[go: up one dir, main page]

WO2024196814A1 - Méthodes de traitement de la dégénérescence maculaire liée à l'âge - Google Patents

Méthodes de traitement de la dégénérescence maculaire liée à l'âge Download PDF

Info

Publication number
WO2024196814A1
WO2024196814A1 PCT/US2024/020294 US2024020294W WO2024196814A1 WO 2024196814 A1 WO2024196814 A1 WO 2024196814A1 US 2024020294 W US2024020294 W US 2024020294W WO 2024196814 A1 WO2024196814 A1 WO 2024196814A1
Authority
WO
WIPO (PCT)
Prior art keywords
herv
subject
rna
nucleic acid
ang
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/020294
Other languages
English (en)
Inventor
Kapil BHARTI
Congxiao Zhang
Andrew Jacob FAUSEY
Ruchi Sharma
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Health and Human Services
Original Assignee
US Department of Health and Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by US Department of Health and Human Services filed Critical US Department of Health and Human Services
Priority to AU2024240009A priority Critical patent/AU2024240009A1/en
Publication of WO2024196814A1 publication Critical patent/WO2024196814A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1891—Angiogenesic factors; Angiogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C12N15/1132—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against retroviridae, e.g. HIV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • Advanced AMD including neovascular age-related macular degeneration (wet) and geographic atrophy (late, dry), is associated with substantial, progressive visual impairment.
  • Major risk factors include cigarette smoking, nutritional factors, cardiovascular diseases, and genetic markers, including genes regulating complement, lipid, angiogenic, and extracellular matrix pathways. More than 50 genetic susceptibility loci have been identified, including the CFH and ARMS2 genes.
  • Accurate diagnosis of AMD uses both clinical examination and vision testing, including retinal photography, angiography, and optical coherence tomography. Dietary anti-oxidant supplementation has been shown to slow progression of the disease, but not eliminate it.
  • Current treatments for neovascular age- related macular degeneration include intraocular injections of anti-VEGF agents.
  • SUMMARY OF THE DISCLOSURE Methods are disclosed for treating or reducing the risk of developing age-related macular degeneration (AMD) in a subject.
  • AMD age-related macular degeneration
  • these methods include selecting a subject having, or at risk of developing, the AMD; and administering to the subject an effective amount of an agent that inhibits HERV-K, for example administration to the eye.
  • An agent that inhibits HERV-K can reduce a) the amount of human endogenous retrovirus (HERV)-K; b) of an mRNA encoding a HERV-K proteins, c) translation of the HERV-K mRNA; d) the amount HERV-K proteins in the subject; e) inhibit activity of HERV-K mRNA and/or proteins, or any combination of a)-e).
  • exemplary agents include, but are not limited to, antibodies, inhibitory RNA molecules, inhibitory peptides, anti-retroviral agents and CRISPR/Cas13 systems targeting HERV-K.
  • the agent that inhibits HERV-K is angiogenin or an agent that increases angiogenin expression and/or activity (such as a viral vector expressing ANG).
  • angiogenin expression and/or activity is increased in the eye, such as RPE cells of the eye, by at least 50%, at least 100%, at least 200%, at least 500%, or more.
  • CRISPR/Cas13 systems and ribonucleoprotein (RNP) complexes for targeting HERV-K, as well as isolated cells containing such.
  • FIG.1A is a set of digital images showing induced pluripotent stem (iPSC)-derived retinal pigment epithelium (iRPE) cells treated with complement incompetent human serum (CIHS, left images) or complement competent human serum (CCHS, right images).
  • iPSC induced pluripotent stem
  • iRPE retinal pigment epithelium
  • CC-HS triggers formation of subRPE deposits (drusen) as shown by change in APOE immunostaining from the apical membrane of iRPE (side views) to basolateral membrane.
  • TW transwell membrane on which iRPE cells are grown.
  • FIG.1B is a graph showing transepithelial resistance (TER) of iRPE monolayers after CIHS (grey bar) and CCHS (white bar) treatments.
  • FIG.2 is a schematic diagram of the genomic structure of retroviruses: endogenous retroviruses containing two long terminal repeats (5’ and 3’LTRs) flanking the internal coding sequence of the three basic retroviral genes (Gag, Pol, and Env).
  • FIGS.3A and 3B are graphs showing RNAseq analysis of CC-HS treated iRPE transcriptome revealed genomic loci that show differential elevation of the mRNA of HERV-K.
  • 5% CCHS was added to both apical and basal media of iRPE grown on trans-well for 48 hours, and HERV- K RNA levels from LTR and ORF containing HERV-K loci ( ⁇ 80), which is displayed as scaled depth values (signal reads) was normalized to length of transcripts.
  • FIGS.4A and 4B are graphs showing sustained low-level CCHS uniquely elevated mRNA of HERV-K components in iRPE.
  • qRT-PCR Quantitative reverse transcriptase polymerase chain reaction quantification of HERV-K components GAG, ENV, POL, in 0.1% CCHS-treated iRPE, shows specifically increases of HERV-K ENV and GAG, not the HERV R in cultured iRPE.
  • FIG.5. shows digital images of iPRE cells.
  • CCHS Sustained low-level complement competent human serum
  • CCHS Sustained low-level complement competent human serum
  • FIG.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 and in eyes of AMD patients.
  • PPIB Peptidylprolyl Isomerase B
  • mRNA probe is included as a positive control and validate the detection procedure.
  • HERV-K was detected specifically in CCHS treated iRPE after 48 hours exposure, (left, bottom), other than in the cells which was treated with CIHS under the same condition (left, Middle).
  • Cross section of eyes obtained from human AMD patient (90 years old, right panel) was subjected to HERV-K RNA detection by RNASCOPETM. The data shows HERV-K RNA was present in RPE and choroid of human patient, and absent in age matched control eye.
  • FIGS.7A-7B are graphs showing reduced CCHS-induced secretion of proinflammatory cytokines upon treatment with anti-viral drug or TLR inhibitors.
  • iRPE grown on transwells were pretreated with antiviral drug (Tenofovir, 20uM), TLR3/dsRNA complex inhibitor (10uM) or TLR4 inhibitor TAK-242 (5uM) overnight, followed by incubation with CIHS (5%) or CCHS (5%) for 48hours.
  • Apical and basal media were collected and were analyzed by the Multiplex Luminex assay to detect IL-6, IL-8 IL-18, IL-1 ⁇ and IFN ⁇ . Levels of IL-18, IL-1 ⁇ and IFN ⁇ are below detection range.
  • FIGS.8A and 8B show lipid staining iRPE treated with CCHS and/or TLR inhibitors. TLR inhibitors reduce CCHS- induced lipid accumulations.
  • FIGS.9A-9C show the generation the lentiviral construct to express HERV-K ENV.
  • A) Schematic diagram illustrates the HERV-K ENV expression cassette of the lentiviral construct.
  • Live imaging of mcherry fluorescent signal shows amount of transduced lentivirus coordinates with the levels of protein expression in fully differentiated iRPE at 14 days after transduction.
  • iRPE was transduced with the lentivirus (MOI – multiplicity of infection - 0.5 or 3) containing 5%RPE media supplemented with 5ug/ml polybrone for 16 hours. The media was replaced with fresh media and continuously incubated for additional 14 days. Media was refreshed every other day.
  • FIGS.10A-10C show that HERV-K ENV is sufficient in inducing RPE degeneration, resembling AMD phenotype.
  • HERV-K-ENV expressed via lentiviral transduction, reduces TER and induces lipid accumulation in iRPE.
  • FIGS.11A-11C are graphs showing that CCHS progressively decreased mRNA and secreted angiogenin (ANG) in iRPE.
  • B) mRNA levels of ANG were progressively decreased by CCHS treatments. CCHS (0.1%) was added to both apical and basal media of iRPE for 2 days, 4 days, and 6 days and refreshed daily. Total RNA was analyzed for ANG and Dicer expression by qRT-PCR using CIHS treated cells as a control. N 3, p ⁇ 0.05.
  • FIG.12 are digital images showing that CCHS progressively decreases intracellular angiogenin (ANG) in iRPE. CCHS (0.1%) decreased intracellular ANG in iRPE. IPRE were treated with CCHS or CIHS (5%) for 48 hours. Total protein lysate was analyzed by Western Blot (top) for the expression ANG.
  • ANG angiogenin
  • FIGS.13A-13B are graphs showing that decreases HERV-K specific ANG targets-transfer RNA fragments (tRFs) in iRPE.
  • tRFs RNA fragments
  • FIGS.14A-14B show a gene therapy strategy (A) to reduce HERV-K levels in CC-HS treated iRPE cells.
  • HERV-K is made from multiple loci within the genome and these loci have slight sequence variations.
  • Cas13 is a nuclease that can target RNA using a complementary guide sequence.
  • three guide sequences SEQ ID NOs: 1-3 (B) were identified in HERV-K that were used to target HERV-K expression in RPE cells. These sequences were designed against the GAG part of HERV-K mRNA because that is the start of the RNA. Degradation of the start of RNA leads to degradation of the remainder of the RNA.
  • FIGS.15A-15C The guide sequences.
  • Sequences showed complementarity across multiple HERV-K loci and also showed good consensuses for CAS13Rx based targeting.
  • the genomic distribution of three guides used is shown in the Table. SEQ ID NOS: 1-3 are shown bold and underlined. The consensus sequences for gRNA1 (SEQ ID NO: 18), gRNA2(SEQ ID NOs: 34 and 51), and gRNA3 (SEQ ID NO: 59) are shown. The various alignments to the genome are shown for gRNA1 (SEQ ID NOs: 19-33), gRNA2(SEQ ID NOs: 35- 50 & 52-58), and gRNA3 (SEQ ID NO: 60-75).
  • FIGS.16A-16B show the induction of Cas13Rx expression using doxycycline.
  • A is a Western blot showing HA-tagged Cas13Rx overexpressed using a lentivirus construct. High expression of HA-tagged Cas13Rx is seen at multiplicity of infection (MOI) of 1.0.
  • B is a digital image showing HA-tag-Cas13Rx enzyme expression in iRPE cells at MOI of 1.0 of the lentivirus vector.
  • FIGS.17A-17E Cas13Rx mediated HERV K knock down reverses complement-induced lipid accumulation.
  • HERV-K levels were measured in cells and monolayer TER and BODIPY® levels were measured in cells.
  • B Shows a TER graph of CIHS (grey bars) and CCHS (black bars) treated samples that were transduced with a scrambled guide RNA (NEG) or no guide RNA (Cas13Rx) or guide RNA #2 (Fig.15). Only guide RNA#2 is able to rescue TER downregulated by CCHS treatment
  • Top panel shows digital images of CIHS or CCHS treated iRPE, transduced either with a scrambled control (NEG) or guideRNA#2.
  • CCHS induced increase in HERV-K expression is downregulated by guide RNA 2 against HERV-K.
  • Bottom panel shows digital of CIHS or CCHS treated iRPE, transduced either with a scrambled control (NEG) or guideRNA#2.
  • CCHS induced increase in lipid deposits is downregulated by guide RNA 2 against HERV-K.
  • D Quantification of HERV-K levels seen in FIG.17C top panel shows guide RNA#2 is able to downregulate HERV-K levels in CCHS treated iRPE cells.
  • FIG.17C bottom panel shows guide RNA#2 is able to downregulate BODIPY® in CCHS treated iRPE cells.
