleic acid encoding Cas9. In non-limiting examples, retinal pigment epithelial cell promoters, such as, but not limited to, RPE65, BEST1, DCT, TYR, or TYRP1 promoters, are used.

[0249] As described above, the Cas9 RNA guide system includes a mature crRNA that base-pairs with a transactivating crRNA (tracrRNA) to form two RNA structures that direct Cas9 to the locus of a desired double-strand (ds) cleavage in the target DNA. In some embodiments, the base-paired tracrRNA:crRNA combination is manipulated to form a single RNA chimera to generate a guide sequence (e.g., gRNA) that retains the ability to direct sequence-specific Cas9 dsDNA cleavage. In some embodiments, the Cas9-guide sequence complex results in cleavage of one or both strands at the target sequence within the safe harbor locus, enabling knock-in. Thus, the Cas9 endonuclease and gRNA molecules are used for sequence-specific target recognition, cleavage, and genome editing of safe harbor loci. In one embodiment, the cleavage site is a specific nucleotide, e.g., the 16th, 17th, or 18th nucleotide (nt) of a 20nt target, but not limited to these. In a non-limited example, the cleavage site is the 17th nucleotide of a 20nt target sequence. The break could be a double-strand break.

[0250] In some embodiments, the gRNA molecule is selected such that the target genome target has a protospacer adjacency motif (PAM). In some embodiments, DNA recognition by the guide RNA, and the resulting cleavage by the endonuclease, requires the presence of a protospacer adjacency motif (PAM) immediately following the target (e.g., 5'-NGG-3'). The PAM is present in the targeted nucleic acid sequence but not in the crRNA generated to target it. In some embodiments, the protospacer adjacency motif (PAM) corresponds to 2-5 nucleotides that begin immediately or near the protospacer at the distal end of the leader. The PAM motif can also be NNAGAA, NAG, NGGNG, AWG, CC, CC, CCN, TCN, or TTC.

[0251] In some embodiments, cleavage occurs approximately 3 base pairs upstream from the PAM. In some embodiments, Cas9 nuclease cleaves the double-stranded nucleic acid sequence.

[0252] In some embodiments, guide sequences are selected to reduce the degree of secondary structure within the sequence. Secondary structure can be determined by any suitable polynucleotide folding algorithm. Some programs are based on the calculation of the minimum Gibbs free energy. One example of such an algorithm is mFold (Zuker and Stiegler, Nucleic Acids Res. 9 (1981), 133-148). Another example of a folding algorithm is the online web server RNAfold, which uses a center-of-mass structure prediction algorithm (see, e.g., Gruber et al., 2008, Cell 106(1): 23-24; and Can and Church, 2009, Nature Biotechnology 27(12): 1151-62). Guide sequences can be designed using the MIT CRISPR design tool found at crispr.mit.edu, Harvard, the University of Bergen CHOPCHOP web tool found at chopchop.cbu.uib.no, or the E-CRISP tool found at www.e-crisp.org / E-CRISP. Additional tools for designing tracrRNA and guide sequences are described in Naito et al., Bioinformatics. 2014 Nov 20, and Ma et al. BioMed Research International, Volume 2013 (2013), Article ID 270805. crRNA can be 18–48 nucleotides long. crRNA can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In one example, the crRNA is 20 nucleotides long.

[0253] The systems disclosed herein introduce double-strand DNA breaks, resulting in a target, e.g., a safe harbor locus, being cleaved by Cas9. This allows for the insertion of nucleic acid sequences encoding ANG, precursors, variants, or fragments, and / or tRNA fragments. In some embodiments, two or more DNA breaks can be introduced by using two or more gRNAs. For example, two gRNAs can be utilized so that two breaks are achieved. When two or more gRNAs are used for two or more break event sites, in some embodiments, it is intended that two or more break events in the target nucleic acid may be carried out by the same or different Cas9 proteins. For example, when two gRNAs are used for two double-strand break sites, a single Cas9 nuclease can be used to produce both double-strand breaks.

[0254] In some embodiments, the disclosed method includes the use of one or more vectors comprising: a) a retina-specific promoter operably ligated to a nucleotide sequence encoding a type II Cas9 nuclease; b) a promoter, e.g., a U6 promoter, operably ligated to one or more nucleotide sequences encoding one or more CRISPR-Cas guide RNAs that hybridize to a safe harbor locus in target cells, e.g., human cells; and c) nucleic acid molecules encoding an ANG, precursor, variant, or fragment, and / or a tRNA fragment, to be introduced into the target cells. These components may be located on the same or different vectors, thereby one or more guide RNAs targeting a non-coding region, and as a result, the Cas9 protein cleaves and is introduced into the DNA and the nucleic acid molecules encoding the ANG, precursor, variant, or fragment, or the tRNA fragment. In specific non-limiting examples, one or more vectors are viral vectors, e.g., lentiviral vectors. In other non-limiting examples, the viral vectors are adenovirus vectors, adeno-associated virus vectors, or retroviral vectors. Cas9 and gRNA can be delivered using AAV, lentivirus, piggybac, episome constructs, or injected as purified nanoparticles consisting of pure Cas9 protein and pure guide RNA (see, for example, Steyer et al., Drug Discov Today Technol 28: 3-12, 2018).

[0255] 4. Chemical compounds Drugs that increase ANG activity include L-minosine (Janjic et al., BMC Oral Health 17:87 (7 pages), DOI 10.1186 / s12903-017-0373-6, 2017), muscone (Zhou et al., Mol. Neurol. 59: 5891-5901, July 9, 2022), opioids, DMOGs, and ML228. Drugs that increase ANG activity may be molecules identified from large libraries of both natural product extracts and synthetic (or semi-synthetic) extracts, or from chemical libraries. Screening methods for detecting increased ANG activity are useful for identifying compounds from various sources regarding their activity. Initial screening may be performed using diverse compound libraries, various other compounds, and compound libraries. In this way, molecules that increase ANG activity can be identified. These small molecules can be identified from combinatorial libraries, natural product libraries, or other small molecule libraries. In addition, ANG agonists can be identified as compounds from commercially available sources and from commercially available analogs of identified agonists.

[0256] The precise origin of the test extract or compound is not critical to the identification of agents that increase ANG activity. Therefore, such agents can be identified from virtually any number of chemical extracts or compounds. Examples of such extracts or compounds include, but are not limited to, plant, fungal, prokaryotic, or animal-based extracts, fermentation broths, and synthetic compounds, as well as modifications of extracted compounds. Numerous methods are also available to result in the random or targeted synthesis (e.g., semi-synthetic or total synthesis) of any number of chemical compounds, including, but are not limited to, saccharides, lipids, peptides, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, Wis.). ANG protein agonists can be identified from synthetic compound libraries commercially available from several companies, including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, Conn.). La protein agonists can be identified from rare chemical libraries, such as those available from Aldrich (Milwaukee, Wis.). La protein agonists can also be identified in libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts, commercially available from several sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, USA (Cambridge, Mass.). Naturally occurring and synthetically produced libraries and compounds are readily modified by conventional chemical, physical, and biochemical means.

[0257] Useful compounds can be found within a number of chemical classes, but typically they are organic compounds, including low molecular weight organic compounds. Low molecular weight organic compounds have molecular weights greater than 50 daltons and even less than about 2,500 daltons, for example, less than about 750 daltons or less than about 350 daltons, which can be utilized in the manner disclosed herein. Exemplary classes include heterocyclic compounds, peptides, saccharides, and steroids. Compounds may be modified to enhance efficacy, stability, pharmaceutically acceptable properties, etc.

[0258] III. Treatment methods and pharmaceutical compositions Methods for treating AMD or reducing the risk of developing AMD are disclosed herein. In some embodiments, the methods inhibit drusen formation. The disclosed agents may be administered systemically (e.g., by peripheral intravenous infusion or orally), locally, or regionally (intraocular). The agents may be agents that inhibit HERV-K. The agents may be agents that increase ANG activity. The target may be early, mid-stage, or advanced stage AMD.

[0259] In some embodiments, the subjects are mammalian subjects, e.g., human or veterinary subjects. In some embodiments, the subjects have exudative AMD. In other embodiments, the subjects have atrophic AMD. In further embodiments, the subjects are at risk of developing AMD. In some embodiments, the disclosed method reduces or prevents vision loss.

[0260] Antiretroviral agents can be administered according to approved drug administration methods; see above. Chemical compounds that increase ANG activity can be administered to the subject. In some embodiments, administration is systemic, e.g., orally or intravenously. However, other routes of administration are considered, e.g., inhalation, vaginal, rectal, and nasal. In some embodiments, antiretroviral agents are formulated for localized administration, e.g., into the eyes.

[0261] Therapeutic compounds can be prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing a polypeptide of desired purity, such as ANG, its precursors, variants, or fragments, an antibody, or another drug, such as an inhibitory nucleic acid molecule or a nucleic acid molecule encoding the CRISPR / Cas13 system, ANG, its precursors, variants, or fragments, or the CRISPR / Cas9 system, with a pharmaceutically acceptable carrier, excipient, or stabilizer as needed (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.

[1980] ). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used, and include buffers, e.g., phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than approximately 10 residues) polypeptides; proteins, e.g., serum albumin Examples include: cellulose, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., TWEEN®, PLURONICS®, or polyethylene glycol (PEG). The pharmaceutical composition can be formulated for localized delivery, for example, to the eye. In one embodiment, the pharmaceutical composition is formulated as eye drops.

[0262] The active ingredient can be encapsulated in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions by microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0263] Formulations used for in vivo administration may be sterile. This is readily achieved by filtration through a sterile filtration membrane. Sustained-release preparations may be prepared. In some embodiments, for example, for the delivery of nucleic acid molecules, antibodies, or their antigen-binding fragments to a target, the drug is administered intraocularly.

[0264] Pharmaceutical compositions may be prepared, packaged, or sold in bulk, as single-dose units, or as multiple single-dose units. As used herein, “unit dose” refers to a distinct amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient administered to a subject, or a convenient fraction of such a dose, such as half or one-third of such a dose. The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional components in a pharmaceutical composition may vary depending on the identity, size, and condition of the subject being treated, and further depending on the route through which the composition is administered. For example, a composition may contain 0.1% to 100% (w / w) of the active ingredient.

[0265] In addition to the main active ingredients, the vehicle and composition may contain various formulation components, such as antimicrobial preservatives and isotonic agents. For example, antimicrobial preservatives include benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, EDTA, sorbic acid, POLYQUAD®, or other agents. Such preservatives, when used, may be used in amounts of about 0.0001 wt.% to 1.0 wt.%. Suitable agents that may be used to adjust the osmotic pressure or osmolality of the composition include mannitol, dextrose, glycerin, and propylene glycol. When used, such agents may be used in amounts of about 0.1 wt.% to 10.0 wt.%. In some embodiments, the composition does not contain preservatives or isotonic agents that adversely affect or irritate the eyes.

[0266] Ophthalmic compositions, such as eye drops, are provided herein, which, in addition to an active ingredient, include a preservative and, optionally, a thickener. Preservatives may include, for example, benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, benzyl alcohol, sodium dehydroacetate, parahydroxybenzoic acid esters, sodium edetate, and boric acid. Thickeners may include, for example, hydroxypropyl methylcellulose (HPMC) or water-soluble cellulose derivatives, such as methylcellulose, hydroxyethylcellulose, and carboxymethylcellulose.

[0267] Other additives include, but are not limited to, isotonic agents such as sodium chloride, potassium chloride, glycerol, mannitol, sorbitol, boric acid, glucose, and propylene glycol; buffers such as phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamic acid, and ε-aminocaproic acid; stabilizers such as sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, and dibutylhydroxytoluene; thickeners such as sodium chondroitin sulfate, sodium hyaluronate, carboxyvinyl polymer, polyvinyl alcohol, polyvinylpyrrolidone, and macrogol; and pH adjusters such as hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid.

[0268] In certain embodiments, the disclosed eye drops may further contain one or more other components or additives that can be included in the artificial tears, namely aminoethylsulfonic acid, sodium chondroitin sulfate, potassium L-aspartate, magnesium L-aspartate, potassium magnesium L-aspartate (equimolar mixture), sodium bicarbonate, sodium carbonate, potassium chloride, calcium chloride, sodium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, anhydrous sodium carbonate, magnesium sulfate, polyvinyl alcohol, polyvinylpyrrolidone, hydroxyethylcellulose, glucose, and methylcellulose. The amount of these additives will vary depending on the type and use of the additive, but such additives should only be added in concentrations that can achieve the purpose of the additive.

[0269] In some embodiments, eye drops are provided that contain agents that may inhibit HERV-K and / or increase ANG activity. The pharmaceutical composition may contain preservatives and / or thickeners. Other components may be included as needed. The concentrations of other components or additives contained in a pharmaceutical composition formulated for ophthalmic administration, e.g., eye drops, are generally, but not limited to, 0.01% w / v or higher, 0.1% w / v or higher, 0.5% w / v or higher, and 20% w / v or lower, for example, 10% w / v or lower, or 7% w / v or lower, or 5% w / v or lower.

[0270] The ophthalmic composition may be prepared as a micronized suspension of isotonic, pH-adjusted sterile saline, or preferably as a solution of isotonic, pH-adjusted sterile saline. The pH of the compositions provided herein may vary over an eye-tolerant range, as is known to those skilled in the art. Preferably, the pH of the formulation is in the range of approximately 4 to 8. Alternatively, the ophthalmic composition may be formulated into an ointment such as petrolatum. In certain embodiments, the ophthalmic formulation comprises a lipophilically modified composition and an implantable carrier.

[0271] Ophthalmic compositions formulated for topical ophthalmic administration may be eye drops, such as aqueous eye drops, such as monophasic aqueous eye drops. Ophthalmic compositions may also be formulated and used in the form of mist, frost, foam, cream, ointment or emulsion for direct application to the eye, or used in ophthalmic implants or injectable ophthalmic therapeutics, or otherwise administered to the eyelid or sclera.

[0272] The ophthalmic composition may also contain compounds such as NF-κB, mTOR, or one or more Rho GTPases, e.g., Nox4 inhibitors which are compounds that modulate CDC42 and / or RACI; compounds that modulate AMPK or compounds that regulate epithelial-mesenchymal transition or dedifferentiation of RPE; cholesterol pathway modulators, complement inhibitors, epigenetic modifiers, mitochondrial activity inducers, oxidative stress inhibitors, and metabolic stress inhibitors. Examples of such compounds, though not limited to them, include aminocapropic acid, L-701,324, Vas2870, L-745,870 hydrochloride, Me3,4-dehostatin, N-methyl-I-deoxynojirimycin, L-750,667 trihydrochloride, (+)-MK-801 hydrogen maleate, hemidine tartrate, (-)-naproxen sodium, raloxifene hydrochloride, SKF 83959 hydrobromide, L-687,384 hydrochloride, 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, SP600125, Ro 41-0960, Ancitabine hydrochloride, Risperidone, Terenzepine dihydrochloride, NO-711 hydrochloride, U-99194A maleate, S(+)-Lacloprid (Rac10pride) L-tartrate, Pirenzepine dihydrochloride, Captopril, Thioperamide maleate, Alprenolol hydrochloride, Ritodrine hydrochloride, Putrescine dihydrochloride, 1-(2-Methoxyphenyl)piperazine hydrochloride, PAPP, U-69593, AG-1478, Riluzole, Phentolamine mesylate, DBO-83, Formestan, Carbamazepine, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, Terbutaline hemisulfate, UK 14304, GR Examples include 113808, leflunomide, acetylthiocholine chloride, spermidine, 5-(N-methyl-N-isobutyl)amyloride, ATPO, acadenisine, or combinations thereof. See U.S. Published Patent Application No. US20220339127A1, incorporated herein by reference.

[0273] The compositions and methods disclosed herein can be used in combination with compounds, or pharmaceutically acceptable salts thereof, that inhibit Nox4 or reactive oxygen species formation, or modulate serine proteases, dopamine receptors, NF-κB, mTOR, AMPK, RPE epithelial-mesenchymal transition, RPE dedifferentiation, or one or more Rho GTPases. In some non-limiting examples, the compounds are Nox4 inhibitors or inhibitors of reactive oxygen species formation. In other embodiments, the compounds modulate NF-κB, mTOR, or one or more Rho GTPases. In further embodiments, the compounds modulate one or more Rho GTPases, where Rho GTPase is CDC42 and / or RACl. In yet another embodiment, the compounds inhibit serine proteases. In more embodiments, the compounds modulate AMPK. In more embodiments, the compounds modulate dopamine receptors. The compounds may be dopamine receptor D4 antagonists.In some embodiments, the compound is aminocaproic acid, L-701,324, Vas2870, L-745,870 hydrochloride, Me-3,4-dehostatin, N-methyl-l-deoxynojirimycin, L-750,667 trihydrochloride, (+)-MK-801 hydrogen maleate, penvidine tartrate, (-)-naproxen sodium, raloxifene hydrochloride, SKF 83959 hydrobromide, L-687,384 hydrochloride, 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, SP-600125, Ro 41-0960, Ancitabine hydrochloride, Risperidone, Terenzepine dihydrochloride, NO-711 hydrochloride, U-99194A maleate, S(+)-Lacloprid L-tartrate, Pirenzepine dihydrochloride, Captopril, Thioperamide maleate, Alprenolol hydrochloride, Ritodrine hydrochloride, Putrescine dihydrochloride, 1-(2-Methoxyphenyl)piperazine hydrochloride, PAPP, U-69593, AG-1478, Riluzole, Pentolamine mesylate, DBO-83, Formestan, Carbamazepine, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, Terbutaline hemisulfate, UK 14304, GR These compounds include 113808, leflunomide, acetylthiocholine chloride, spermidine, 5-(N-methyl-N-isobutyl)amyloride, ATPO, akadesin or metformin, or a combination thereof. In some non-limiting examples, the compounds are aminocaproic acid; Vas2870, L-745,870; riluzole; akadesin; metformin or pharmaceutically acceptable salts thereof. Preferred compounds are disclosed, for example, in PCT Publication WO2021 / 050980A1, which is incorporated herein by reference.

[0274] The drug administration may be a single-dose schedule or a multi-dose schedule that ultimately delivers the amount specified above. Doses may be intermittent. The subject may also be administered as multiple doses, where appropriate. Individual doses are typically greater than or equal to the amount required to produce a measurable effect on the subject and may be determined based on the pharmacokinetics and pharmacology of the absorption, distribution, metabolism, and excretion ("ADME") of the composition of the subject or its by-products, and therefore based on the disposal of the composition within the subject. This includes consideration of dosage as well as route of administration and may be adjusted for localized application. The effective dose and / or dose regimen can be readily determined empirically from preclinical assays, safety and dose escalation and dose range studies, individual clinician-patient relationships, and in vitro and in vivo assays.

[0275] The administration may be provided as a single dose, a periodic bolus, or a continuous infusion (e.g., from an implant placed in an intraocular location). Intraocular injections of nucleic acid molecules, antibodies, and CRISPR / Cas13 systems disclosed herein may be administered once or repeatedly, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times, or more. Administration may be given every other week, weekly, every other week, monthly, or every 2, 3, 4, 5, or 6 months.

[0276] Localized administration methods include intraocular routes, which are discussed below. In some embodiments, a considerably smaller amount (compared to a systemic approach) may be more effective when administered locally (e.g., intraocularly) than when administered systemically (e.g., intravenously). Localized administration methods can reduce or eliminate the incidence of potential side effects. In some embodiments, the method involves administering a drug, e.g., but not limited to, antiretroviral agents, inhibitory RNA, or agents that increase ANG activity, into the eye. The amount of ophthalmic preparation administered into the eye may depend on the individual being treated and is preferably the optimal amount to achieve the desired treatment without significant side effects.

[0277] Medication may include, for example, administration to the eye in the form of eye drops. These eye drops may be administered, for example, once, twice, three times, four times, five times, or more times a day (e.g., every hour, every two hours, every four hours, every six hours, every twelve hours, or every twenty-four hours). Eye drops may be administered once a day, every other day, every other week, or every week.

[0278] The visual perception of an object can be evaluated as discussed below.

[0279] A. Intraocular administration Intraocular administration may be by subretinal, direct retinal, choroidal, or intravitreal injection. The volume of the pharmaceutical composition to be injected may be, for example, about 10 to 500 μL, for example, about 50 to 500, 100 to 500, 200 to 500, 300 to 500, 400 to 500, 50 to 250, 100 to 250, 200 to 250, or 50 to 150 μL. The volume may be, for example, about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μL. In some embodiments, the volume of the pharmaceutical composition to be injected is 100 μL.

[0280] Subretinal injection is an injection into the subretinal space, i.e., beneath the retinal neurosensory epithelium. During subretinal injection, the injected material is directed towards the photoreceptor cells and retinal pigment epithelium (RPE) layer, creating a space between the photoreceptor cells and the RPE layer. If the injection is performed through a small retinal incision, retinal detachment may be created. The detached, raised layer of the retina caused by the injected material is called a "bleb." The hole created by subretinal injection can be small enough that the injected solution does not significantly backflow into the vitreous cavity after administration. Such backflow can be particularly problematic when the drug is injected, as the effect of the drug may be directed away from the target zone. Preferably, the injection creates a self-sealing entry point in the retinal neurosensory epithelium, i.e., when the needle is removed, the hole created by the needle closes again, and as a result, the injected material is released little to substantially through the hole.

[0281] Subretinal injection needles are commercially available (e.g., DORC 41 G Teflon® subretinal injection needle, Dutch Ophthalmic Research Center International BV, Zuidland, The Netherlands).

[0282] As long as no damage to the retina occurs during the injection, and as long as a sufficiently small needle is used, substantially all of the injected material remains localized (i.e., does not flow into the vitreous cavity) between the detached retinal neurosensory epithelium and the RPE at the site of localized retinal detachment. Indeed, typical persistence of blebs over short timeframes usually indicates the presence of slight escape of the injected material into the vitreous. Blebs can dissipate over longer timeframes as the injected material is absorbed.