  • FIG.18. Cas13Rx mediated HERV K knock down reverses complement-induced lipid accumulation by all three guides selected in FIG.15. Digital images of CCHS treated AMD patient 1-iRPE, transduced either with a scrambled control (NEG) or three guide RNAs #1, 2, 3 alone or in combination. CCHS induced increase in lipid deposits (measured as BODIPY® signal). BODIPY® signal is downregulated by all three guides against HERV-K when transduced with CCHS treated cells. Left graph shows quantification of digital imaging data.
  • FIG.19 Cas13Rx mediated HERV K knock down reverses complement-induced lipid accumulation by guides selected in FIG.15 (example shown for two guides).
  • CCHS induced increase in lipid deposits (measured as BODIPY® signal).
  • BODIPY® signal is downregulated by guides against HERV-K when transduced with CCHS treated cells.
  • CIHs treatment is a negative control for the study.
  • Left graph shows quantification of digital imaging data.
  • FIG.20 Schematic diagram of method for testing the role of exogenously expressed angiogenin (ANG) in protection of CCHS-induced iRPE injury.
  • ANG angiogenin
  • FIG.21 A bar graph showing ANG overexpression by lentivirus.
  • FIG.22 A digital image showing that ANG prevented the CCHS-induced HERVK increase.
  • FIG.23 A digital image showing that ANG reduced the CCHS-induced lipid accumulation.
  • FIG.24 A bar graph showing ANG overexpression prevents CCHS induced TER decrease in iRPE.
  • FIGS.25A-25C (A) Schematic illustrates the transgene domain structure in expression vector, where HERV-K ENV is under RPE specific human VMD2 promoter (also known as best1 promoter).
  • B qPCR data of genotyping of six HERV-K ENV founder mice.
  • C qRT-PCR identified two founder mice lines- founder1 and founder6 showing significantly higher expression of HERV-K ENV. These founder mice lines (1&6) were backcrossed to C57BL/6J to generate pure to C57BL/6.
  • iPSC-RPE induced pluripotent stem cell derived-RPE
  • CC-HS complement competent human serum
  • HERV-K ENV increased HERV-K ENV, coinciding with increased lipid accumulation, cytokine release and loss of epithelial cell morphology, mimicking pathogenic changes during early AMD (Sharma R, et.al., Nat Commun.2021 Dec 15;12(1):7293. doi: 10.1038/s41467-021-27488-x.PMID: 34911940, 36550275).
  • FIGS.26A-26H HERV-K ENV overexpression induces lipid accumulation in transgenic RPE.
  • A-B Immunostaining images show higher expression of HERV-K ENV in flat mounts of RPE isolated from 1–2-month-old transgenic mice eye (A) and quantification of the HERV-K ENV signal is shown in (B).
  • C-D Immunostaining images show higher of HERV-K ENV in flat mounts of RPE isolated from 7–8-month-old transgenic eye (C) quantification data is shown in (D).
  • E- F Immunostaining images show higher lipid droplets stained with BODIPY®, in HERV-K ENV over- expression mice aged 1–2-month (E) and corresponding quantification data show significant higher lipid droplet signal in homo HERV-K ENV mice (F).
  • G-H Immunostaining images show higher lipid droplets stained with BODIPY®, in HERV-K ENV over-expression mice aged 7-8 months old (G) and corresponding quantification data shows significantly higher lipid droplet levels in homo HERV-K ENV mice (H). Four images per sample was captured under laser scanning according to an established method and normalized to size of imaging area in the view.
  • FIGS.27A and 27B Lipid droplets co-localize with HERV-K ENV protein in RPE of transgenic mice.
  • A En-face view of images show the localization of HERV-K ENV (magenta) with lipid droplets (stained with BODIPY®, green) in 7–8-month-old transgenic mice compared to wild type mice.
  • B 3D rendered images of panel A shows the sub-cellular localization of HERV-K ENV and lipid droplets.
  • FIGS.28A and 28B Lipid droplets co-localize with HERV-K ENV protein in RPE of transgenic mice.
  • the anatomic layers of the retina are labeled including the ganglion cell layer (GCL); inner plexiform layer (IPL); inner nuclear layer (INL); outer plexiform layer (OPL); outer nuclear layers (ONL); inner segment / outer segment layer (IS/OS) and the neighboring RPE and choroid.
  • GCL ganglion cell layer
  • IPL inner plexiform layer
  • IPL inner nuclear layer
  • OPL outer plexiform layer
  • ONL outer nuclear layers
  • INL inner segment / outer segment layer
  • IS/OS inner segment / outer segment layer
  • FIGS 29A-29D Appearance of subretinal cells and RPE intracellular vacuoles in HERV-K ENV overexpressing eye.
  • FIGS.30A-30F Transmission electron (TEM) micrograph images of the photoreceptor-RPE- choroid complex in the 7-8 month wildtype +/+ (A) and heterozygous HERV-K ENV tg/+ (B) or homozygous HERV-K ENV tg/tg transgenic mice (C,D), show lipid aggregates (white arrows, B ), an abnormal subretinal cell (horizontal black arrow, C), vacuoles (white arrow, C), and disrupted tight junctions (black arrowheads, D) in transgenic eye.
  • FIGS.30A-30F Combination of antiviral drugs exerted greater effect on blocking CCHS induced lipid accumulation effect.
  • A) Flow chart of experimental process and assay.
  • FIGS.31A and 31B Application of AAV8-shRNA HERV-K ENV as therapeutics to treat AMD, in vitro and in vivo studies.
  • A) Schematic diagram illustrates the workflow of in Vitro experiments with iRPE to test the effect of AAV8-shRNA HERV-K ENV in rescuing the cell defects caused by anaphylatoxin (CC-HS).
  • Either na ⁇ ve iRPE or HERV-K ENV overexpressing iRPE are transduced with AAV HERV-K ENV or AAV control at MOI 10 4 to 10 5 4 days before the addition of CC-HS (0.1%) or control CIHS (0.1%).
  • the anaphylatoxin containing media is refreshed daily for 3 days. Samples are collected at day 4 and corresponding assays will be performed to detect the rescuing effect.
  • SEQ ID NOs: 1-3 are exemplary nucleic acid sequence of gRNA.
  • SEQ ID NO: 4 is an exemplary nucleic acid sequence of HERV-K.
  • SEQ ID NO: 5 is an exemplary nucleic acid sequence encoding a Cas13Rx.
  • SEQ ID NO: 6 is an exemplary HERV-K Gag amino acid sequence (GENBANK® Accession No.
  • SEQ ID NO: 7 is an exemplary HERV-K Gag amino acid sequence (GENBANK® Accession No. AAL60056.1).
  • SEQ ID NO: 8 is an exemplary HERV-K Env amino acid sequence (GENBANK® Accession No. AAL16780.1).
  • SEQ ID NO: 9 is an exemplary Cas13Rx protein (without the signal peptide) (GENBANK® Accession No. QMT62609.1).
  • SEQ ID NO: 10 is an exemplary nucleic acid sequence encoding a Cas13Rx (GENBANK® Accession No. MN934322.1).
  • SEQ ID NO: 11 is an exemplary amino acid sequence of human ANG.
  • SEQ ID NOs: 12 and 13 are exemplary genomic DNA corresponding to tRNA fragments, tRNA- CTT and tRNA-TTT.
  • SEQ ID NO: 14 is an exemplary nucleic acid sequence encoding human ANG.
  • SEQ ID NOs: 15-16 are nucleic acid sequences of primers.
  • SEQ ID NO: 17 is the nucleic acid sequence of a reporter.
  • SEQ ID NO 18 is the consensus nucleic acid sequence for gRNA1.
  • SEQ ID NOs 19-33 are the nucleic acid alignment sequences for gRNA1.
  • SEQ ID NOs 34 & 51 are the consensus nucleic acid sequences for gRNA2.
  • SEQ ID NOs: 35-50 & 52-58 are the nucleic acid alignment sequences for gRNA2.
  • SEQ ID NO 59 is the consensus nucleic acid sequence for gRNA3.
  • SEQ ID NOs 60-75 are the nucleic acid alignment sequences for gRNA3.
  • DETAILED DESCRIPTION OF SEVERAL ASPECTS Human endogenous retroviruses (HERVs) and elements containing long terminal repeat-like sequences comprise up to 8% of the human genome, see U.S. Published Patent Application No. 2008/0019979A1.
  • HERVs Human endogenous retroviruses
  • HERVs have lost infectivity due to mutations, and in general they are largely noninfectious retroviral remnants.
  • ORFs open reading frames have been observed for ERV3, HERV-E 4-1, and HERV-K.
  • HERV-K The members of the HERV-K superfamily, which is characterized by the presence of primer binding sites for lysine tRNA, are the most biologically active. Only HERV-K seems to have the full complement of open reading frames typical of replication competent mammalian retroviruses.
  • the K family contains a central open reading frame (cORF) and is comparable to HIV-1 Rev protein.
  • HERV-K was originally identified by its homology to the mouse mammary tumor virus (MMTV), and is transcriptionally active in several human cancer tissues, including breast cancer tissues, as well as tumor cell lines, such as the human breast cancer cell line T47D and the teratocarcinoma cell line GH.
  • HERV-K env mRNA is frequently expressed in human breast cancer and HERV-E mRNA is expressed in prostate cancer and ovarian cancer, see U.S. Published Patent Application No.2008/0019979A1, which discloses antibodies that specifically bind an HERV-K protein, see also U.S. Patent No.10,723,787 and U.S. Patent No.10,981,976. It is disclosed herein that HERV-K expression is associated with the AMD phenotype of human cells in the retinal pigment epithelium (RPE).
  • RPE retinal pigment epithelium
  • inhibition of HERV-K transcription and translation can reduce pathological phenotype of RPE cells.
  • an agent that increases ANG activity prevented CCHS-induced HERVK increase, reduced CCHS-induced lipid accumulation, and prevented a CCHS induced TER decrease in RPE cells.
  • Methods are disclosed for treating or reducing the risk of developing age-related macular degeneration (AMD) in a subject. These methods include selecting a subject having, or at risk of developing, AMD, and administering to the subject an effective amount of an agent that inhibits HERV-K.
  • CRISPR/Cas13 systems and RNPs which can be used in the methods for treating AMD. Methods are also disclosed for treating a subject having, or at risk of developing, the AMD, by administering to the subject an effective amount of an agent that increases ANG activity.
  • a protein includes single or plural proteins and can be considered equivalent to the phrase “at least one protein.”
  • the term “comprises” means “includes.” Unless otherwise indicated “about” indicates within five percent. It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
  • Administration The introduction of a composition (such as one containing an agent that increases or decreases HERV-K transcription or translation) into a subject by a chosen route.
  • Administration can be local or systemic. For example, if the route is intravenous, the composition is administered by introducing the composition into a vein of the subject. Similarly, if the route is intramuscular, the composition is administered by introducing the composition into a muscle of the subject. If the chosen route is oral, the composition is administered by ingesting the composition.
  • Exemplary routes of administration of use in the methods disclosed herein include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intraosseous, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.
  • Administration can also be local, such as to the eye of a subject.
  • administration is topical to the eye, such as via an eye drop solution.
  • administration is intraocular, such as subretinal, direct retinal, suprachoroidal or intravitreal injection.
  • co-administer refers to administration of two or more agents within about 2 hours of each other, for example, as part of a clinical treatment regimen. In other aspects, “co-administer” refers to administration of two or more agents within 1 hour of each other. In other aspects, “co-administer” refers to administration of two or more agents within 30 minutes of each other. In other aspects, “co-administer” refers to administration of two or more agents within 15 minutes of each other. In other aspects, “co-administer” refers to administration of two or more agents at the same time, either as part of a single formulation or as multiple formulations that are administered by the same or different routes.