[0283] For example, optical coherence tomography can be used to visualize the eye, particularly the retina. Because the retina is thin and transparent, and the underlying background of damaged, heavily pigmented epithelium makes it difficult to visualize, identifying the retina is difficult under certain circumstances, such as during end-stage retinal degeneration. The use of blue vital dyes (e.g., BRILLIANT PEEL®, Geuder; MEMBRANEBLUE-DUAL®, Dore) can facilitate the identification of retinal holes created for retinal detachment procedures (i.e., step (a) in the two-step subretinal injection method of the present invention), allowing the drug to be administered through the same hole without the risk of backflow into the vitreous cavity. The use of blue vital dyes also identifies any areas of the retina where a thickened internal limiting membrane or epithelial membrane exists, as injection through any of these structures would prevent clean access to the subretinal space. Furthermore, contraction of any of these structures immediately after surgery can lead to widening of the retinal entry hole, which can lead to the flow of the drug into the vitreous cavity.

[0284] A suprachoroidal injection can be used. In this method, the drug is delivered into the suprachoroidal space using a microcatheter-assisted incision approach (see, e.g., Peden et al. (2011) PLoS One 6(2): e17140). In this method, a limbal periconjunctival incision is performed to expose the bare sclera, followed by a scleral incision to expose the bare choroid. A microcatheter (e.g., iTrack 250A from iScience Interventional, connected to an illumination system such as an iLumin laser-diode-based micro-illumination system (iScience Interventional) if necessary) is introduced into the suprachoroidal space and advanced posteriorly toward the optic disc. After manipulating the microcatheter tip to the desired position, the injection of the product, polynucleotide, or vector forms a bleb in the retina and choroid. In some embodiments, the drug is delivered to the choroid by a method comprising (i) introducing a microcatheter into a space above the choroid; (ii) advancing the microcatheter within the space until the tip is in close proximity to the affected area of ​​the retina; and (iii) injecting a product, polynucleotide, or vector from the microcatheter tip to create a bleb.

[0285] In some embodiments, a therapeutically effective dose of the drugs disclosed herein is administered by intraocular injection, e.g., intravitreal injection. A general method for intravitreal injection can be described by the following brief summary. This example is intended to illustrate certain features of the method and not to limit it in any way. Procedures for intravitreal injection are known in the art (see, for example, Peyman, et al. (2009) Retina 29(7):875-912 and Fagan and Al-Qureshi, (2013) Clin. Experiment. Ophthalmol. 41(5):500-7). Other methods of intraocular administration are known in the art and include subretinal administration.

[0286] In short, a subject for intravitreal injection can be prepared for the procedure by pupillary dilation, eye disinfection, and administration of an anesthetic. Any suitable mydriatic agent known in the art may be used for pupillary dilation. Proper pupillary dilation may be confirmed before the procedure. Disinfection may be achieved by applying an iodine-containing solution to the eye to be disinfected, e.g., povidone-iodine (BETADINE®). Similar solutions may also be used to cleanse the eyelids, eyelashes, and any other nearby tissues (e.g., skin). Any suitable anesthetic, e.g., lidocaine or propalacaine, may be used at any suitable concentration. The anesthetic may be administered by any method known in the art, including, but not limited to, topical eye drops, gels or jelly, and subconjunctival application of the anesthetic.

[0287] Before the injection, the eyelashes may be cleaned from the area using a sterilized ophthalmoscopy. The injection site may be marked with the syringe. The injection site may be selected based on the patient's lens. For example, the injection site may be 3–3.5 mm from the edge in patients with pseudophakia or aphakia, and 3.5–4 mm from the edge in patients with phakia. The patient may look in the opposite direction from the injection site. During the injection, the needle may be inserted perpendicular to the sclera and positioned in the center of the eye. The needle may be inserted so that the tip ends in the vitreous humor and not in the subretinal space. Any suitable volume known in the art for injection may be used. After the injection, the eye may be treated with an antiseptic such as an antibiotic. The eye may also be rinsed to remove any excess antiseptic.

[0288] Intravitreal injection of the drugs disclosed herein may be performed as a single dose or repeatedly, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. Administration may be performed every other week, weekly, every other week, monthly, or every 2, 3, 4, 5, or 6 months.

[0289] In some embodiments, the method may include administering a therapeutically effective dose of a drug to inhibit unwanted angiogenesis, for example, to prevent new choroidal vessels (CNVs) growing submacula in patients with AMD. Exemplary therapeutic agents can reduce the activity of vascular endothelial growth factor (VEGF), for example, by binding to the receptor site of the active form of VEGF and preventing the interaction between VEGF and its receptor. Others suppress VEGF expression by inhibiting pathways that lead to therapeutically effective doses and VEGF secretion, for example, STAT3, NF-κB, and HIF-1α. Other drugs can prevent RPE cell atrophy by targeting the complement pathway, autophagy, or NF-κB pathway. Treatments that may be helpful for AMD include drug therapies directed to halt the growth of new vessels, such as bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), and aflibercept (EYLEA®); photodynamic therapy; photocoagulation; and low vision rehabilitation.

[0290] In a further embodiment, the method comprises administering to a subject a therapeutically effective amount of ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), or pigment epithelial-derived factor (PEDF) which can be used to promote the development or function of nerve cells, such as photoreceptor cells. Other exemplary and impractical embodiments include administering to a subject a therapeutically effective dose of thrombospondin 1, anti-inflammatory cytokines (e.g., interleukin (IL)-1, IL-6, Fas ligand or tumor growth factor (TGF)-beta, neurotrophic / neuroprotective growth factors, e.g., glial cell lineage growth factors, brain-derived neurotrophic factors, neurogrowth factors, neurotrophin-3, -4 / 5, -6, and vitamin E). Such agents may be provided individually or in combination. Implants are also useful in the methods disclosed herein. Implants can be inserted into the eye by various methods, including by forceps or by replacement with a sheath needle after making an incision in the sclera (e.g., a 2-3 mm incision) or another suitable site. In some cases, the implant is inserted by another incision. The device can be implanted using a sheath needle without opening the eye, or it can be implanted by creating a hole directly in the eye using the sheath needle. The method of replacement may affect the release kinetics. For example, implanting the device in the vitreous humor or posterior chamber using a sheath needle may result in deeper device placement within the vitreous humor than with forceps placement, which may result in implantation closer to the edge of the vitreous humor. The location of the implanted device may affect the concentration gradient of the therapeutic agent around the device, and thus may affect the release rate (for example, a device placed closer to the edge of the vitreous humor may result in a slower release rate; see U.S. Patents 5,869,079 and 6,699,493). In one embodiment, the implant is formulated using a biodegradable polymer matrix.

[0291] Generally, when implants are used, the therapeutic agent is uniformly distributed through the polymer matrix, and as a result, it is distributed sufficiently uniformly so that adverse variations in the rate of release do not occur due to the non-uniform distribution of the immunosuppressant in the polymer matrix. The choice of polymer composition used depends on the desired release kinetics, the implant site, patient tolerance, and the characteristics of the implantation procedure. The polymer may be included as at least about 10 weight percent of the implant. In one example, the polymer is included as at least about 20 weight percent of the implant. In another embodiment, the implant contains two or more polymers. These factors are described in detail in U.S. Patent No. 6,699,493. Common characteristics of polymers include biodegradability at the implantation site, compatibility with the drug of interest, ease of encapsulation, and water insolubility. Generally, the polymer matrix does not completely decompose until the drug load is released. Suitable polymer chemical compositions are known in the art (see, for example, U.S. Patent No. 6,699,493).

[0292] Topical administration to the eye is also useful in the manner disclosed. Topical preparations may include eye drops, ointments, sprays, etc. Eye drops or sprays may be provided in unit-dose dispensers (e.g., eye drop bottles that dispense a fixed unit dose). These may include, for example, a wetting agent and an inert matrix. As an example of an inert matrix, liposomes may be prepared from dipalmitoylphosphatidylcholine (DPPC), for example, egg phosphatidylcholine (PC). Liposomes can be applied topically or injected intraocularly, either in the form of eye drops or as an aqueous cream. In formulations for topical application, the active agent is released slowly over time as the liposomal capsule degrades due to abrasion and tear fluid from the surface of the eye. In formulations for intraocular injection, the liposomal capsule degrades due to cellular digestion. Both of these formulations offer the advantages of a delayed-release drug delivery system, allowing the subject to be exposed to a substantially constant concentration of the active agent over time. For example, the active agent may be soluble in an organic solvent, such as DMSO or alcohol, as previously described, and may contain polyacid anhydride, poly(glycol) acid, poly(lactic) acid, or polycaprolactone polymer.

[0293] B. Pharmaceutical composition containing nucleic acid molecules Pharmaceutical compositions containing nucleic acid molecules can be formulated and administered in various ways (see, for example, U.S. Publication No. 2005 / 0054567, which discloses pharmaceutical compositions and the administration of such compositions and is incorporated herein by reference). Pharmaceutical compositions may contain nanoparticles or dendrimers. These pharmaceutical compositions are useful in the methods disclosed herein.

[0294] Pharmaceutical compositions comprising nucleic acid molecules, formulated for localized delivery to the eye, are provided. These include antibodies and their antigen-binding fragments, inhibitory RNA molecules, Cas13 proteins, and nucleic acid molecules encoding gRNA. The nucleic acid molecules can be administered in vivo to the target by, for example, but not limited to, oral, intravenous, or intraocular (e.g., intravitreous) administration.

[0295] Generally, it is desirable to prepare a composition as a pharmaceutical composition suitable for the intended application. Therefore, methods for preparing pharmaceutical compositions containing the nucleic acid molecules or vectors described above are included herein. Typically, the preparation of a pharmaceutical composition involves preparing a pharmaceutical composition that is essentially free not only from pyrogens but also from any other impurities that may be harmful to humans or animals. Typically, the pharmaceutical composition contains appropriate salts and buffers that stabilize the composition and allow for the uptake of nucleic acids or viruses by target cells.

[0296] Pharmaceutical compositions containing nucleic acid molecules can be formulated for injection, for example, for intraocular or intravenous administration. Such compositions are generally formulated by mixing a disclosed nucleic acid molecule of desired purity in a unit dose injection form (solution, suspension, or emulsion) with a pharmaceutically acceptable carrier, e.g., one that is nontoxic to the recipient at the dose and concentration used and compatible with other components of the formulation. The pharmaceutical composition may contain an effective amount of nucleic acid molecules dispersed (e.g., dissolved or suspended) in a pharmaceutically acceptable carrier or excipient. pharmaceutically acceptable carriers and / or pharmaceutically acceptable excipients are known in the art and are described, for example, in Remington's Pharmaceutical Sciences by EW Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995). The characteristics of the carrier depend on the specific mode of administration used. For example, the formulation typically contains an injectable liquid as a vehicle, such as a pharmaceutically and physiologically acceptable liquid, e.g., water, physiological saline, equilibrium salt solution, aqueous dextrose, glycerol, etc. In addition, the administered pharmaceutical composition may contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffers, e.g., sodium acetate or sorbitan monolaurate. The disclosed nucleic acid molecules can be suspended in an aqueous carrier, e.g., an isotonic or hypotonic buffer solution with a pH of about 3.0 to about 8.5, e.g., about 4.0 to about 8.0, about 6.5 to about 8.5, or about 7.4. Useful buffers include physiological saline buffered phosphate or ionic borate buffer. The active ingredient may also be in the form of a lyophilized product, together with excipients as needed, and may be dissolved before administration by the addition of a suitable solvent.

[0297] Examples of pharmaceutically acceptable carriers include all kinds of solvents, dispersions, coatings, isotonic agents, and absorption retarders. The use of such media and agents for pharmaceutically active substances is well known in the art. Auxiliary active ingredients can also be incorporated into the composition. For example, certain pharmaceutical compositions may contain a vector or virus in water, mixed with a suitable surfactant, e.g., hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth. Pharmaceutically acceptable salts, e.g., mineral salts, e.g., hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids, e.g., acetate, propionate, malonate, benzoate, etc., may be included therein. In addition, auxiliary substances, e.g., wetting agents or emulsifiers, pH buffers, etc., may be present in such vehicles.

[0298] In some embodiments, the excipients confer a protective effect to viruses containing nucleic acid molecules, such as AAV virions or lentiviral virions, thereby minimizing the loss of AAV virions or lentiviral virions and the loss of transdependency resulting from formulation procedures, packaging, storage, and transport. These excipient compositions are therefore considered "virion-stabilizing" in the sense that they provide higher virion titers and higher transdependency levels than their unprotected counterparts, as measured using standard assays; see, for example, Published U.S. Application No. 2012 / 0219528.

[0299] Examples of excipients that can be used to protect virions from active decomposition conditions include, but are not limited to, detergents, proteins, e.g., ovalbumin and bovine serum albumin, amino acids, e.g., glycine, polyhydric and dihydric alcohols, e.g., but are not limited to, polyethylene glycol (PEG), propylene glycol (PG), sugar alcohols, e.g., carbohydrates, preferably sorbitol, with molecular weights of 1500 to 6000, e.g., PEG-200, PEG-400, PEG-600, PEG-1000, PEG-1450, PEG-3350, PEG-6000, PEG-8000 and any molecular weight within these values. One preferred type of nonionic detergent is sorbitan esters, such as polyoxyethylene sorbitan monolaurate (TWEEN®-20), polyoxyethylene sorbitan monopalmitate (TWEEN®-40), polyoxyethylene sorbitan monostearate (TWEEN®-60), polyoxyethylene sorbitan tristearate (TWEEN®-65), polyoxyethylene sorbitan monooleate (TWEEN®-80), and polyoxyethylene sorbitan trioleate (TWEEN®-85), for example, TWEEN®-20 and / or TWEEN®-80. These excipients are commercially available from several suppliers, such as Sigma, St. Louis, and Mo.

[0300] The amounts of various excipients in any of the disclosed compositions, including viruses such as AAV or lentiviruses, vary and are readily determined by those skilled in the art. For example, protein excipients, such as BSA, if present, may be present at concentrations between 1.0 wt.% and about 20 wt.%, for example, 10 wt.%. If amino acids, such as glycine, are used in the formulation, they may be present at concentrations between about 1 wt.% and about 5 wt.%. If carbohydrates, such as sorbitol, if present, may be present at concentrations between about 0.1 wt% and about 10 wt.%, for example, about 0.5 wt.% and about 15 wt.%, or between about 1 wt.% and about 5 wt.%. If polyethylene glycol is present, it may generally be present at concentrations on the order of about 2 wt.% to about 40 wt.%, for example, about 10 wt.% to about 25 wt.%. When propylene glycol is used in the formulation in question, it is typically present at concentrations of about 2 wt.% to about 60 wt.%, for example, about 5 wt.% to about 30 wt.%. When a detergent, such as sorbitan ester (TWEEN®), is present, it may be present at concentrations of about 0.05 wt.% to about 5 wt.%, for example, about 0.1 wt.% to about 1 wt%, see U.S. Published Patent Application No. 2012 / 0219528 incorporated herein by reference. For example, an aqueous virion-stabilizing formulation contains a carbohydrate, such as sorbitol, in a concentration between 0.1 wt.% and about 10 wt.%, for example between about 1 wt.% and about 5 wt.%, and a detergent, such as sorbitan ester (TWEEN®), in a concentration between about 0.05 wt.% and about 5 wt.%, for example between about 0.1 wt.% and about 1 wt.%. The virion is generally present in the composition in an amount sufficient to provide a therapeutic effect when given in one or more doses as defined above.

[0301] In some embodiments, the composition can be formulated into unit dosage forms suitable for individual administration of precise dosages. The amount of active compound administered depends on the subject being treated, the severity of the distress, and the mode of administration, and is best left to the discretion of the prescribing clinician. Within these boundaries, the administered formulation contains an amount of active compound effective in achieving the desired effect in the subject being treated.

[0302] Nucleic acid molecules can be incorporated into an inert matrix. As an example of an inert matrix, liposomes may be prepared from dipalmitoylphosphatidylcholine (DPPC), for example, egg phosphatidylcholine (PC). Liposomes, including cationic and anionic liposomes, can be prepared and used in this method.

[0303] In formulations for intrahepatic injection, the liposome capsule is degraded due to cellular digestion. These formulations can provide a delayed-release drug delivery system that exposes the target to a substantially constant concentration of nucleic acid molecules over time. In one example, the nucleic acid molecules may be soluble in an organic solvent, such as DMSO or alcohol, as previously described, and may contain polyacid anhydrides, poly(glycol) acids, poly(lactic) acids, or polycaprolactone polymers.

[0304] Nucleic acid molecules may be formulated to allow release over a specified period. The release system may include a biodegradable material or a matrix of material that releases nucleic acid molecules incorporated by diffusion. Nucleic acid molecules may be distributed uniformly or heterogeneously within the release system. Various release systems may be useful, however, the selection of the appropriate system depends on the rate of release required by the particular application. Both non-degradable and degradable release systems can be used. Preferred release systems include polymers and polymer matrices, non-polymer matrices, or inorganic and organic excipients, as well as diluents, e.g., calcium carbonate and sugars (e.g., trehalose), but are not limited to these. The release system may be natural or synthetic. However, synthetic release systems are generally preferred because they produce more reliable, more reproducible, and more defined release profiles. The release system material may be selected so that active ingredients with different molecular weights are released by diffusion through that material or by degradation of the material.

[0305] Typical synthetic biodegradable polymers include, for example, polyamides, e.g., poly(amino acids) and poly(peptides); polyesters, e.g., poly(lactic acid), poly(glycolic acid), poly(lactide-co-glycolic acid), and poly(caprolactone); poly(anhydride); polyorthoesters; polycarbonates; and their chemical derivatives (chemical groups, e.g., alkyl and alkylene substitution, addition, hydroxylation, oxidation, and other modifications routinely performed by those skilled in the art), copolymers, and mixtures thereof. Representative synthetic non-degradable polymers include, for example, polyethers, e.g., poly(ethylene oxide), poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymers—polyacrylates and polymethacrylates, e.g., methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic acid, and methacrylic acid, e.g., poly(vinyl alcohol), poly(vinylpyrrolidone), and poly(vinyl acetate); poly(urethane); cellulose and its derivatives, e.g., alkyl, hydroxyalkyl, ether, ester, nitrocellulose, and various cellulose acetates; polysiloxanes; and any chemical derivatives thereof (chemical group substitution, addition, hydroxylation, oxidation, and other modifications routinely performed by those skilled in the art), copolymers, and mixtures thereof.

[0306] Poly(lactide-co-glycolide) microspheres can also be used for intrahepatic injection. Typically, microspheres are composed of polymers of lactic acid and glycolic acid, which are structured to form hollow spheres. The spheres may have a diameter of approximately 15–30 microns and can be loaded with the biological molecules described herein.

[0307] Implants can be used that can be inserted into the eye by various methods, which may affect the release kinetics. The location of the implanted device may affect the concentration gradient of nucleic acid molecules around the device, and therefore may affect the release rate. Generally, when implants are used, nucleic acid molecules are uniformly distributed through the polymer matrix, and as a result, they are distributed sufficiently uniformly so that adverse fluctuations in the release rate do not occur due to heterogeneous distribution in the polymer matrix. The choice of polymer composition used depends on the desired release kinetics, the location of the implant, patient tolerance, and the characteristics of the implantation procedure. The polymer may be included as at least about 10 weight percent of the implant. In one example, the polymer is included as at least about 20 weight percent of the implant. In another embodiment, the implant contains two or more polymers. These factors are described in detail in U.S. Patent No. 6,699,493. Characteristics of the polymer may include, among others, biodegradability at the implantation site, compatibility with the drug of interest, ease of encapsulation, and water insolubility. Generally, the polymer matrix does not completely degrade until the drug load is released. Suitable polymer chemical compositions are known in the art (see, for example, U.S. Patent No. 6,699,493). Nucleic acid molecules can be formulated into implantable forms using other carriers and solvents. For example, buffers and preservatives can be used. The size and shape of the implant may also vary for use in specific areas of the liver (see, for example, U.S. Patent No. 5,869,079). In some embodiments, nanoparticles or dendrimers are used.

[0308] Nucleic acid molecules can be delivered by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, nanoparticle-mediated delivery (e.g., lipid or polymeric nanoparticle-mediated delivery), dendrimer-mediated delivery, as a conjugate with GalNAc, as mRNA modified by a base linker sugar, in association with a degradable polymer, as an mRNA-lipoplex, as a PEG-10 mRNA cargo, or by other methods known in the art. In some embodiments, the appropriate dose of mRNA depends on several factors, including the subject being treated (e.g., human or non-human primate or other mammal), the age and general condition of the subject being treated, the severity of the condition being treated, and the method of administration. An appropriate effective dose can be readily determined by those skilled in the art. Therefore, the "therapeutic effective dose" is within a relatively broad range that can be determined by clinical trials.

[0309] In some embodiments, a viral vector is used. In some embodiments, the therapeutically effective dose is approximately 10 5 ~10 16 virions (e.g., AAV virion, lentivirus or baculovirus), for example, 10 8 ~10 14 The order is on the order of billions. The dose may depend on the efficiency of transduction, promoter strength, stabilization of the messenger and the protein it encodes, as well as clinical factors. The effective dosage can be readily established by those skilled in the art through routine trials to establish a dose-response curve.

[0310] In some embodiments, when nucleic acid molecules are contained in the AAV vector, the effective amount administered to the recipient is approximately 1 × 10⁶ 8 The vector genome or more, and in some cases, approximately 1 × 10⁶ 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , or approximately 1 x 10 13 A vector genome or more, and in certain cases, approximately 1 × 10⁶ 14A vector genome or more, typically about 1 × 10⁶ 15 The vector is smaller than the genome. In some embodiments, the amount of vector delivered is approximately 1 × 10⁶, administered to the recipient. 14 A vector or smaller, for example, about 1 × 10⁻¹⁶ 13 , about 1×10 12 , about 1×10 11 , about 1×10 10 , or approximately 1 x 10 9 A vector or smaller, in a particular case, approximately 1 × 10⁻⁶ 8 A vector, and typically 1 × 10 8 It is more than a vector. In some non-limiting cases, the amount of vector genome delivered is approximately 1 × 10⁻⁶. 10 ~Approx. 1×10 11 It is a vector. In an additional, non-limiting example, the amount of vector delivered is approximately 1 × 10⁻⁶. 10 ~Approx. 1×10 12 It is a vector genome.