  • AMD Age Related Macular Degeneration
  • AMD AMD is caused by damage to the macula of the retina. Onset of AMD may be asymptomatic, but AMD gradually worsens over time and generally results in blurred or no vision in the center of the visual field in one or both eyes. It can be hard to recognize faces, drive, read, or perform other activities of daily life, and visual hallucinations may also occur. AMD typically occurs in older people, such as subjects about 50 years and older. Genetic factors and smoking can play a role. Diagnosis includes a complete eye exam, and severity can range from early, intermediate, and late types, in which the late type can further include "dry" and "wet” forms.
  • Dry AMD also called atrophic AMD
  • Symptoms can include visual distortions, reduced central vision in one or both eyes, a need for brighter light for reading or close work, increased difficulty adapting to low light levels, increased blurriness of printed words, decreased intensity or brightness of colors, and difficulty recognizing faces.
  • Dry AMD is diagnosed by examining the back of the eye for drusen; testing for defects in the center of the vision (such as using an Amsler grid to identify whether straight lines in the grid to look faded, broken, or distorted, indicating the presence of dry AMD); fluorescein or indocyanine green angiography (examining for abnormal blood vessel or retinal changes); and/or optical coherence tomography (examining for retinal thinning, thickening, or swelling).
  • Currently available treatments include rehabilitation for adapting to the loss of central vision (low vision rehabilitation) and implanting a telescopic lens.
  • Wet AMD also called advanced neovascular AMD
  • wet AMD symptoms can also include a well-defined blurry spot or blind spot in the field of vision, general haziness in overall vision, and abrupt onset and rapid worsening of symptoms.
  • AVASTIN® bevacizumab
  • LUCENTIS® ranibizumab
  • EYLEA® aflibercept
  • Agent Any substance or any combination of substances that is useful for achieving an end or result; for example, a substance or combination of substances useful for treating AMD.
  • Agents include proteins, nucleic acid molecules (such as gRNAs), compounds, small molecules, organic compounds, inorganic compounds, or other molecules of interest.
  • An agent can include a therapeutic agent (such as an anti-retroviral agent), a diagnostic agent or a agent.
  • Angiogenin (ANG) A protein, also as ribonuclease 5, that is stimulator of the formation of new blood vessels in vivo. ANG can hydrolyze cellular RNA, resulting in modulated levels of protein synthesis and interacts with DNA causing a promoter-like increase in the expression of ribosomal (r)RNA.
  • ANG enhances rRNA transcription by binding to a CT-rich angiogenin binding element.
  • ANG has a similar catalytic activity to RNase A, it preferentially binds on the 3' side of pyrimidines and follows a transphosphorylation/hydrolysis mechanism. Unlike RNase A, which has no base specificity, ANG usually cleaves the 3′-side of cytidylic or uridylic acid residues when the pyrimidine is followed by adenine, but not restrict to all the potential cleavage sites. ANG shows preferential cleavage of single-stranded RNA as the substrate, and cleaves tRNAs.
  • ANG has been shown to bind DNA in vivo, it does not cleave DNA, see Sheng and Zu, Acta Biochimica et Biophysica Sinica 48: 3990-410, 2016. ANG overexpression selectively cleaves a subset of tRNAs, including tRNA Glu , tRNA Gly , tRNA Lys , tRNA Val , tRNA His , tRNA Asp , and tRNA SeC to tRNA halves and tRF-5s that are 16–30 bases called tiRNAs Zu et al. mammals and birds.
  • the term mammal includes both human and non-human mammals.
  • the term “subject” includes both human and veterinary subjects.
  • Antibody A polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope (e.g., an antigen, such as an HERV-K protein (for example, Gag, Pol, or Env, or fragment thereof).
  • an antigen such as an HERV-K protein (for example, Gag, Pol, or Env, or fragment thereof).
  • a scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
  • the term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 rd Ed., W.H. Freeman & Co., New York, 1997.
  • an immunoglobulin has a heavy and light chain.
  • Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”).
  • the heavy and the light chain variable regions specifically bind the antigen.
  • Light and heavy chain variable regions contain a "framework" region interrupted by three regions, also called “complementarity- determining regions” or “CDRs”. The extent of region and CDRs has been 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). The Kabat database is now 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 serves to position and align the CDRs in three-dimensional space.
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N- terminus, and are also typically identified by the chain in which the particular CDR is located.
  • a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found
  • a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
  • references to “VH” or “VH” refer to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.
  • References to “VL” or “VL” refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
  • a “monoclonal antibody” is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected.
  • Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells.
  • Monoclonal antibodies include humanized monoclonal antibodies.
  • a "humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin.
  • the non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.”
  • all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin.
  • Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences.
  • a “humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs.
  • the acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework.
  • Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.
  • Humanized immunoglobulins can be constructed by means of genetic engineering (e.g., see U.S. Patent No.5,585,089).
  • Anti-retroviral agent An agent that specifically reduces or inhibits, expression or replication activity of a retrovirus (or endogenous retrovirus viral elements in the human genome) in cells, or a retrovirus from infecting cells.
  • Non-limiting of antiretroviral agents that are drugs include fusion (e.g., enfuvirtide), 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 (NNRTI) (e.g.
  • fusion e.g., enfuvirtide
  • entry inhibitors e.g., maraviroc
  • nucleoside and nucleotide reverse transcriptase inhibitors e.g., lamivudine, zidovudine, abacavir, tenofovir, TAF, TDF, FTC
  • NRTI non-nucleoside reverse transcriptase inhibitors
  • DAPY diarylpyrimidine
  • protease inhibitors e.g., indinavir, ritonavir, darunavir, atazanavir
  • integrase inhibitors e.g., elvitegravir, raltegravir, dolutegravir.
  • Anti-retroviral agents of use in the present methods are disclosed, for example, in Tyagi et al., Inhibition of human endogenous retrovirus-K by antiretroviral drugs, Retrovirology 14: 21 (13 pages), 2017, incorporated herein by reference.
  • Anti-retroviral therapy A therapeutic treatment for inhibiting a retrovirus involving administration of at least one anti-retroviral agent (e.g., one, two, three or four anti-retroviral agents) to an individual (such as one infected with a retrovirus) during a course of treatment.
  • at least one anti-retroviral agent e.g., one, two, three or four anti-retroviral agents
  • an individual such as one infected with a retrovirus
  • ART includes Highly Active Anti-Retroviral Therapy (HAART).
  • Caspase Caspase (Cas): An enzyme that is that a cysteine-aspartic protease, cysteine aspartase or cysteine- dependent aspartate-directed protease.
  • Caspases are a family of protease enzymes playing essential roles in programmed cell death. They are named caspases due to their specific cysteine protease activity, wherein a cysteine in its active site nucleophilically attacks and cleaves a target protein only after an aspartic acid residue.
  • 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.
  • Cobicistat 1,3-thiazol-5-ylmethyl (2R,5R)-(5- ⁇ [(2S)-2-[(methyl ⁇ [2-(propan-2-yl)-1,3- thiazol-4-yl]methyl ⁇ carbamoyl)amino]]-4-(morpholin-4-yl)butanamido ⁇ -1,6-diphenylhexan-2-yl)carbamate.
  • Cobicistat is a cytochrome P4503A inhibitor that acts as pharmacokinetic enhancer to increase the effectiveness of HIV anti-retroviral drugs. It is used to increase the bioavailability of other anti-retroviral agents.
  • Cobicistat is marketed as TYBOST® and is also known as GS-9350.
  • Control A reference standard.
  • the control is a negative control sample obtained from a healthy patient (such as one without AMD), or a subject treated with a carrier, non-targeted nucleic acid sequences, scrambled nucleic acid/amino acid sequences or untreated cells from a healthy patient.
  • the control is a positive control sample obtained from a patient that has been treated with an active agent.
  • the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients with known prognosis or outcome, or group of samples that represent baseline or normal values).
  • a difference between a test sample and a control can be an increase or conversely a decrease.
  • the difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference.
  • a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at 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%.
  • CRISPR Clustered Regularly InterSpaced Short Palindromic Repeats
  • Cas CRISPR-associated protein Editing System
  • An engineered nuclease system based on a bacterial system that is used for genome engineering. It is based in part on the adaptive immune response of many bacteria and archaea. Such methods can be used to allow genetic material to be added, removed, or altered at particular locations, for example in a target DNA or RNA sequence (such as a La nucleic acid sequence).
  • CRISPR/Cas systems can be used for nucleic acid targeting (such as DNA or RNA), for example to detect a target DNA or RNA, modify a target DNA or RNA at any desired location, or cut the target DNA or RNA at any desired location.
  • such methods can be used to modify expression of a HERV-K Gag, Pol or Env, for example by introducing a mutation to silene expression, such as knocking out the La gene.
  • the method edits DNA, such as a genome, and uses a Cas9 nuclease. Cas9 nuclease cleaves the DNA to generate blunt ends at the double-strand break at sites specified by a 20-nucleotide complementary strand sequence contained within the crRNA transcript.
  • a CRISPR/Cas system can be engineered to create a double-strand break at a desired target in a genome of a cell, and harness the cell's endogenous mechanisms to repair the induced break by homology-directed repair (HDR) or nonhomologous end-joining (NHEJ).
  • the method edits RNA, such as a an HERV-K Gag, Pol or Env RNA, and uses a Cas13 nuclease.
  • Exemplary Cas13 nucleases include Cas13a, Cas13b, Cas13c, Cas13Rx, Cas13x, and Cas13y (see for example WO 2019/040664, US 11,293,011, and US 10,392,616).
  • a CRISPR array includes at least a DR-spacer-DR-spacer. This feature was used to identify the Cas13Rx protein family. In bacteria, the array is transcribed as one single transcript (containing multiple crRNA units), which is then processed by the Cas13Rx protein and other RNases into individual crRNAs. CRISPRs are often associated with cas genes that code for proteins related to CRISPRs (such as the Cas13Rx proteins).
  • a CRISPR/Cas system can be used for RNA targeting, for example, to modify a target HEV-K RNA at any desired location.
  • Degenerate variant A polynucleotide encoding a peptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in this disclosure as long as the amino acid sequence of the polypeptide encoded by the nucleotide sequence is unchanged.
  • Downregulated or knocked down When used in reference to the expression of a molecule, such as a target RNA, refers to any process which results in a decrease in production of the target RNA, but in some examples not complete elimination of the target RNA product or target RNA function. In one example, downregulation or knock down does not result in complete elimination of detectable target RNA expression or target RNA activity.
  • the target RNA is a coding HERV-K RNA.
  • the target RNA is non-coding HERV-K RNA. Downregulation or knock down includes any decrease in the target HERV-K RNA.
  • detectable target HERV-K RNA in a cell or cell free system decreases 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% (such as a decrease of 40% to 90%, 40% to 80% or 50% to 95%) as compared to a control (such an amount of target HERV-K RNA detected in a corresponding normal cell or sample).
  • a control is a relative amount of expression in a cell that does not include Cas13 or guide RNA).
  • Emtricitabine 2'-deoxy-5-fluoro-3'thiacytidine (FTC).
  • FTC is sold under the trade name EMTRIVA® (emtricitabine) formerly COVIRACIL®), is a nucleoside reverse transcriptase inhibitor (NRTI) used in the treatment of HIV infection in adults and children.
  • Emtricitabine is also marketed in a fixed-dose combination with tenofovir disproxil fumerate (Viread) under the brand name TRUVADA®.
  • TRUVADA® tenofovir disproxil fumerate
  • a fixed-dose triple combination of emtricitabine, tenofovir and efavirenz was approved by the U.S.