[0311] In some embodiments, the amount of pharmaceutical composition administered may be measured using the Multiplicity of Infection (MOI). In some embodiments, MOI refers to the ratio or multiplicity of the nuclei of the vector or viral genome to the cells to which it can be delivered. In some embodiments, MOI is approximately 1 × 10⁻⁶ 6 This is also possible. In some cases, the MOI is approximately 1 × 10⁻⁶. 5 ~Approx. 1×10 7 This is possible. In some cases, the MOI is approximately 1 × 10⁻⁶. 4 ~Approx. 1×10 8 This may also apply. In some cases, the recombinant viruses of this disclosure are at least about 1 × 10⁻⁶ 1 , about 1×10 2 , about 1×10 3 , about 1×10 4 , about 1×10 5 , about 1×10 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×1013 , about 1×10 14 , about 1×10 15 , about 1×10 16 , about 1×10 17 , and about 1×10 18 MOI. In some cases, the recombinant virus of the present disclosure is about 1×10 8 ~1×10 14 MOI. In some, the amount of pharmaceutical composition to be delivered is about 1×10 8 ~about 1×10 15 particles of recombinant virus, about 1×10 9 ~about 1×10 14 particles of recombinant virus, about 1×10 10 ~about 1×10 13 particles of recombinant virus, or about 1×10 11 ~about 1×10<0000:100>particles of recombinant virus.

[0312] The dosing regimen can be a single-dose schedule or a multiple-dose schedule that ultimately delivers the amounts specified above. Also, the subject may, where appropriate, be administered multiple doses. Thus, the recipient may, for example, receive 10 5 ~10 16 AAV virions as a single dose, or, for example, 10 5 ~10 16 AAV virions delivered collectively in 2, 4, 5, 6 or more doses. One of ordinary skill in the art can readily determine the appropriate number of doses to administer.

[0313] In some embodiments, AAV is administered to the recipient and / or donor liver (e.g., in an ex vivo perfusion system) at a dose of about 1×10 11 ~about 1×10 14 virus particles (vp) / kg. In some examples, AAV is administered to the recipient at a dose of about 1×10 12 ~about 8×10 13 vp / kg. In other examples, AAV is about 1×10 13 ~about 6×10 13It is administered to the recipient's liver at a dose of vp / kg. In specific, non-limiting examples, AAV is administered at least approximately 1 × 10⁶ times. 11 , at least about 5 × 10 11 , at least about 1 × 10 12 , at least about 5 × 10 12 , at least about 1 × 10 13 , at least about 5 × 10 13 , or at least about 1 × 10 14 It is administered to the recipient at a dose of vp / kg. In other non-limiting cases, AAV is approximately 5 × 10⁻⁶. 11 Below, approximately 1×10 12 Below, about 5×10 12 Below, approximately 1×10 13 Below, about 5×10 13 The following, or approximately 1 × 10 14 It is administered to the recipient at doses of less than vp / kg. In one non-specific example, AAV is approximately 1 × 10⁶ 12 AAV is administered to the recipient at a dose of vp / kg. AAV can be administered as a single dose or in multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses, or more) as needed to achieve the desired therapeutic effect.

[0314] In some embodiments, lentiviruses are approximately 1 × 10⁻⁶ 11 ~Approx. 1×10 14 It is administered at a dose of viral particles (vp) / kg. In some cases, lentiviruses are approximately 1 × 10⁶ 12 ~Approx. 8×10 13 The recipient is administered a dose of vp / kg. In other cases, the lentivirus is approximately 1 × 10⁶ 13 ~Approx. 6×10 13 The recipient is administered a dose of vp / kg. In a specific, non-limiting example, the lentivirus is at least approximately 1 × 10⁶ 11 , at least about 5 × 10 11 , at least about 1 × 10 12 , at least about 5 × 10 12 , at least about 1 × 10 13 , at least about 5 × 10 13 , or at least about 1 × 1014 The recipient is administered a dose of vp / kg. In other non-limiting cases, the lentivirus is approximately 5 × 10⁻⁶ 11 Below, approximately 1×10 12 Below, about 5×10 12 Below, approximately 1×10 13 Below, about 5×10 13 The following, or approximately 1 × 10 14 The recipient is administered a dose of less than vp / kg. In one non-specific example, the lentivirus is approximately 1 × 10⁶ 12 The recipient is administered a dose of vp / kg. Lentivirus can be administered in a single dose or in multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses, or more) as needed to achieve the desired therapeutic effect.

[0315] C. Rating After administration of one or more therapeutic agents provided herein, the subject may be evaluated for response. Exemplary methods, but not limited to, include ophthalmoscopic examination, microperimetry, gonioscopy, corneal thickness measurement, optical coherence tomography, reading speed, electroretinography, adaptive optics, ETDRS reading chart, or nerve fiber analysis. In some embodiments, the number and / or viability of retinal ganglion cells can be assessed. Those skilled in the art can readily determine the effectiveness of the disclosed methods. For example, this can be determined by whether the cupped-optic disc diameter ratio has been stabilized. Retinal nerve fiber layer analysis can be performed, for example, using scanning laser optical rotation analysis or optical coherence tomography. Progression can be monitored using visual field testing. For any of the disclosed methods, the therapeutic effectiveness in treating visual field defects may be as a substitute for the individual's vision. [Examples]

[0316] (Example 1) HERV-K RNA expression observed in the eyes of cadavers with AMD. Age-related macular degeneration (AMD) is caused by the death and degeneration of retinal pigment epithelial (RPE) cells. While several factors, including aging and the complement pathway, are associated with AMD, the precise mechanism of RPE degeneration remains unknown. An in vitro model was developed using induced pluripotent stem (iPSC)-derived RPE cells, which replicated the pathogenesis of AMD. Treatment of iPSC-RPE cells with activated complement (CC-HS) induced prominent features of AMD in iPSC-RPE cells, including the formation of RPE-subcutaneous drusen deposits that stain for antibodies against lipoprotein APOE and the formation of lipid-staining dyes (BODIPY® fluorophores) (Figure 1). 48-hour treatment with CC-HS resulted in a significant change in APOE localization from the apical to the basal side of the cells (Figure 1). BODIPY® fluorophore staining revealed higher staining for the dye in CC-HS-treated cells. Drusens are not formed when cells are treated with inactive complement (CI-HS). Furthermore, treatment with CC-HS resulted in degeneration of the RPE monolayer, as confirmed by reduced F-actin staining marking cell boundaries (Figure 1) and loss of monolayer transepithelial resistance (TER). Monolayer TER is formed by functional tight junctions between nearby RPE cells. As cells degenerate in the diseased state, the tight junctions break down, as seen under CC-HS treatment conditions, reducing the cell's TER (Figure 1B).

[0317] Activation of endogenous retroviruses can induce cellular degeneration. Endogenous retroviral RNA (Figure 2), as well as three translated proteins, gag, pol, and env, can induce cellular degeneration. Using comparative RNA-seq analysis, we identified that the expression of several members of the HERV-K family of endogenous retroviruses was significantly increased in CC-HS-treated cells compared to CI-HS-treated cells (Figures 3A-3B). Approximately 80 different loci were found to exhibit differential expression of HERV-K retroviruses (Figure 3A). We highlighted 16 of the most differentially expressed HERV-K loci between CI-HS and CC-HS and determined their exact locations in the genome (Figure 3B).

[0318] Temporal analysis of CC-HS treatment showed increased expression of different components of the endogenous retrovirus HERV-K family (ENV and GAG) in CC-HS-treated cells compared to CI-HS-treated cells (Figures 4A, 4B, and 5). Expression of other retroviruses, such as HERV-R, did not increase in this context (Figures 4A, 4B). A similar increase in HERV-K RNA expression was noted in the eyes of cadaveric AMDs, confirming a physiological relevance (Figure 6). Overall, this analysis revealed that the expression of endogenous retrovirus-specific HERV-K classes increased in CC-HS-treated iRPEs, and that this increase in HERV-K expression occurred concurrently with RPE deposition and the formation of RPE monolayer denaturation.

[0319] (Example 2) Reduction of HERV-K RNA expression in the eyes of cadavers with AMD. CC-HS treatment increased the expression of inflammatory cytokines (IL6 and IL8) secreted by RPE cells in both the apical and basal directions (Figures 7A, 7B). We determined whether this increase in inflammatory cytokine expression was mediated by increased HERV-K expression in CC-HS-treated cells. To suppress HERV-K expression, we used tenofovir, an antiretroviral drug that blocks the transcription of HERV-K mRNA. Co-treatment of iRPE cells with CC-HS and tenofovir reduced apical and basal secretion of both IL6 and IL8 (Figures 7A, 7B).

[0320] HERV-K ENV protein and RNA are thought to induce intracellular inflammatory changes by activating their receptors, TLR3 and TLR4. Therefore, we determined whether blocking the activity of both of these receptors reduced the expression of IL6 and IL8. Blocking the activity of these receptors also reduced the expression of pro-inflammatory cytokines in CC-HS-treated RPE cells, suggesting that the ENV protein acts in RPE cells through these receptors (Figures 7A, 7B). Furthermore, blocking TLR3 and TLR4 activity along with co-treatment of iRPE with CC-HS also reduced the formation of drusen deposits under RPE cells stained with the lipid stain BODIPY® (Figures 8A, 8B). These data suggest that the degenerative phenotype observed in iRPE cells is driven by alterations in HERV-K-mediated signaling, which may be mediated by the TLR3 and TLR4 receptors.

[0321] To confirm that viral activation in RPE cells could induce the AMD phenotype without CC-HS treatment, the HERV-K ENV protein was overexpressed in healthy cells (Figures 9A-9C). The HERV-K ENV protein, which is thought to drive HERV-K pathology in cells, was fused to a V5 tag, allowing for the detection of the overexpressed protein and its separation from the endogenously produced ENV protein. The red fluorescent protein mcherry was driven from the same expression cassette that drives ENV protein expression. However, once transcribed, the two proteins were produced separately due to the presence of an intrasequential ribosome entry site (IRES) between ENV and mcherry. Expression of the entire cassette was driven by the CMV promoter, enabling high expression. This construct was packaged in a lentivirus, and iRPE cells were transduced with it. Western blotting performed on cell lysates from iRPE transduced with ENV-expressing lentivirus, compared to untransduced cells, showed that the construct was able to drive the expression of the ENV protein in HERV-K. Immunostaining for the ENV protein in cells transduced at different MOIs (0.5 and 3.0) showed that the lentivirus increased ENV protein expression in a dose-dependent manner (Figure 9A-9C). Overexpression of the ENV protein in wild-type iRPE was sufficient to induce the AMD phenotype without CC-HS treatment, as measured by loss of cell barrier resistance (TER) and increased BODIPY® staining (Figure 10A-10C). The decrease in monolayer TER was dose-dependent for the ENV protein. For example, higher MOI resulted in higher amounts of ENV protein (Figure 10C) and a greater decrease in monolayer TER (Figure 10A), but the increase in lipid deposits under RPE stained by BODIPY® was not a dose-dependent phenomenon (Figure 10B). Overall, this indicates that the ENV protein of HERV-K was sufficient to induce the AMD phenotype in iRPE cells.