  • Expression Control Sequences Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
  • expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct gene reading frame to permit proper translation of mRNA, and stop codons.
  • control sequences includes, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • Expression control sequences can include a promoter.
  • a promoter is a minimal sequence sufficient to direct transcription.
  • promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell- type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene.
  • constitutive and inducible promoters are included (see e.g., Bitter et al., 1987, Methods in Enzymology 153, 516-544).
  • inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used.
  • promoters derived from the genome of mammalian cells such as the metallothionein promoter
  • mammalian viruses such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter
  • Promoters produced by recombinant DNA or synthetic techniques can also be used to provide for transcription of the nucleic acid sequences.
  • Heterologous Originating from a different source, so that the biological components are not found together in nature. The components may cells, genes, or regulatory regions, such as promoters.
  • heterologous components are not found together in nature, they can function together, such as when a promoter heterologous to a gene is operably linked to the gene.
  • Host cells Cells in which a vector can be propagated and its DNA expressed.
  • the cell may be prokaryotic or eukaryotic.
  • the cell can be mammalian, such as a human cell.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used. In one example a host cell is a retinal cell.
  • HERV Human Endogenous Retrovirus
  • ORFs open reading frames
  • HERV-K open reading frames
  • HERV-K seems to have the full complement of open reading frames typical of replication competent mammalian retroviruses.
  • the K family contains a central open reading frame (cORF) and is comparable to HIV-1 Rev protein.
  • HERV-K is transcribed during embryogenesis from the eight cell stage up to the stem cell derivation.
  • HERV-K also is transcriptionally active in several human cancer tissues, including breast cancer tissues, as well as tumor cell lines, such as the human breast cancer cell line T47D and the teratocarcinoma cell line GH, see U.S. Published Patent Application No.2008/0019979A1.
  • Inhibiting or treating a disease refers to inhibiting the full development of a disease.
  • inhibiting a disease refers to lessening symptoms of the particular disease.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition related to the disease.
  • Treatment can be measured using success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject’s physical state.
  • the treatment may be assessed by objective or subjective parameters; including the results of a physical examination or tests, such as vision tests.
  • Inhibitory nucleic acid molecules Includes inhibitory RNA and DNA molecules, such as an antisense oligonucleotide, a siRNA, a microRNA (miRNA), a shRNA or a ribozyme.
  • antisense compound that specifically targets and regulates of a nucleic acid encoding Gag, Pol or Env of HERV-K is contemplated for use.
  • An antisense is one which specifically hybridizes with and modulates expression of a Gag, Pol or Env nucleic acid molecule.
  • These compounds can be introduced as single-stranded, double-stranded, circular, branched or hairpin compounds and can contain structural elements such as internal or terminal bulges or loops.
  • Double-stranded antisense compounds can be two strands hybridized to form double-stranded compounds or a single strand with sufficient self- complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.
  • an antisense oligonucleotide is a single stranded antisense compound, such that when the antisense oligonucleotide hybridizes to a mRNA encoding Gag, Pol or Env protein and the resulting duplex is recognized by RNaseH, resulting in cleavage of the mRNA.
  • a miRNA is a single- stranded RNA molecule, such as about 21-23 nucleotides in length that is at least partially complementary to an mRNA molecule that regulates gene expression through an RNAi pathway.
  • a shRNA is an RNA oligonucleotide that forms a tight hairpin, which is cleaved into siRNA.
  • siRNA molecules are generally about 15-40 nucleotides in length, such as 20-25 nucleotides in length, and may have a 0 to 5 nucleotide overhang on the 3' or 5’ end, or may be blunt ended.
  • one strand of a siRNA is at least partially complementary to a nucleic acid molecule encoding Gag, Pol or Env.
  • Antisense compounds specifically targeting a Gag, Pol or Env gene can be prepared by designing compounds that are complementary to a target nucleotide sequence, such as an mRNA sequence. Antisense compounds need not be 100% complementary to the nucleic acid molecule encoding Gag, Pol or Env to specifically hybridize and regulate expression of the target.
  • the antisense compound, or antisense strand of the compound if a double-stranded compound can be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% complementary to a nucleic acid molecule encoding Gag, Pol or Env.
  • Methods of screening antisense compounds for specificity are known (see, for example, U.S.
  • Inhibitory peptide A blocking peptide competitively inhibits protein-protein interaction by mimicking one of their binding domains, or “binding epitopes.”
  • the term includes peptides that competitively interrupt protein-protein interactions by binding to one of the partner’s binding domains, including those that interfere with protein-protein interactions.
  • Isolated An “isolated” biological component (such as a nucleic acid molecule or protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins.
  • Label A detectable compound or that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule.
  • labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.
  • Mammal This term includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects.
  • Modulate To alter in a statistically significant manner. Modulation can be an increase or a decrease. One of skill in the art can identify an appropriate assay to determine a statistically significant increase or decrease in a parameter. These include, but are not limited to, a student’s t-test or a paired ratio t test.
  • Nucleic acid molecule A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof.
  • nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like.
  • oligonucleotide typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.” Conventional notation is used herein to describe nucleotide sequences: the left-hand end of a single- stranded nucleotide sequence is the 5'-end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5'-direction.
  • RNA transcripts The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • the DNA strand having the same sequence as an mRNA is referred to as the “coding strand;” sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5'-end of the RNA transcript are referred to as “upstream sequences;” sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as “downstream sequences.”
  • cDNA refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA.
  • a sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. “Recombinant nucleic acid” refers to a nucleic acid having nucleotide sequences that are not naturally joined together.
  • nucleic acid vectors comprising an amplified or assembled nucleic acid which can be used to transform a suitable host cell.
  • a host cell that comprises the recombinant nucleic acid is referred to as a “recombinant host cell.”
  • the gene is then expressed in the recombinant host cell to produce, such as a “recombinant polypeptide.”
  • a recombinant nucleic acid may serve a non-coding function (such as a promoter, origin of replication, ribosome-binding site, etc.) as well.
  • a first sequence is an “antisense” with respect to a second sequence if a polynucleotide whose sequence is the first sequence specifically hybridizes with a polynucleotide whose sequence is the second sequence.
  • Terms used to describe sequence relationships between two or more nucleotide sequences or amino acid sequences include “reference sequence,” “selected from,” “comparison window,” “identical,” “percentage of sequence identity,” “substantially identical,” “complementary,” and “substantially complementary.” For sequence comparison of nucleic acid sequences, typically one sequence acts as a reference sequence, to which test sequences are compared.
  • sequence comparison algorithm When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are used. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, 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.
  • NRTIs Nucleoside analog reverse-transcriptase inhibitors
  • NRTIs include zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, and emtricitabine (also called FTC).
  • Nucleotide analog reverse-transcriptase inhibitors NtRTIs: NTARTIs and NtRTIs are nucleotide analogues of cytidine, guanosine, thymidine, and adenosine that are of use in treatment of HIV infections. For example, tenofovir (and its related prodrugs) is an NtRTI adenosine analogue.
  • Open reading frame A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a protein.
  • Operably linked A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence, such as a sequence that encodes a polypeptide.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • compositions and formulations suitable for pharmaceutical delivery of the therapeutic agents herein disclosed are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the therapeutic agents herein disclosed. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Pharmacoenhancer A substance that increases the bioavailability and bioefficacy of active substances with which they are combined without having any activity of their own at the dose used.
  • Increased bioavailability means increased levels of an agent, such as in the blood.
  • Increased bioefficacy means the increased effectiveness of the drug due to, at least in part, to increased bioavailability.
  • COBI is a pharmacoenhancer.
  • Polypeptide Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). With regard to polypeptides and proteins, the word “about” indicates integer amounts. Thus, in one example, a polypeptide “about” 29 amino acids in length is from 28 to 30 amino acids in length. Thus, a polypeptide “about” a specified number of residues can be one amino acid shorter or one amino acid longer than the specified number.
  • a fusion polypeptide includes the amino acid sequence of a first polypeptide and a second different polypeptide (for example, a heterologous polypeptide), and can be synthesized as a single amino acid sequence.
  • a recombinant polypeptide has an amino acid sequence that is not naturally occurring or that is made by two otherwise separated segments of an amino acid sequence.
  • Promoter An array of nucleic acid control which direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
  • a promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active/"ON” state), an inducible promoter (i.e., a promoter whose state, active/"ON” or inactive/"OFF", is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein.), a spatially restricted promoter (e.g., tissue specific promoter, cell type specific promoter, etc.), or it may be a temporally restricted promoter (i.e., the promoter is in the "ON" state or "OFF” state during specific stages of embryonic development or during specific stages of a biological process).
  • RPE specific promoters can be used to drive expression specifically in the RPE.
  • Some examples include promoters for genes like DCT (ID#1638), tyrosinase (ID#7299), VMD2 (ID #7439), RPE65 (ID#6121). Promoters specific for choroid can also be used, examples include Carbonic Anhydrase 4 (ID# 762), PLVAP (ID# 83483). Photoreceptor specific promoter examples include – Rhodopsin (ID# 6010), NR2E3 (ID# 10002) and NRL (ID# 4901). Purified: The term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein referred to is purer than the protein in its natural environment within a cell.
  • a preparation of a protein is purified such that the protein represents at least 50% of the total protein content of the preparation.
  • a purified nucleic acid molecule preparation is one in which the nucleic acid molecule is purer than in an environment including a complex mixture.
  • a purified population of nucleic acids or proteins is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure, or free other nucleic acids or proteins, respectively.
  • RNA Editing A type of genetic engineering in which an RNA molecule (or ribonucleotides of the RNA) is inserted, deleted or replaced in the genome of an organism using engineered nucleases (such as Cas13Rx proteins), which create site-specific strand breaks at desired locations in the RNA. The induced breaks are repaired resulting in targeted mutations or repairs.
  • engineered nucleases such as Cas13Rx proteins
  • the CRISPR/Cas methods disclosed herein such as those that use a Cas13Rx protein and a gRNA specific for HERV-K, can be used to edit the sequence of one or more target HERV-K RNAs.
  • Sequence identity The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity.
  • Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
  • Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are known t. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl.
  • NCBI 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) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet. Homologs and variants of a polypeptide are typically characterized by possession of at least 75%, for example at least 80%, sequence identity counted over the full length alignment with the amino acid sequence of a polypeptide using the NCBI Blast 2.0, gapped blastp set to default parameters.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • homologs and variants When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. These sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
  • variants of a polypeptide or nucleic acid sequence are typically characterized by possession of at least about 75%, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid or nucleotide sequence of interest. Sequences with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • homologs and variants When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids (or 30-60 nucleotides), and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet.
  • reference to “at least 90% identity” refers to “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” to a specified reference sequence.
  • Subject Living multi-cellular vertebrate a category that includes human and non- human mammals, such as non-human primates, rats, dogs, cats, horses, cows and pigs.
  • a subject is a human.
  • a subject is selected that has AMD.
  • Tenofovir Prodrugs 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.
  • Tenofovir is sequentially phosphorylated in the cell by AMP kinase and nucleoside diphosphate kinase to the active species, tenofovir diphosphate, which acts as a competitive inhibitor of HIV-1 reverse transcriptase that terminates the growing viral DNA chain.
  • Tenofovir disoproxil fumarate (TDF) is an oral prodrug of tenofovir, marketed as VIREAD®, that has received marketing authorization in many countries as a once-daily tablet (300 mg) in combination with other antiretroviral agents for the treatment of HIV-1 infection.
  • 9-[(R)-2-[[(S)-[[(S)-1- (isopropoxycarbonyl)ethyl]amino] phenoxyphosphinyl]-methoxy]propyl]adenine 16 is an isopropylalaninyl phenyl ester prodrug of tenofovir.
  • Tenofovir alafenamide (TAF) is also known as GS-7340.
  • TAF has been marketed under the name VEMLIDY®. The hemifumarate form of TAF is also of use in the methods disclosed herein.
  • TAF exhibits potent anti-HIV activity 500- to 1000-fold enhanced activity relative to tenofovir against HIV-1 in T cells, activated peripheral blood mononuclear lymphocytes (PBMCs), and macrophages. TAF also has enhanced ability to deliver and increase the accumulation of the parent tenofovir into PBMCs and other lymphatic tissues in vivo. TAF can be prepared as described in U.S. Pat. No. 7,390,791, incorporated herein by reference.
  • FTC/TAF/EVG/COBI also called GENVOYA® (which contains 150 mg EVG, 150 mg COBI, 200 mg FTC, and 10 mg TAF) is approved for the treatment of an existing HIV infections in subjects. EVG, FTC and TAF have been shown to suppress viral reproduction.
  • Cobicistat increases the effectiveness of the combination, such as by inhibiting the liver and gut wall enzymes that metabolize EVG.
  • the use of FTC/TAF/EVG/COBI for treatment of an existing HIV infection is disclosed, for example, in U.S. Patent Publication U.S.2015/0105350 entitled “Combination Therapy Comprising Tenofovir Alafenamide Hemifumarate and Cobicistat for Use in the Treatment of Viral Infections,” which is incorporated herein by reference.
  • a FTC/TAF/EVG/COBI combination drug is manufactured, and is commercially available from, Gilead Sciences.
  • Therapeutically effective amount A quantity of a composition or a cell to achieve a desired effect in a subject being treated.
  • this can be the amount necessary to inhibit vision loss and/or retinal degeneration.
  • a dosage When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve an in vitro effect.
  • a therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the AMD, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the beneficial therapeutic effect can include enablement of diagnostic determinations; amelioration of the AMD symptoms, improvement of vision, or delay in reducing or preventing the onset of AMD symptoms.
  • an “effective amount” is an amount sufficient to reduce symptoms of AMD, for example by at least 10%, at least 20%, at least 50%, at least 70%, or at least 90% (as compared to no administration of the therapeutic agent), or that delays onset or progression.
  • the term also applies to a dose that will allow for expression of a Cas13 and/or gRNA herein, and that allows for targeting (e.g., detection or modification) of a target HERV-K RNA.
  • Transfer (t)RNA An adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length, that carries an amino acid to the ribosome.
  • RNA interference specifically in the suppression of retroviruses and retrotransposons that use tRNA as a primer for replication.
  • tiRNA-derived stress-induced RNAs tiRNAs
  • tRFs tRNA fragments
  • Unit dosage form A physically discrete unit, such as a capsule, tablet, or solution, that is suitable as a unitary dosage for a human patient, each unit containing a predetermined quantity of one or more active ingredient(s) calculated to produce a therapeutic effect, in association with at least one pharmaceutically acceptable diluent or carrier, or combination thereof.
  • Unit dosage formulations contain a daily dose or an appropriate fraction thereof, of the active ingredient(s).
  • tRNA-derived fragments are short molecules that emerge after cleavage of the mature tRNAs or the precursor transcript.
  • up regulation refers to any process which results in an increase in production of an RNA of interest. In one example, upregulation increases detectable RNA expression or RNA activity.
  • a “knock-in” is an increase in expression due to the introduction of a nucleic acid molecule encoding a protein of interest, such as ANG, tRNA, or a tRNA fragment. Upregulation includes any detectable increase in the RNA.
  • detectable RNA in a cell or cell free system 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% (such as a decrease of 40% to 90%, 40% to 80% or 50% to 95%) as compared to a control (such an amount of ANG RNA detected in a corresponding non-treated cell or sample).
  • Vector A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker gene and other genetic elements known in the art.
  • Vectors include plasmid vectors, including plasmids for expression in gram- negative and gram-positive bacterial cell. Exemplary vectors include those for expression in E. coli and Salmonella.
  • Vectors also include viral vectors, such as, but are not limited to, retrovirus, lentiviral, adeno- associated virus (AAV), orthopox, avipox, fowlpox, capripox, suipox, adenoviral, herpes virus, alpha virus, baculovirus, Sindbis virus, vaccinia virus and poliovirus vectors.
  • Vectors also include vectors for expression in yeast cells or mammalian cells.
  • the vector is a lentivirus (such as an integration-deficient lentiviral vector) or adeno-associated viral (AAV) vector.
  • lentivirus such as an integration-deficient lentiviral vector
  • AAV adeno-associated viral
  • Certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors.”
  • Common expression vectors are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid provided herein (such as a guide RNA (which can be expressed from an RNA sequence or a RNA sequence), nucleic acid encoding a Cas13Rx protein) in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • a nucleic acid provided herein such as a guide RNA (which can be expressed from an RNA sequence or a RNA sequence), nucleic acid encoding a Cas13Rx protein
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc.
  • a vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).
  • Virus Microscopic infectious organism that reproduces inside living cells. A virus consists essentially of a core of a single nucleic acid surrounded by a protein coat and has the ability to replicate only inside a living cell. “Viral replication” is the production of additional virus by the occurrence of at least one viral life cycle.
  • “Retroviruses” are RNA viruses wherein the viral genome is RNA.
  • a host cell When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells.
  • the integrated DNA intermediate is referred to as a provirus.
  • a wild-type retrovirus genome encodes a polymerase, glycosaminoglycan, and an envelope protein.
  • the term "lentivirus" is used in its conventional sense to describe a genus of viruses containing reverse transcriptase.
  • the lentiviruses include the “immunodeficiency viruses” which include human immunodeficiency virus (HIV) type 1 and type 2 I and HIV-II), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV).
  • HIV human immunodeficiency virus
  • SIV simian immunodeficiency virus
  • FV feline immunodeficiency virus
  • II. Agents that Inhibit HERV-K Methods are disclosed herein for treating AMD in a subject, and for reducing the risk of developing AMD.
  • the method can reduce retinal degeneration, and/or halt vision loss or improve vision, for example by 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 at least 500%.
  • These methods use one or more agents that reduce or inhibit HERV-K activity, for example by 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 aspects, 100% inhibition of HERV-K activity is not required for the disclosed methods of treating AMD.
  • An agent that treats, or reduces the risk of AMD in a subject can reduce one or more of a) the amount of human endogenous retrovirus (HERV)-K, b) production of an mRNA encoding a HERV-K protein, c) translation of the mRNA; d) the amount of an HERV-K protein in the subject ; e) inhibit activity of HERV-K mRNA and/or proteins, or any combination of a, b, c, d, and e. Exemplary agents are disclosed below, and can be used in any combination.
  • an agent that inhibits HERV-K reduces the amount of HERV-K in a subject, such as locally in the retina of the subject.
  • the agent is administered systemically. Suitable agents are discussed in more detail below.
  • the treatments listed below can be used in any combination.
  • an anti-retroviral agent is administered in combination with CRISPR/Cas13 system.
  • the treatment reduces the amount of, activity of proteins, and/or reduces virus mRNA.
  • Inhibitory peptides are of use. Inhibitory peptides are also disclosed, for example, in Hoffman et al., J. Virol.94(23) e01682-20, 2000, incorporated herein by reference. Inhibitory peptides are also available for HIV (see Egerer et al., Mol.
  • the agent that inhibits KERV-K is ANG, such as a vector expressing ANG.
  • ANG such as a vector expressing ANG.
  • the agent is an antibody or an antigen binding fragment thereof, that specifically binds an HERV-K protein, such as, but not limited to, a monoclonal antibody that specifically binds Gag, Pol or Env of HERV-K.
  • An exemplary complete nucleic acid sequence for HERV-K is: TGTGGGGAAAAGCAACAGAGGTCAGATTGTTACTGTGTCTGTATAGAAAGAAGTAGACATAGGAGACTCCATTTTGT TCTGTACTAAGAAAAATTATTCTGCCTTGAGATGCTGTTAATCTATGACCTTACCCCCAACCCCGTGCTCTCTGAAACA TGTGCTGTGTCAAACTCAGGGTTAAATGGATTAAGGGCGGTGCAAGATGTGCTTTGTTAAACAGATGCTTGAAGGCAG CATGCTCATTAAGAGTCATCACCACTCCCTAATCTCAAGTACCCAGGGACACAAAAACTGCGGAAGGCTGCAGGGGC CTCTGCCTAGGAAAGCCAGGTATTGTCCAAGGTTTCTCCCCATGTGAGAGTCTGAAATATGGCCTCGTGGGAAGGGAA AGACCTGACCGTCCCCCAGCCCGACACCCATAAAGGGTCTGTGCTGAGGAGGATTAGTATAAGAGGAAAGCATGCCT CTTGCAGTTGAGACAAGAGG
  • a nucleic acid molecule encoding an antibody or antigen binding fragment is delivered locally to the eye or systemically to the subject.
  • Antibodies to HERV-K envelope protein are commercially available from MyBioSource, Catalog number MBS602670 and MBS603725, and from United States Biological, Catalog number E2286-13-100ug and E2286-14-100ug.
  • Antibodies to HERV-K envelope protein are commercially available from LSBio, catalog number LS-C65286-100, and from antibodies-online, catalog number ABIN472658.
  • Antibodies that specifically bind and substantially reduce or inhibit Gag, Pol or Env activity are of use in the methods disclosed herein.
  • Antibodies include monoclonal antibodies, human antibodies, humanized antibodies, and immunoglobulin (Ig) fusion proteins. Fully human and humanized antibodies can also be produced using methods known to those of skill in the art.
  • Polyclonal antagonistic antibodies can be prepared, such as by immunizing a suitable subject (such as a human subject or a veterinary subject) with an HERV-K Gag, Pol or Env protein.
  • the anti-HERV-K Gag, Pol or Env antibody titer in the immunized subject can be monitored over time, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized HERV-K Gag, Pol or Env or an epitope thereof.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules that specifically bind HERV-K Gag, Pol or Env can be isolated from a mammal (such as from serum) and further purified, for example using protein A chromatography to isolate IgG antibodies.
  • the antibody can also be selected using a functional assay, such as to detect inhibition of HERV-K function.
  • Antibody-producing cells can be obtained from a subject, such as an immunized subject, and used to prepare monoclonal antibodies (see Kohler and Milstein Nature 256:49549, 1995; Brown et al., J. Immunol. 127:53946, 1981; Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.7796, 1985; Gefter, M. L. et al. (1977) Somatic Cell Genet.3:23136; Kenneth, R. H. in Monoclonal Antibodies: A New Dimension In Biological Analyses. Plenum Publishing Corp., New York, N.Y. (1980); Kozbor et al. Immunol.
  • an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with HERV-K Gag, Pol or Env, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that specifically binds to the polypeptide of interest and inhibits a function of the polypeptide.
  • lymphocytes typically splenocytes
  • an immortal cell line (such as a myeloma cell line) is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with HERV-K Gag, Pol or Env, or an epitope thereof, with an immortalized mouse cell line.
  • a mouse myeloma cell line is utilized that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • myeloma cell lines can be used as a fusion partner, including, for example, P3-NS1/1-Ag4-1, P3-x63- Ag8.653 or Sp2/O-Ag14 myeloma lines, which are available from the American Type Culture Collection (ATCC), Rockville, MD.