[0322] To determine the mechanism of increased HERV-K expression in CC-HS-treated cells, the expression of angiogenin (ANG), a ribonuclease enzyme thought to degrade HERV-K RNA via the use of Lys-specific tRNA halves, was determined using RNA-seq. The analysis revealed that ANG expression was significantly reduced in CC-HS-treated cells, concurrently with increased HERV-K expression, compared to untreated or CC-HS-treated cells (Figure 11A). Temporal qRT-PCR analysis of CC-HS-treated cells after 2, 4, and 6 days of treatment showed that ANG expression progressively decreased in the lysates of CC-HS-treated cells. In contrast to the expression of another RNA-degrading enzyme, ANG expression did not decrease (Figure 11B). Next, we determined whether the concentration of ANG protein secreted into the cell culture medium by RPE cells was also reduced after 2, 4, and 6 days of CC-HS treatment of RPE cells, according to different analyses. Consistent with RNA-seq and qRT-PCR analyses, ELISA revealed a progressive decrease in secreted ANG levels in CC-HS-treated cells compared to CI-HS-treated cells (Figure 11C). The reduction in ANG expression was consistent across three biological replicates. Similar findings regarding lower ANG expression in CC-HS-treated cells were observed by Western blotting and immunostaining (Figure 12). We determined whether ANG degrades HERV-K RNA using Lys-specific tRNA expression as a template. qRT-PCR confirmed that all Lys-specific tRNA fragments (LysCTT, LysTTT, pro-AGG) decreased within 24 hours of CC-HS treatment of iRPE cells, but the expression of another tRNA fragment for leucine-specific tRNA (LeuTAA), which does not act as a template for HERV-K degradation by ANG, did not decrease in CC-HS-treated iRPE cells (Figures 13A, 13B). Overall, this analysis shows that HERV-K levels increase CC-HS-treated iRPE through downregulation of ANG expression and tRNA fragments required for HERV-K degradation.

[0323] A gene therapy strategy was developed to reduce HERV-K levels in CC-HS-treated iRPE cells. HERV-K is generated from multiple loci in the genome, and these loci have slight sequence variations between them. Therefore, to develop gene therapy for HERV-K, RNA generated from most of these different loci of HERV-K was targeted. CAS13 is a nuclease that can target and degrade RNA using complementary guide sequences. Using bioinformatics analysis, three guide sequences were identified in HERV-K to be used to target HERV-K expression in RPE cells. These sequences were designed against the GAG ​​region of HERV-K mRNA, which is the RNA start. Degradation of the RNA start results in the degradation of the entire RNA. Using bioinformatics analysis, it was found that the three guide sequences in HERV-K were used to target HERV-K expression in RPE cells (Figures 14A-14B).

[0324] The selected sequences showed complementarity across multiple HERV-K loci and also demonstrated good consensus for CAS13-based targeting (Figures 15A-15B). First, we confirmed the detection of high levels of Cas13Rx enzyme in iRPE cells using lentiviral-based overexpression. HA-tagged Cas13Rx was overexpressed using a lentiviral construct. Western blotting and immunostaining for the HA tag confirmed enzyme expression in iRPE cells with a vector of 1.0 MOI (Figures 16A-16B).

[0325] Figures 17A–17E show reverse complement-induced lipid accumulation in Cas13Rx-mediated HERV-K knockdown. As shown in Figure 17A, iRPE cells were transduced with lentivirally expressed HA-tagged CAS13Rx under the control of a doxycycline-inducible promoter to overexpress Cas13Rx. Seven days of doxycycline treatment induced CAS13Rx expression (Figure 16). At this stage, iRPE cells were transduced with another lentiviral construct expressing one of three guide RNAs alone or together, and a control guide RNA. After another seven days, allowing high levels of guide RNA expression, the cells were then treated with CC-HS. Four days after CC-HS or CI-HS treatment, HERV-K levels were measured in the cells, and monolayer TER and BODIPY® levels were measured in the cells. Figure 17B shows TER graphs for CIHS (gray bars) and CCHS (black bars) treated samples transduced with either scrambled guide RNA (NEG) or guide RNA (Cas13Rx) or guide RNA #2 (Figure 15). Only guide RNA #2 is able to rescue TER downregulated by CCHS treatment. In Figure 17C, the upper panel shows digital images of CIHS or CCHS treated iRPE transduced with either scrambled control (NEG) or guide RNA #2. CCHS-induced increase in HERV-K expression (comparing CIHS NEG samples with CCHS NEG samples) is downregulated by guide RNA 2 for HERV-K. The lower panel shows digital images of CIHS or CCHS treated iRPE transduced with either scrambled control (NEG) or guide RNA #2. The CCHS-induced increase in lipid deposition in CCHS (measured as a BODIPY® signal) is downregulated by guide RNA 2 for HERV-K. Figure 17D shows the quantification of HERV-K levels observed in Figure 17C, and the upper panel shows that guide RNA #2 can downregulate HERV-K levels in CCHS-treated iRPE cells.Figure 17E shows the quantification of BODIPY® levels observed in Figure 17C, and the lower panel shows that guide RNA #2 can downregulate BODIPY in CCHS-treated iRPE cells.

[0326] Figure 18 shows reverse complement-induced lipid accumulation of Cas13Rx-mediated HERV K knockdown by all three guides selected in Figure 15. Digital images of CCHS-treated iRPE transduced with either a scrambled control (NEG) or one or a combination of the three guide RNAs #1, 2, and 3. The CCHS-induced increase in lipid deposition in CCHS (measured as a BODIPY® signal) is downregulated by all three guides for HERV-K. The graph on the left shows quantification of the digital imaging data. Additional results are provided in Figure 19.

[0327] Overall, the data showed that physiological stressors such as CC-HS increase the expression of the HERV-K endogenous retrovirus in iRPE cells, and that this virus is required and sufficient for the induction of the AMD phenotype in iRPE cells. Downregulation of HERV-K transcription by anti-HIV drugs or gene therapies targeting the expression of HERV-K and other similar endogenous retroviruses can be used to treat AMD and other neurodegenerative diseases. Furthermore, three target sites were identified for gene therapy targeting of HERV-K.

[0328] (Example 3) material and method This embodiment provides the materials and methods used for the data described in Examples 1 and 2 above.

[0329] Immunofluorescence or BODIPY® fluorescence staining: Fully differentiated iRPE monolayers, 6-8 weeks old and grown on Transwells, were used for all experiments. iRPE cells were treated for 48 hours with either 5% CC-HS (S1-LITER, EMD Millipore) or 5% CI-HS (heat-inactivated CC-HS) supplemented in RPE maintenance medium (RPEMM), with daily medium changes. For chronic treatment, 0.1% CC-HS (or CI-HS) was added to both the apical and basal media of the iRPE cells and left for up to 6 days. The medium was changed daily.

[0330] Immunostaining was performed as previously described (see Sharma et al., Nature Comm. 12: 7293, doi.org / 10.1038 / s41467-021-27488-x, 2021). iRPEs, prepared either as cross-sections or monolayers on Transwell membranes (TWMs), were fixed in 4% paraformaldehyde and blocked for 1 hour at RT in immunocytochemistry (ICC) buffer containing 1× phosphate-buffered saline (PBS) (10010-023, ThermoFisher), 1% bovine serum albumin (BSA) (160069, MP Biomedicals), 0.25% TWEEN® 20 (900-64-5, Affymetrix), and 0.25% TRITON® X-100 (9002-64-5, Sigma). The cells were incubated overnight at 4°C with primary antibodies diluted in ICC buffer. Primary antibodies were used against the following proteins: APOE (approx. 250 ng / mL; AB947, Millipore, Figure 1), HERV-K (approx. 1 μg / mL, HERM-1811-5, Austral Biologicals), angiogenin (1 ug / mL, AF265, R&D systems, Figures 13, 17), HA tag (2 μg / mL, SAB2702217, Sigma Aldrich), and ZO-1 (5 μg / mL; MA3-39100-A488, ThermoFisher). Cells were then washed with ICC buffer and incubated with secondary antibodies diluted in ICC buffer in the dark at RT for 1 hour.Secondary antibodies include: ALEXA FLUOR® 555 goat anti-rabbit (1:200) (5 μg / mL; A21429, ThermoFisher), ALEXA FLUOR® 488 goat anti-rabbit (1:1000) (5 μg / mL; A11078, ThermoFisher), ALEXA FLUOR® 555 goat anti-mouse (1:500) (5 μg / mL; A-21422, ThermoFisher), ALEXA FLUOR® 488 goat anti-mouse (1:200) (5 μg / mL; A32723, ThermoFisher), Hoechst-33542 (1:1000) (H3570, ThermoFisher), and phalloidin (1:300) (ALEXA FLUOR® 488, A12379, ThermoFisher) or (1:400) (Phalloidin-iFluor 647 reagent, ab176759, Abcam). Cells were washed with ICC buffer and mounted on glass slides with Fluoromount-G aqueous mounting medium (0100-01; Southern tech) and glass coverslips. Samples were imaged using a Zeiss 880 confocal microscope (Carl Zeiss). Images were processed using Zenblue 3.2 software (Carl Zeiss) and exported as TIFF files.

[0331] Lipid deposits were stained using BODIPY® dyes (D3922 or D3835, ThermoFisher, below Figure 1, 9a, 11b, and 17C). Cells were fixed in 4% paraformaldehyde at RT for 20 minutes and washed with 1×PBS. BODIPY® stock solution was prepared in DMSO at a concentration of 1 mg / mL (3.8 mM), and then diluted 1:1000 in 1×PBS to prepare diluted standard solutions. Both the stock solution and the diluted standard solution were brought to RT and then filtered through a 0.22 μm filter before use. Cells were incubated in the diluted standard solution at RT for 30 minutes, washed with 1×PBS, mounted, and imaged using the procedure described above. Lipid droplets (BODIPY® dyes) were quantified in ImageJ (v1.8.0, Bethesda, USA). For HERV-K, particles were counted using a maxima prominence 8000, and for BODIPY® dyes, particles were counted using a maxima prominence 2900.

[0332] RNAscope probes and reagents for localizing HERV-K mRNA were obtained from Advanced Cell Diagnostics (acdbio.com), unless otherwise noted.