  • HAT-sensitive mouse myeloma cells can be fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused (and unproductively fused) myeloma cells.
  • Hybridoma cells producing a monoclonal antibody of interest can be detected, for example, by screening the hybridoma culture supernatants for the production antibodies that bind a HERV-K Gag, Pol or Env, such as by using an immunological assay (such as an enzyme-linked immunosorbant assay (ELISA) or radioimmunoassay (RIA).
  • an immunological assay such as an enzyme-linked immunosorbant assay (ELISA) or radioimmunoassay (RIA).
  • a monoclonal antibody that specifically binds HERV-K Gag, Pol or Env can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (such as an antibody phage display library) with HERV-K Gag, Pol or Env, or an epitope thereof, to isolate immunoglobulin library members that specifically bind the polypeptide.
  • Library members can be selected that have particular activities, such as binding to HERV-K Gag, Pol or Env, or inhibiting HERV-K in an in vitro assay.
  • Kits for generating and screening phage display libraries are commercially available (such as, but not limited to, Pharmacia and Stratagene).
  • Examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No.5,223,409; PCT Publication No. WO 90/02809; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/18619; PCT Publication WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 92/01047; PCT Publication WO 93/01288; PCT Publication No. WO 92/09690; Barbas et al., Proc. Natl. Acad. Sci.
  • each CDR is determined. Residues outside the SDR (specificity determining region, e.g., the non-ligand contacting sites) are substituted. For example, in any of the CDR sequences, at most one, two or three amino acids can be substituted. Chimeric antibodies, which include a framework region from one antibody and the CDRs from a different antibody, can be produced. For example, humanized antibodies can be produced.
  • the antibody or antibody fragment can be a humanized immunoglobulin having CDRs from a donor monoclonal antibody that binds HERV-K Gag, Pol or Env, or an epitope thereof, and immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks.
  • Humanized monoclonal antibodies can be produced by transferring CDRs from heavy and light variable chains of the donor mouse immunoglobulin (that specifically binds HERV-K Gag, Pol or Env) into a human variable domain, and then substituting human residues in the framework regions when required to retain affinity.
  • the antibody may be of any isotype, but in several aspects the antibody is an IgG, including but not limited to, IgG1, IgG2, IgG3 and IgG4.
  • the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework.
  • the sequence of the humanized immunoglobulin heavy chain variable region framework can 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.
  • Human framework regions, and mutations that can be made in humanized antibody framework regions see, for example, in U.S. Patent No.5,585,089).
  • Exemplary human antibodies are LEN and 21/28 CL.
  • the sequences of the many human heavy and light chain frameworks are known.
  • an antibody such as a human or humanized antibody specifically binds to HERV-K Gag, Pol or Env, and/or an epitope thereof, with an affinity constant of at least 10 7 M -1 , such as at least 10 8 M -1 at least 5 X 10 8 M -1 or at least 10 9 M -1 .
  • the antibody specifically binds HERV-K Gag, Pol or Env , or an epitope thereof, with an affinity constant of at least 10 8 M -1 at least 5 X 10 8 M -1 or at least 10 9 M -1 .
  • the antibody can be a fully human antibody.
  • Antibodies such as murine monoclonal antibodies, chimeric antibodies, and humanized antibodies, include full length molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which include a heavy chain and light chain variable region and are capable of binding specific epitope determinants. These antibody fragments retain some ability to selectively bind with their antigen or receptor.
  • fragments include: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole 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 the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable
  • variable region includes the variable region of the light chain and the 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 per each heavy chain and each light chain.
  • the VH and the VL can be expressed from two individual nucleic acid constructs in a host cell. If the VH and the VL are expressed non-contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions.
  • the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light chain variable region are chemically linked disulfide bonds.
  • dsFv disulfide stabilized Fv
  • the Fv V H and V L chains connected by a peptide linker are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide.
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing scFvs 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, supra.
  • Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Patent No.4,036,945 and U.S.
  • Patent No.4,331,647 and 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).
  • Other methods of cleaving antibodies such as separation of heavy chains to form monovalent light- heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. Any of the antigen binding fragments described herein are of use.
  • Conservative variants of the antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the VH and the VL regions, and will retain the charge characteristics of the residues to preserve the low pI and low toxicity of the molecules. Amino acid substitutions (such as at most one, at most two, at most three, at most four, or at most five amino acid substitutions) can be made in the V H and the V L regions to increase yield. In some aspects, these substitutions are made in the framework regions, and are not made in the CDRs. A table of conservative amino acid substitutions is provided above.
  • the amino acid sequence of an antibody of interest can be reviewed to locate one or more of the amino acids in a HERV-K Gag, Pol or Env sequence, identify a conservative substitution, and produce the conservative variant using molecular techniques.
  • Effector molecules such as detectable or therapeutic moieties can be linked to an antibody that specifically binds HERV-K Gag, Pol or Env, using any number of means. Both covalent and noncovalent attachment means may be used.
  • the procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector.
  • the antibody typically contain a variety of functional groups; such as carboxylic acid (COOH), free NH 2 ) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule.
  • the antibody is derivatized to expose or attach additional reactive functional groups.
  • the derivatization may involve 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 join the antibody to the effector molecule.
  • the linker is capable of forming covalent bonds to both the antibody and to the effector molecule.
  • Suitable linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids. Nucleic acid sequences encoding the antibodies can be prepared, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol.68:90-99, 1979; the phosphodiester method of Brown et al., Meth.
  • Nucleic acids can also be prepared by amplification methods.
  • Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence (3SR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • 3SR self-sustained sequence
  • cDNA encoding a detectable marker (such as an enzyme) is ligated to a scFv so that the marker is located at the carboxyl terminus of the scFv.
  • a detectable marker is located at the amino terminus of the scFv.
  • cDNA encoding a detectable marker is ligated to a heavy chain variable region of an antibody that specifically binds HERV-K Gag, Pol or Env, so that the marker is located at the carboxyl terminus of the heavy chain variable region.
  • the heavy chain-variable region can subsequently be ligated to a light chain variable region of the antibody that specifically binds HERV-K Gag, Pol or Env using disulfide bonds.
  • cDNA encoding a marker is ligated to a light chain variable region of an antibody that binds HERV-K Gag, Pol or Env, so that the marker is located at the carboxyl terminus of the light chain variable region.
  • the light chain-variable region can subsequently be ligated to a heavy chain variable region of the antibody that specifically binds HERV-K Gag, Pol or Env using disulfide bonds.
  • the protein can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells.
  • a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells.
  • One or more DNA sequences encoding the antibody or functional fragment thereof can be expressed in vitro by DNA transfer into a suitable host cell.
  • the cell may be prokaryotic or eukaryotic.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication.
  • Polynucleotide sequences encoding the antibody or functional fragment thereof (such as an scFV) can be operatively linked to expression control sequences.
  • An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences.
  • the expression control sequences include, but are not limited to appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • RNA encoding the disclosed antibodies are also of use.
  • the polynucleotide sequences encoding the antibody or functional fragment thereof can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes.
  • Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes can be used.
  • Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host can be used. Transformation of a host cell with recombinant DNA may be carried out. Where the host is prokaryotic, such as E.
  • competent cells are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl2 method. Alternatively, MgCl2 can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.
  • the host is a eukaryote
  • such methods of transfection of DNA as calcium phosphate coprecipitates, mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used.
  • Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding the antibody of functional fragment thereof and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
  • Expression systems such as plasmids and vectors, can be used to produce proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines. Isolation and purification of a recombinantly expressed polypeptide can be performed, for example using preparative chromatography and immunological separations. Once expressed, the recombinant antibodies can be purified, or example using ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y., 1982).
  • compositions of at least about 90 to 95% homogeneity are disclosed herein, and 98 to 99% or more homogeneity can be used for pharmaceutical purposes.
  • the polypeptides should be substantially free of endotoxin.
  • An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).
  • Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena et al., Biochemistry 9: 5015-5021, 1970, Buchner et al., supra. Renaturation is typically accomplished by dilution (for example, 100-fold) of the denatured and reduced protein into refolding buffer.
  • An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.
  • the heavy and light chain regions are separately solubilized and reduced and then combined in the refolding solution.
  • an exemplary yield is obtained when these two proteins are mixed in a molar ratio such that a 5 fold molar excess of one protein over the other is not exceeded. It is desirable to add excess oxidized glutathione or other oxidizing low molecular weight compounds to the refolding solution after the redox-shuffling is completed.
  • the antibodies and functional fragments thereof that are disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of the polypeptides of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence.
  • Methods of forming peptide bonds by activation of a carboxyl terminal end can be used.
  • Pharmaceutical compositions for delivery of nucleic acid molecules encoding antibodies are disclosed below.
  • B. Inhibitory Nucleic Acid Molecules An agent that inhibits HERV-K can be an inhibitory nucleic acid molecule. Inhibitory nucleic acids that decrease the expression and/or activity of HERV-K Gag, Pol or Env can also be used in the methods disclosed herein.
  • inhibitor nucleic acid molecules decrease HERV-K Gag, Pol or Env expression or activity 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%.
  • RNAi RNA interference
  • small inhibitory RNA (siRNA) or short hairpin RNA small inhibitory RNA (siRNA) or short hairpin RNA, which can be used for interference or inhibition of expression of a target.
  • siRNA small inhibitory RNA
  • short hairpin RNA short hairpin RNA
  • WO2017/059122 discloses the use of a polynucleotide sequence in the LTR of the HERV-K genome, wherein the polynucleotide sequence contains eight or more contiguous pyrimidine bases, to reduce HERV-K transcription
  • Exemplary commercially available RNAi sequences specific for HERV-K Gag, Pol or Env that can be used with the disclosed methods include those that target any part of the HERV-K open reading frame.
  • siRNAs are generated by the cleavage of relatively long double-stranded RNA molecules by Dicer or DCL enzymes (Zamore, Science, 296:1265-1269, 2002; Bernstein et al., Nature, 409:363-366, 2001).
  • siRNAs are assembled into RISC and guide the sequence specific ribonucleolytic activity of RISC, thereby resulting in the cleavage of mRNAs or other RNA target molecules in the cytoplasm.
  • siRNAs also associated histone and DNA methylation, resulting in transcriptional silencing of individual genes or large chromatin domains.
  • inhibitory RNA includes double stranded RNA of about 19 to about 40 nucleotides with the sequence that is substantially identical to a portion of an mRNA or transcript of a target gene, such as HERV-K Gag, Pol or Env, or any regulatory sequence, such as the LTR, for which interference or inhibition of expression is desired.
  • a sequence of the RNA “substantially identical” to a specific portion of the mRNA or transcript of the target gene for which interference or inhibition of expression is desired differs by no more than about 30%, and in some aspects no more than about 10% or no more than 5% from the specific portion of the mRNA or transcript of the target gene.
  • the sequence of the RNA is exactly identical to a specific portion of the mRNA or transcript of the target gene (e.g., HERV-K Gag, Pol or Env transcripts.
  • Target sequences are provided in FIG.15A-15B.
  • siRNAs disclosed herein include double-stranded RNA of about 15 to about 40 nucleotides in length and a 3’ or 5’ overhang having a length of 0 to 5-nucleotides on each strand, wherein the sequence of the double stranded RNA is substantially identical to (see above) a portion of a mRNA or transcript of a nucleic acid encoding HERV-K Gag, Pol or Env.
  • the double stranded RNA contains about 19 to about 25 nucleotides, for instance 20, 21, or 22 nucleotides substantially identical to a nucleic acid encoding HERV-K Gag, Pol or Env.