[0333] Fully matured confluent monolayers of iRPE grown on Transwell were treated with CI-HS or CC-HS (5%) for 48 hours. Cells were fixed in 10% formalin. Transwell membranes were punched out, embedded in paraffin, and sectioned transversely into 5 μM cross-sections in an RNA-free environment. The assay was performed using the RNAscope 2.5 HD Chromogenic Detection Kit (PN332360) according to the manufacturer's protocol. In short, the slides are dried at 60°C for 1 hour (HybEZ oven), immersed in xylene 2x for 5 minutes with stirring, then in 100% ETOH 2x for 1 minute, air-dried, and then the pigment is removed for 20 minutes in a buffer containing (250 ul of deionized formamide, 4 ml of water, 250 ul of 20x SSC (1x final), 817 ul of 30% hydrogen peroxide) in a humidified chamber about 3-5 inches away from a fluorescent light (checking periodically for pigment disappearance). The slides are then washed with 1x PBS, sequentially dehydrated in 70% ETOH for 1 minute, then in 100% ETOH for 1 minute, and air-dried for 5 minutes at RT. The slides were then treated with H2O2 at room temperature (RT) for 10 minutes, washed three times with distilled water, and subjected to targeted recovery in 1× recovery buffer at 99C for 15 minutes in a Black and Decker Steamer HS3000, followed by a thorough rinse with distilled water for 15 minutes. The slides were then transferred to 100% ETOH for 3 minutes and dried at RT. The slides were then treated with protease plus in a preheated humidified tray at 40°C, incubated in a 40°C oven for 15 minutes, and immediately washed five times with distilled water. The slides were air-dried and incubated with the probes in a hybEZ rack located in a humidified tray in the oven at 40°C for 2 hours, followed by three washes with 1× wash buffer at room temperature (RT) for 2 minutes. The slides were stored overnight at RT in 5× SSC.The following day, the slides were washed twice with 1x washing buffer for 2 minutes at RT, then incubated with AMP 1 at 40°C for 30 minutes, AMP 2 at 40°C for 15 minutes, AMP 3 at 40°C for 30 minutes, AMP 4 at 40°C for 15 minutes, AMP 5 at RT for 30 minutes, and AMP 6 at RT for 15 minutes, each added to the same washing step after each AMP. The signal was detected by incubation with Fast RED-B solution (prepared as a 1:60 ratio of Fast RED-B to Fast RED-A, used within 5 minutes, avoiding direct sunlight or UV light) at RT for 10 minutes, and then washed with tap water. The slides were then counterstained in 50% hematoxylin solution (Gill's Hematoxylin I, HXGHE1LT, American MasterTech Scientific (1:4)) at RT for 20 minutes, washed three times with tap water, rapidly immersed in 0.02% aqueous ammonia, then washed five times with tap water, and dried for 15 minutes in a drying oven at 60°C. The slides were dehydrated in pure xylene (not pure ETOH) and immediately mounted on the slides using EcoMount medium (EM897L, Biocare medical) before drying the xylene. Images were acquired using a Zeiss Axio Imager M2 microscope with a 40x oil immersion objective and an AxioCamIcc1 digital camera, and Zen software (Carl Zeiss AG, Oberkochen, Germany, zeiss.com).

[0334] The probes included either the human positive control probe Hs PPIB C1 (PN 321641) or the RNASCOPE® probe V-HERV-K-pol (469831).

[0335] Eye slides from human cadavers were obtained through the Advancing Sight Network (Alabama Eye Bank). Diagnoses of AMD and non-AMD were based on the donor's medical records. The time from death to preservation was 3.3 hours for non-AMD eyes and 3.6 hours for AMD eyes. The slides were processed in the same manner as described above.

[0336] TER Measurement: Transepithelial resistance (TER) in iRPE was measured using a commercially available Epithelial Volt / Ohm Meter (EVOM2, WPI). Chopstick-shaped electrodes (STX2, WPI) were simultaneously placed on the apical and basal media of the iRPE. Resistance values ​​(Ohm) were noted. Actual resistance (Ohm·cm) 2 The TER value is calculated by multiplying the raw TER value by the measurement area (Transwell with a diameter of 12 mm).

[0337] RNA-seq analysis to identify CC-HS-induced locus-specific expression of HERV-K: First, the dataset was analyzed for CCHS-induced transposon element (TE) expression using TEToolkit GTF (github.com / mhammell-laboratory / tetoolkit). Then, a custom index for RNA quantification of the established RNA-seq data (nature.com / articles / s41467-021-27488-x) was constructed using the following method: 1) Assign users who provided transposition elements (TEs) to the file. Use a set of coordinates to orient / count mapping events at genomic locations. Two types of reads were counted: 1) "Unique" reads, where part of the read is in a specific part of the genome and the remainder is in a TE. 2) "Multiple" reads that cannot be mapped to specific locations (because they are large / overall within the TE). 3) The reads are then aggregated into the type / class of the TE (resulting in a HERV type, and then hundreds to thousands of unique entries are folded into a single value). 4) These AGGREGATED reads (again, by TE class such as "HERV15-int ERV1 LTR") are used in differential expression analysis.

[0338] Next, locus-specific HERV-K expression was performed as follows: following the annotated loci of HERV-K in the human genome (see retrovirology.biomedcentral.com / articles / 10.1186 / s12977-020-00519-z#MOESM1 and retrovirology.biomedcentral.com / articles / 10.1186 / 1742-4690-8-90) and locus annotations in GENBANK® for the version hg38 human genome. The combined dataset of approximately 80 HERV-K loci in the human genome consisted of loci containing either gag (complete or nearly complete loci) or partial loci (no gag and / or other parts of HERV-K missing), as well as sequence identifiers for each locus. Site-specific expression data for all HERV-K loci were narrowed down to the 20 most differentially expressed loci (CI-HS and CC-HS). The data was expressed as adjusted depth for each locus, calculated by taking the total reads of a region and dividing it by the number of base pairs in that region. For visualization of the RNA-seq data, sample Bam files were created that allowed the data to be viewed using the Integrative Genomics Viewer (IGV) (ncbi.nlm.nih.gov / pmc / articles / PMC3346182 / ), which enables real-time examination of RNA-seq datasets across the entire genome.

[0339] The established RNA-seq datasets CI-HS and CC-HS (nature.com / articles / s41467-021-27488-x) were queried via a customized RNA sequencing (RNA-seq) transcriptome database of healthy human eye tissue (pubmed.ncbi.nlm.nih.gov / 31343654, eyeIntegration.nei.nih.gov) to read differential expression data for a set of RNASEs (including angiogenin, dicer, and RNase I, 2, 3, and 4). The data were plotted as box plots.

[0340] qRTPCR (mRNA or tiRNA quantification): Total RNA was isolated from fully differentiated RPE monolayers grown on transwells using the mirVana miRNA isolation Kit (AM1561, Invitrogen / ThermoFisher) as previously described (academic.oup.com / hmg / article / 28 / 20 / 3355 / 5537027). RNA samples were treated with DNASE I to remove carried-over genomic DNA (RapidOut DNA Removal Kit, K2981, THERMO SCIENTIFIC). Quantification and RNA integrity were assessed using nanodrop (see nature.com / articles / s41467-021-27488-x#Sec11).

[0341] Regarding mRNA qRT-PCR: cDNA was prepared using the ISCRIPT® cDNA Synthesis Kit (1708891, Bio-Rad) according to the manufacturer's protocol. The PCR reaction was performed in 10 μl of reaction mixture containing 5 ng of cDNA and 5 μL of SSOADVANCED® Universal SYBR® Green Supermix (1725274, Bio-Rad), and amplified in three replicates using a Viia7 Real-Time PCR System (ThermoFisher Scientific). The HERV-K primer sequence was synthesized according to publicly available data (Li et al., 2015 Sep 30;7(307):307ra153. doi: 10.1126 / scitranslmed.aac8201. PMID: 26424568; PMCID: PMC6344353).

[0342] Regarding tiRNA quantification: Fully mature iRPEs grown on Transwell were treated with CI-HS or CC-HS (5%) for 3, 24, or 48 hours. Total RNA was pretreated to remove post-transcriptional modifications according to an established protocol (RTSTAR® tRF&tiRNA pretreatment Kit (catalog #: AS-FS-005)). Briefly, 3'-end deacylation of 1 μg of total RNA was carried out in 15 μl of reaction mix at 37°C for 40 minutes, and the reaction was stopped by incubation at RT with 19 μL of deacylation stop buffer for 5 minutes. The reaction was continued by adding the final treatment mix to remove the 3'-phosphate and add the 5'-phosphate to the RNA in 50 μl of reaction mix at 37°C for 40 minutes. The terminal enzyme was inactivated by incubation at 70°C for 5 minutes. The RNA was then purified by magnetic bead-based RNA purification (SEQ-STAR® RNACLEAN® and SMALLENRICH® beads, AS-MB-009, Arraystar, Inc., see below). The purified RNA samples were treated with a demethylation reaction product, which was obtained by incubating the ligated RNA with a demethylation mix at 37°C for 2 hours using an adapter, followed by the addition of stop buffer to terminate the reaction. The RNA was then purified again using the bead-based RNA purification method described above.

[0343] RNA purification was performed using the SEQ-STAR® RNACLEAN® and SMALLENRICH® bead assays. Pre-treated RNA products were thoroughly mixed with a 1.8× (1.8:1 v:v ratio) bead suspension and 4.2× 100% isopropanol (RNA > 17 bp collected) by vortexing for 30 minutes. The mixture was then incubated at RT for 10 minutes to bind the RNA to the beads. The mix tube was placed on a magnetic stand (DYNAMAG®, 12321D, Invitrogen) for 3 minutes until the supernatant was completely clear. The supernatant was carefully aspirated and discarded. The tube was kept on the magnetic stand, washed twice with 200 μL of freshly prepared 80% ethanol, and the beads were air-dried. RNA was eluted by resuspending the beads in TE buffer and incubated at RT for 2 minutes. The tube was tapped on a magnetic stand until the supernatant was completely clear from the beads, and then the supernatant containing the purified RNA was transferred to a new tube.

[0344] Reverse transcription was performed using the RTSTAR® First-Strand cDNA Synthesis Kit for tiRNA Detection (AS-FS-003, ARRAYSTAR). Briefly, the treated RNA was attached using a 3-minute Adapter by incubation with a ligation mix at 25°C for 1 hour, followed by hybridization with RT primers in a thermocycler at 75°C for 5 minutes, 37°C for 15 minutes, and 25°C for 15 minutes. The product with the 3' adapter ligated was then incubated with a 5' adapter ligation mixture at 25°C for 1 hour. For CDNA synthesis, the RNA with the adapter ligated was then incubated with a reverse transcription mixture at 45°C for 1 hour.

[0345] PCR was performed using pre-designed tiRNA primers from ARRASTAR Inc. (RTSTAR® Pre-designed tRF&tiRNA Primer Sets (H / M): 3'tiR_088_LysCTT(n), AS-NR-002-1-013; 3006B-LysTTT, AS-NR-002-1-086; 3002A pro-AGG, AS-NR-002-1-091; 3016 / 18 / 22B LysCTT(n), AS-NR-002-1-099; LeuTAA 3009B, AS-NR-002-1-097; Housekeeping SNORD43, AS-NR-002-1-187) following the same protocol as above (cDNA synthesis from mRNA).