  • the double stranded RNA contains about 19 to about 25 nucleotides 100% identical to a nucleic acid encoding HERV-K Gag, Pol or Env. It should be noted that in this context “about” refers to integer amounts only. In one example, “about” 20 nucleotides refers to a nucleotide of 19 to 21 nucleotides in length. Regarding the overhang on the double-stranded RNA, the length of the overhang is independent between the two strands, in that the length of one overhang is not dependent on the length of the overhang on other strand.
  • the length of the 3’ or 5’ overhang is 0-nucleotide on at least one strand, and in some cases it is 0-nucleotide on both strands (thus, a blunt dsRNA).
  • the length of the 3’ or 5’ overhang is 1-nucleotide to 5-nucleotides on at least one strand. More particularly, 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.
  • the dsRNA molecule has 3’ overhangs of 2-nucleotides on both strands.
  • the double-stranded RNA contains 20, 21, or 22 nucleotides, and the length of the 3’ overhang is 2-nucleotides on both strands.
  • the double-stranded RNA contains about 40-60% adenine+uracil (AU) and about 60-40% guanine+cytosine (GC). More particularly, in specific examples the double-stranded RNA contains about 50% AU and about 50% GC. Also disclosed herein are RNAs that at least one modified ribonucleotide, for instance in the sense strand of the double-stranded RNA.
  • the modified ribonucleotide is in the 3’ overhang of at least one strand, or more particularly in the 3’ overhang of the sense strand.
  • modified ribonucleotides include ribonucleotides that include a detectable label (for instance, a fluorophore, such as rhodamine or FITC), a thiophosphate nucleotide analog, a deoxynucleotide (considered modified because the base molecule is ribonucleic acid), a 2’-fluorouracil, a 2’-aminouracil, a 2’-aminocytidine, a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3- aminoallyl)-uracil, an inosine, or a 2’O-Me-nucleotide analog.
  • a detectable label for instance, a fluorophore, such as
  • Nucleotide analogs may also include modifications to the sugar portion of the nucleotides.
  • the 2' OH-group may be replaced by H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, or COOR, wherein R is substituted or unsubstituted C.sub.l-C.sub.6 alkyl, alkenyl, alkynyl, aryl, etc.
  • R is substituted or unsubstituted C.sub.l-C.sub.6 alkyl, alkenyl, alkynyl, aryl, etc.
  • Other possible modifications include those described in U.S. Pat. Nos.5,858,988, and 6,291,438.
  • a locked nucleic acid (LNA) often referred to as inaccessible RNA, is a modified RNA nucleotide.
  • Antisense and ribozyme molecules for HERV-K Gag, Pol or Env are of use in the methods disclosed herein.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American 262:40, 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate an mRNA that is double- stranded.
  • Antisense oligomers of about 15 nucleotides can be used, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target cell producing HERV-K Gag, Pol or Env (see, for example, Marcus-Sakura, Anal. Biochem.172:289, 1988).
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions.
  • an antisense nucleic acid molecule can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, such as phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridin- e, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, amongst others.
  • oligonucleotide Use of an oligonucleotide to stall transcription is a triplex strategy where an oligonucleotide winds around double-helical DNA, forming a three-strand helix. Therefore, these triplex compounds can be designed to recognize a unique site on a chosen gene (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 of use in the methods disclosed herein. Ribozymes, which are RNA molecules the ability to specifically cleave other single- stranded RNA in a manner analogous to DNA restriction endonucleases, are also of use.
  • RNA molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it. Because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • ribozymes There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, Nature 334:585, 1988) and “hammerhead”-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while “hammerhead”-type ribozymes recognize base sequences 11-18 bases in length.
  • RNA delivery systems can be used to administer the siRNAs and other inhibitory nucleic acid molecules as therapeutics. Such systems include, for example, encapsulation in liposomes, microparticles, microcapsules, nanoparticles, recombinant cells capable of expressing the therapeutic molecule(s) (see, e.g., Wu et al., J. Biol.
  • CRISPR/Cas13 As disclosed in PCT Publication No. WO 2019/040664, incorporated herein by reference, Cas13 proteins (and coding sequences), and guide molecules (e.g., gRNA and coding sequences) can be used in a CRISPR/Cas system to target one or more RNA molecules, such as HERV-K.
  • a CRISPR/Cas system for RNA targeting includes two general components: (1) a Cas13 protein or its coding sequence (whose expression can be driven by a promoter) and (2) a guide nucleic acid molecule, such as RNA (gRNA), which is specific for the target RNA (whose expression can also be driven by promoter).
  • gRNA guide nucleic acid molecule
  • the guide molecule guides the Cas13 to the target RNA (such as HERV-K).
  • the Cas13 is Cas13a, Cas13b, Cas13c, Cas13Rx, Cas13x, or Cas13y.
  • the Cas13 is Cas13Rx.
  • Exemplary Cas13 sequences are found in PCT Publication No. WO 2019/040664, US 11,293,011, and US 10,392,616, and are also provided below.
  • administration of a CRISPR/Cas system for HERV-K RNA targeting is into the eye.
  • the Cas 13 is Cas13Rx.
  • An exemplary nucleic acid sequence encoding Cas13Rx is: ATGAGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCATCGAAAAAAAAAAGTCCTTCGCCAAGGGCATGGGCGTGA AGTCCACACTCGTGTCCGGCTCCAAAGTGTACATGACAACCTTCGCCGAAGGCAGCGACGCCAGGCTGGAAAAGATC GTGGAGGGCGACAGCATCAGGAGCGTGAATGAGGGCGAGGCCTTCAGCGCTGAAATGGCCGATAAAAACGCCGGCT ATAAGATCGGCAACGCCAAATTCAGCCATCCTAAGGGCTACGCCGTGGTGGCTAACAACCCTCTGTATACAGGACCC TATTTGTATCCAGGTGATCCATAACATCCTGGACATTGAAAAAATCCTCGCCGAATACATTACCAACGCCGCCTACGC CGTCAACAATATCTCCGGCCTGGATAAGGACATTATTGGATTCGGCAAGTTCTCCACAGTGTATACCTACGACGAATT CAAAGACCCCGAGCACCATAGGGCCGCTCT
  • RNA sequences are easily targeted, for example edited or detected, optionally with an effector domain.
  • a Cas13Rx protein 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 SEQ ID NO: 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, 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,
  • the Cas13 protein is expressed in a recombinant cell and purified.
  • the resulting purified Cas13 protein, along with an appropriate guide molecule specific for the target RNA, is then introduced into the retina where one or more RNAs can be targeted.
  • the Cas13 protein and guide nucleic acid molecule are introduced as components into the retina.
  • the purified Cas13 protein is complexed with the nucleic acid (e.g., gRNA) specific for HERV-K, and this ribonucleoprotein (RNP) complex is introduced into cells in the retina (e.g., using transfection or injection).
  • the nucleic acid e.g., gRNA
  • RNP ribonucleoprotein
  • the Cas13 protein and HERV-K guide nucleic acid molecule are in cells in the retina, one or more HERV-K RNAs can be targeted.
  • the Cas13 is Cas13a, Cas13b, Cas13c, Cas13Rx, Cas13x, or Cas13y.
  • the Cas13 is Cas13Rx.
  • the Cas13 protein is expressed from a nucleic acid molecule in a retinal cell containing a target HERV-K.
  • the Cas13 protein is expressed from a vector, such as a viral vector, for example an adenovirus vector, lentivirus vector, or baculovirus vector, or plasmid introduced into cells in the retina.
  • a viral vector for example an adenovirus vector, lentivirus vector, or baculovirus vector
  • plasmid introduced into cells in the retina.
  • these nucleic acid molecules can be co-expressed in cells in the retina with the guide nucleic acid molecule (e.g., gRNA) specific for HERV-K.
  • gRNA guide nucleic acid molecule
  • multiple plasmids or vectors are used for RNA targeting.
  • the nucleic acid molecule encoding the Cas13 can be provided for example on one vector or plasmid, and the guide nucleic acid molecule (e.g., gRNA) on another plasmid or vector. Multiple plasmids or viral vectors can be mixed and introduced into retinal cells at the same time, or separately. In some examples, multiple nucleic acid molecules are expressed from a single vector or plasmid.
  • a single vector can include the nucleic acid molecule encoding the Cas13, and a separate vector can include the guide molecule.
  • a plurality of different guide molecules are present on a single array and/or vector.
  • the method includes delivering a plurality of gRNAs (such as at least 2, at least 3, at least 4, at least 5, different gRNAs), which are part of an array (which can be part of a vector, such as a viral vector or plasmid).
  • the array is processed by the Cas13 protein, such as a Cas13Rx protein, into the individual mature gRNAs.
  • the nucleic acid molecules expressed from the vector can be under the control of a promoter and optionally contain selection markers (such as antibiotic resistance).
  • the method of targeting the RNA results in editing the sequence of a target RNA.
  • a Cas13Rx protein with a non-mutated HEPN domain e.g., SEQ ID NO: 1, 3, 42, 62, 70, 82, 83, or 92 of PCT Publication No. WO 2019/040664
  • the target RNA can be cut or nicked at a precise location.
  • such a method is used to decrease expression of a target HERV-K RNA, which will decrease translation of Gag, Pol and/or Env. Combination of gRNAs for these targets are also of use.
  • targeting the target HERV-K RNA (such as Gag, Pol and/or Env) allows for decreasing expression of the protein encoded by the RNA.
  • a Cas13Rx fusion protein with a mutated HEPN domain and a translational repression domain such as Pumilio or FBF PUF proteins, deadenylases, CAF1, Argonaute proteins, and and a guide RNA containing at least one spacer sequence specific for the target HERV-K RNA, of a target HERV-K RNA, such as Gag, Pol, and/or Env can be decreased.
  • Cas13Rx can be fused to a ribonuclease (such as a PIN endonuclease domain, an NYN domain, an SMR domain from SOT1, or an RNase domain from Staphylococcal nuclease) or a domain that affects RNA stability (such as tristetraprolin or domains from UPF1, EXOSC5, and STAU1).
  • RNA aptamer sequences can be appended to or inserted within the gRNA molecule, such as MS2, PP7, Q ⁇ , and other aptamers.
  • Proteins that specifically bind to these aptamers can be fused to a translational repression domain, a ribonuclease, or a domain that affects RNA stability.
  • This aptamer-effector domain fusion can be used to target the target RNA because the Cas13 and gRNA complex will guide the aptamer protein-effector domain in proximity to the target RNA.
  • the target RNA is an HERV-K RNA, such as encoding Gag, Pol or Env.
  • a Cas13 protein and a guide RNA containing at least one spacer sequence specific for a HERV-K RNA can be used.
  • the gRNA that hybridizes with the one or more target HERV-K RNA molecules includes one or more direct repeat (DR) sequences, one or more spacer sequences, or one or more sequences comprising DR-spacer-DR-spacer.
  • DR direct repeat
  • the one or more DR sequences have 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 SEQ ID NO: 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, 205, 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 of PCT Publication No.
  • the gRNA includes additional sequences, such as an aptamer sequence.
  • a plurality of gRNAs are processed from a single array transcript, wherein each gRNA can be different, for example to target different RNAs (such as two or more of HERV-K Gag, Pol and Env) or target multiple regions of a single RNA (such as HERV-K Gag, Pol or Env).
  • a guide sequence can also include one or more direct repeats (DRs). The DR is the constant portion of the guide, which contains strong secondary structure, which facilitates interaction between a Cas13 protein and the guide molecule.