[0346] Relative quantification was performed using GAPDH (for mRNA) and nuclear small RNA (SNoR043) as a normalization control. -ΔΔCT The calculation was performed using the method (Schmittgen and Livak Nat Protoc. 2008;3(6):1101-8. doi: 10.1038 / nprot.2008.73. PMID: 1854660). Statistical significance was determined using Student's t-test (two-tailed, independent) at a significance level of P<0.05. Data were compared with controls (2 n This is expressed as twice the change compared to ), and is the average of 3 replicates ± SEM.

[0347] Western blot: Total protein lysates of iRPE cells were prepared as previously described (nature.com / articles / s41467-021-27488-x) by lysing cells on Transwell membranes with RIPA buffer (89900, ThermoFisher) supplemented with an inhibitor cocktail (1× HALT® protease and phosphatase inhibitor cocktail (100×), 78440, ThermoFisher). The lysates were clarified by centrifugation at 4°C and 16,000×g for 20 minutes. Protein concentrations were quantified by BCA protein assay (23227, ThermoFisher) according to the manufacturer's protocol. SDS-PAGE for Western blot analysis was performed using 4-16% pre-prepared gels (AnykD® CRITERION® TGX® Precast Midi Protein Gel, 12+2 wells, 45 μl, catalog #5671123, Bio-Rad) and equal amounts of loaded protein (30 μg / well). Proteins were transferred to polyvinylidene fluoride (PVDF) membranes using a semi-dry transfer device (Trans-Blot Turbo Transfer System, 1704150, Bio-Rad). The blots were blocked at RT for 1 hour in a buffer containing 5% BSA in PBST (1× PBS and 1% Tween 20), and then incubated overnight at 4°C with the primary antibody on a shaker. The blots were washed with 1× phosphate-buffered saline - 1% TWEEN® (PBST), and then incubated at RT for 1 hour wit...

Claims

1. A method for treating age-related macular degeneration (AMD) or reducing the risk of developing it in a subject, Selecting subjects who have AMD or are at risk of developing it; and The subject is given an effective dose of a drug that inhibits HERV-K. A method comprising, by means of, treating AMD in the subject or reducing the risk of developing it.

2. Selecting the object having the aforementioned AMD; and The subject is administered an effective amount of the drug that inhibits HERV-K. The method according to claim 1, comprising, and thereby treating the AMD in the subject.

3. The method according to claim 1 or claim 2, wherein the subject has exudative AMD.

4. The method according to claim 1 or claim 2, wherein the subject has atrophic AMD.

5. The method according to any one of claims 1 to 4, wherein the drug comprises an antiretroviral agent.

6. The method according to claim 5, wherein the antiretroviral agent is a reverse transcriptase inhibitor, a protease inhibitor, an integrase inhibitor, a viral fusion inhibitor, or a viral entry inhibitor.

7. The method according to claim 6, wherein the antiretroviral agent is a reverse transcriptase inhibitor.

8. The method according to claim 7, wherein the reverse transcriptase inhibitor comprises lamivudine, zidovudine, abacavir, tenofovir, tenofovir prodrug, emtricitabine (FTC), delavirdin, efavirenz, nevirapine, or a pharmaceutically acceptable salt thereof.

9. The method according to claim 8, wherein the reverse transcriptase inhibitor comprises tenofovir or the tenofovir prodrug, and the tenofovir prodrug is tenofovir disoproxil fumarate, tenofovir alafenamide, or a pharmaceutically acceptable salt thereof.

10. The method according to any one of claims 1 to 9, comprising administering to the subject a) an effective amount of FTC or a pharmaceutically acceptable salt thereof, and b) an effective amount of tenofovir disoproxil fumarate (TDF) and / or tenofovir alafenamide (TAF) or a pharmaceutically acceptable salt thereof.

11. The method according to any one of claims 5 to 10, further comprising administering an effective amount of a drug efficacy enhancer to the subject.

12. The method according to claim 11, wherein the drug effect enhancer is cobicistat.

13. The method according to any one of claims 5 to 12, further comprising administering an effective amount of elvitegravir (EVG) to the subject.

14. The method according to any one of claims 7 to 9, further comprising administering a second antiretroviral agent to the subject, wherein the antiretroviral agent is a protease inhibitor.

15. The method according to claim 14, wherein the protease inhibitor is darunavir.

16. The method according to any one of claims 1 to 15, further comprising administering an effective amount of an additional drug to the subject, wherein the additional drug is metformin hydrochloride or L-745,870 trihydrochloride.

17. The method according to any one of claims 1 to 16, wherein the agent comprises an inhibitory oligonucleotide or an inhibitory peptide.

18. The method according to claim 17, wherein the inhibitory oligonucleotide is an antisense RNA, a small inhibitory RNA, a ribozyme, or a small hairpin (sh) RNA.

19. The method according to any one of claims 1 to 18, wherein the drug comprises a CRISPR / Cas13 system.

20. The method according to claim 19, wherein the CRISPR / Cas13 system comprises a) a guide RNA (gRNA) or a nucleic acid molecule encoding the gRNA that specifically hybridizes to RNA encoding the glycosaminoglycan (Gag) protein of HERV-K; b) a gRNA or a nucleic acid molecule encoding the gRNA that specifically hybridizes to RNA encoding the polymerase (Pol) protein of HERV-K; or c) a gRNA or a nucleic acid molecule encoding the gRNA that specifically hybridizes to RNA encoding the envelope (Env) protein of HERV-K.

21. The method according to claim 20, wherein the gRNA comprises one or more of sequence numbers 1 to 3.

22. The method according to any one of claims 19 to 21, wherein the CRISPR / Cas13 system comprises a lentiviral vector encoding the Cas13 protein.

23. The method according to any one of claims 19 to 21, wherein the CRISPR / Cas13 system comprises an adenovirus vector, a lentiviral vector, a baculovirus vector, or nanoparticles.

24. The method according to claim 22 or 23, wherein the lentiviral vector, the adenovirus vector, or the baculovirus vector, or nanoparticles comprises an inductive promoter, a constitutive promoter, or a tissue-specific promoter operably linked to a nucleic acid molecule encoding the Cas13 protein.

25. a) the constitutive promoter is CMV; b) the inducible promoter is doxycycline or another inducible promoter; or c) the tissue-specific promoter is a retinal pigment epithelial cell-specific promoter.

26. The method according to any one of claims 19 to 25, wherein the CRISPR / Cas13 system comprises a ribonucleoprotein complex containing the Cas13Rx protein and the gRNA.

27. The method according to any one of claims 19 to 26, wherein Cas13 is Cas13a, Cas13b, Cas13c, Cas13Rx, Cas13x, or Cas13y.

28. The method according to any one of claims 1 to 27, wherein the drug comprises an antibody that specifically binds to Gag, Pol, or Env of HERV-K, or an antigen-binding fragment thereof.

29. The method according to any one of claims 1 to 28, wherein administering an effective amount of a drug that inhibits HERV-K to the subject includes administering the drug that inhibits HERV-K to the eye of the subject.

30. (a) at least one Cas13 protein, or a nucleic acid molecule encoding the at least one Cas13 protein; and (b) At least one CRISPR-Cas system guide RNA (gRNA) comprising SEQ ID NOs. 1, 2, and / or 3 that hybridizes with a HERV-K RNA molecule, or at least one nucleic acid molecule encoding the at least one gRNA. A naturally occurring or manipulated clustered, regularly arranged short palindrome repeat (CRISPR) association (Cas) system for targeting HERV-K, comprising: The Cas13 protein forms a complex with the at least one gRNA, and the at least one gRNA directs the complex toward the HERV-K RNA molecule. system.

31. At least one Cas13 protein; and One or more guide RNAs A ribonucleoprotein (RNP) complex comprising, wherein at least one of the one or more guide RNAs comprises SEQ ID NOs: 1, 2, and / or 3.

32. Isolated cells comprising the system described in claim 30 or the RNP complex described in claim 31.

33. The isolated cells according to claim 32, which are retinal cells.

34. The isolated cells according to claim 33, wherein the retinal cells are retinal pigment epithelial cells, photoreceptor cells, or choroidal cells.

35. A method for treating age-related macular degeneration (AMD) or reducing the risk of developing it in a subject, Selecting subjects who have AMD or are at risk of developing it; and The subject is given an effective amount of a drug that increases angiogenin (ANG) activity. A method comprising, by means of, treating AMD in the subject or reducing the risk of developing it.

36. Selecting the object having the aforementioned AMD; and The subject is administered an effective amount of the drug that increases the ANG activity. The method according to claim 35, comprising, and thereby treating the AMD in the subject.

37. The method according to claim 35 or claim 36, wherein the subject has exudative AMD.

38. The method according to claim 35 or claim 36, wherein the subject has atrophic AMD.

39. The method according to any one of claims 35 to 38, wherein the agent comprises ANG or a nucleic acid molecule encoding ANG.

40. The aforementioned ANG is a) Amino acid sequence that is 95% identical to SEQ ID NO: 11; or b) Amino acid sequence of SEQ ID NO: 11 The method according to claim 39, including the method described in claim 39.

41. The method according to claim 39 or 40, wherein the agent comprises the nucleic acid molecule encoding ANG.

42. The method according to claim 41, comprising administering an expression vector containing the nucleic acid molecule encoding ANG to the subject.

43. The method according to claim 42, wherein the expression vector is an adenovirus vector, a lentivirus vector, or an adeno-associated virus vector.

44. The method according to claim 42 or 43, wherein the expression vector comprises a retinal pigment epithelial cell-specific promoter operably linked to the nucleic acid molecule encoding ANG.

45. The method according to claim 44, wherein the retinal pigment epithelial cell-specific promoter is the RPE65, BEST1, DCT, TYR, or TYRP1 promoter.

46. The method according to claim 42 or 43, wherein the expression vector comprises a constitutive promoter operably linked to the nucleic acid molecule encoding ANG.

47. The method according to claim 46, wherein the constitutive promoter is a human β-actin promoter, a human elongation factor-1α promoter, a β-actin promoter, a monkey virus 40 promoter, or a herpes simplex virus thymidine kinase promoter.

48. The method according to any one of claims 35 to 47, wherein the agent comprises a CRISPR / Cas9 system that increases the expression of ANG in the subject.

49. The method according to any one of claims 35 to 38, wherein the agent comprises a low molecule that increases ANG activity.

50. The method according to claim 49, wherein the low molecular weight is L-minosine, muscone, opioid, DMOG, or ML228.

51. The method according to any one of claims 35 to 50, wherein the drug comprises a tRNA fragment complementary to HERV-K, or a nucleic acid molecule encoding the tRNA fragment.

52. The method according to claim 51, wherein the tRNA fragment comprises GCCCCACGTGGGCGCCA (SEQ ID NO: 12) or GTCCCCTGTTCGGGCGCCA (SEQ ID NO: 13).

53. The method according to claim 51 or 52, wherein the drug comprises the nucleic acid molecule encoding the tRNA fragment.

54. The method according to claim 53, comprising administering to the subject an expression vector containing the nucleic acid molecule encoding the tRNA fragment.

55. The method according to claim 54, wherein the expression vector is an adenovirus vector, a lentivirus vector, or an adeno-associated virus vector.

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