  • Targeting an RNA molecule can include one or more of cutting or nicking one or more target HERV-K RNA molecules, such as encoding Gag, Pol and/or Env, deactivating or downregulating one or more target HERV-K RNA molecules, deactivating or suppressing translation the one or more target HERV-K RNA molecules, visualizing, labeling, or detecting the one or more target HERV-K RNA molecules, binding the one or more target HERV-K RNA molecules, editing the one or more target HERV-K RNA molecules, trafficking the one or more target HERV-K RNA molecules, and masking the one or more target HERV-K RNA molecules.
  • modifying one or more target HERV-K RNA molecules includes one or more of an RNA base substitution, an RNA base deletion, an RNA base insertion, or a break in the target HERV-K RNA.
  • gRNA molecules can include naturally occurring or non-naturally occurring nucleotides or ribonucleotides (such as LNAs or other chemically modified nucleotides or ribonucleotides, for example to protect a guide RNA from degradation).
  • the guide sequence is RNA.
  • the guide nucleic acid can include modified bases or chemical modifications (e.g., see Latorre et al., Angewandte Chemie 55:3548-50, 2016).
  • a guide sequence directs a Cas13 protein to a target HERV-K RNA, thereby targeting the HERV-K RNA (e.g., modifying or detecting the RNA).
  • the ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target HERV-K RNA may be assessed by any suitable assay.
  • the components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested may be provided to a host cell having the corresponding target HERV-K RNA molecule, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence.
  • cleavage of a target HERV-K RNA sequence may be evaluated in a test tube by providing the target HERV-K RNA, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target HERV-K RNA between the test and control guide sequence reactions.
  • Other assays are possible.
  • vectors such as a viral vector or plasmid (e.g., retrovirus, lentivirus, adenovirus, adeno-associated virus, or herpes simplex virus), that includes a guide nucleic acid molecule that targets HERV-K.
  • the guide nucleic acid molecule is operably linked to a promoter or expression control element (specific examples of which are provided elsewhere in this application).
  • a promoter or expression control element can include other elements, such as a gene encoding a selectable marker, such as an antibiotic, such as puromycin, hygromycin, or a detectable marker such as GFP or other fluorophore.
  • the vector can be an adenovirus vector.
  • the vector can be a lentiviral vector.
  • the vector can be a baculovirus vector.
  • the lentiviral vector, the adenoviral vector, or the baculovirus vector comprises an inducible (such as a doxycycline inducible promoter) or constitutive (e.g., CMV) or tissue- specific (e.g., RPE-specific) promoter operably linked to a nucleic acid molecule encoding the Cas13, such as Cas13Rx.
  • Guide molecules can include one or more regions referred to as spacers.
  • a spacer has sufficient complementarity with a target HERV-K RNA sequence to hybridize with the target HERV-K RNA and direct sequence-specific binding of a Cas13 protein, such as Cas 13d, to the target HEV-K RNA.
  • the spacer is the variable portion of the guide sequence.
  • a spacer has 100% complementarity to a target HERV-K RNA (or region of the HERV-K RNA to be target), but a spacer can have less than 100% complementarity to a target RNA, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% complementarity to a target RNA.
  • the gRNA specifically hybridizes to a) an RNA encoding glycosaminoglycan (Gag) protein of HERV-K or a nucleic acid molecule encoding the gRNA; b) an RNA encoding (Pol) protein of HERV-K or a nucleic acid molecule encoding the gRNA; or c) an RNA encoding (Env) protein of HERV-K or a nucleic acid molecule encoding the gRNA.
  • Exemplary guide molecules s are provided as SEQ ID NOs: 1-3 of the present disclosure. Delivery of nucleic acid molecules is described below.
  • Host cells are also provided that are transduced with the disclosed vectors. These host cells can be in vitro.
  • the cell is as a retinal pigment epithelial cell, a photoreceptor cell, or a choroidal cell.
  • D. Anti-Retroviral Agents It is disclosed herein that one or more anti-viral agents can inhibit HERV-K, and thus are of use for treating AMD.
  • an effective amount of reverse transcriptase inhibitor such as lamivudine, zidovudine, abacavir, tenofovir, a tenofovir prodrug, emtricitabine (FTC), or a pharmaceutically acceptable salt thereof is administered to a subject that has AMD, or is at risk of developing AMD.
  • 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 one non-limiting example, the reverse transcriptase inhibitor is nevirapine.
  • and effective amount of an integrase inhibitor is administered to the subject.
  • the integrase inhibitor can be raltegravir.
  • an effective amount of a protease inhibitor can be administered to the subject.
  • the protease inhibitor can be darunavir.
  • the disclosed methods can use an effective amount of a NRTI, a NtRTI, an integrase inhibitor, and/or a protease inhibitor. Combinations of these agents are also of use.
  • NtRTI operative in the treatment of AMD, or reducing the risk of developing AMD include tenofovir, tenofovir prodrugs such as tenofovir disproxil fumerate (TDF) or Tenofovir alafenamide (TAF), adefovir; 2′,3′-dideoxy-3′-fluoroadenisine; 2′,3′-dideoxy-3′-fluoroguanasine; 3′deoxy-3′-fluoro-5-O-[2-(L- valyloxy)-propionyl]guanosine and include pharmaceutically acceptable salts, esters, ester salts, nitrile oxides, and other prodrugs of any of the active agents.
  • TDF tenofovir disproxil fumerate
  • TAF Tenofovir alafenamide
  • An A NRTI operative in the treatment of AMD, or of use in reducing the risk of developing AMD includes FTC, lamivudine, zalcitabine, zidovudine, azidothymidine, didanosine, stavudine, and abacavir.
  • Other antiretrovirals such as nonnucleoside reverse transcriptase inhibitors, protease inhibitors, fusion inhibitors, and combinations thereof, are also of use.
  • Representative non-nucleoside reverse transcriptase inhibitors operative herein illustratively include delavirdine, efavirenz, nevirapine, and other diarylpyrimidine (DAPY) derivatives.
  • protease inhibitors operative herein illustratively include amprenavir, tipranavir, indinavir, saquinavir, lopinavir, ritonavir, fosamprenavir calcium, ritonavir, atazanavir sulfate nelfinavir mesylate, darunavir, and combinations thereof.
  • An entry inhibitor operative herein as an optional active ingredient in an inventive composition illustratively includes enfuvirtide, Schering C (Schering Plough), S-1360 (Shionogi), and BMS806 (Bristol Myers Squibb).
  • Derivates and salts of use in the disclosed methods include salts such as alkali metal salts; esters such as acetate, butyrate, palmitate, chlorobenzoates, benzoates, C1-C6 benzoates, succinates, and mesylate; salts of such esters; and nitrile oxides.
  • Pharmaceutically acceptable salts, esters, ester salts, nitrile oxides, and prodrugs of any of the active agents are also of use in the disclosed methods.
  • both a NRTI and/or an NtRTI is/are administered to the subject.
  • the NRTI and/or the NtRTI can be administered as a prodrug.
  • prodrug includes a compound that when administered to a primate host generates an active NRTI or NtRTI as a result of spontaneous reaction under physiological conditions, enzymatic catalysis, metabolic clearance, or combinations thereof.
  • An exemplary NtRTI prodrug currently FDA approved for HAART use is tenofovir disoproxil fumarate (TDF) and is detailed in U.S. Patent 5,935,946, TAF and succinate salts of tenofovir.
  • the methods include the administration of tenofovir, TAF and/or TDF.
  • 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.
  • a protease inhibitor such as amprenavir, tipranavir, indinavir, saquinavir, lopinavir, ritonavir, fosamprenavir calcium, ritonavir, atazanavir sulfate nelfinavir mesylate, and darunavir
  • an effective amount of darunavir is administered to the subject.
  • 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.
  • an effective amount of a) tenofovir, TAF, and/or TDF is administered to the subject, and b) darunavir is administered to the subject.
  • Other analogs of pharmaceutically active NRTIs, NtRTIs, or integrase inhibitors are also suitable for use.
  • a pharmaceutically acceptable carrier or diluent includes agents that are compatible with other ingredients of a dosage and not injurious to the subject with AMD, or at risk for AMD.
  • the methods include the administration of an effective amount of a) tenofovir, TAF and/or TDF, and b) FTC.
  • the methods include the administration of an effective amount of a) tenofovir, TAF and/or TDF, and b) darunavir.
  • an NRTI, NtRTI and optionally an integrase inhibitor are administered concurrently, such as in a single formulation, to the subject. This combination can be co-administered with or without a pharmacoenhancer, such as, but not limited to, cobistat (COBI).
  • the methods include the administration of an effective amount of a) tenofovir, TAF and/or TDF, b) FTC; and c) COBI.
  • the methods utilize a combination of at least one NRTI, at least one NtRTI, and optionally at an integrase inhibitor.
  • the integrase inhibitor can be elvitegravir (EVG).
  • the methods include the administration of an effective amount of a) tenofovir, TAF and/or TDF, b) FTC; c) COBI; and d) EVG.
  • effective amount of a protease inhibitor such as, but not limited to darunavir, is administered to the subject.
  • these can be compounded into a dosage form suitable for delivery by a route with administration by intraocular (e.g., into the eye), oral, rectal, topical, vaginal or parenteral routes of administration.
  • intraocular e.g., into the eye
  • oral, rectal, topical, vaginal or parenteral routes of administration e.g., Compositions and compounding methods 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.
  • administration is oral.
  • the administration can be into the eye as a suspension, slow release implant or eye drops.
  • the disclosed methods include co-administering to a subject, such as a human having or at risk of developing AMD, a combination of a pharmacologically effective amount of the nucleoside reverse transcriptase inhibitor, such as, but not limited to, FTC, an effective amount of the nucleotide reverse transcriptase inhibitor, such as, but not limited to, tenofovir or a tenofovir prodrug such as, but not limited to, a tenofovir salt such as TDF, TAF, or another salt form of tenofovir.
  • the subject can also be administered an effective amount of an integrase inhibitor, such as, but not limited to, EVG.
  • the subject can be administered an effective amount of a protease inhibitor, such as darunavir.
  • a protease inhibitor such as darunavir.
  • these combinations can be co-administered with or without a pharmacoenhancer, such as, but not limited to, COBI.
  • the co-administration is oral. In more aspects, the co-administration is simultaneous.
  • an effective amount of a nucleoside reverse transcriptase inhibitor such as, but not limited to, FTC
  • an effective amount of a nucleotide reverse transcriptase inhibitor such as, but not limited to, tenofovir or a tenofovir prodrug such as TDF or TAF
  • an effective amount of an integrase inhibitor such as EVG
  • a single composition such as in a unit dose
  • an effective amount of a pharmacoenhancer, such as COBI is included in this same composition.
  • the composition can be formulated for oral administration.
  • these active agents can be combined into a single unit dose and administered to a subject having, or at risk of developing, AMD.
  • the doses of individual active components are administered in effective amounts, to create a therapeutic concentration of the active composition at the retina.
  • establishing an effective concentration for a given active agent in the target cells, such as the retina includes factors for the agent such as the route of administration, pharmacokinetics, absorption rate based on administration route, effects of food on oral absorption, in vivo distribution, metabolic pathways, elimination route, race, gender, and age of the subject, single dose incident side effects, long term administration side effects, and synergistic effects with co-administered active agents. Information related to these factors considered in dosing are available from the United States Food and Drug Administration (fda.gov/oashi/aids/virals.html).
  • the dosing according to the present methods utilize as a starting point the maximal recommended tolerated dosing levels for the given active agent combination associated with HAART treatment protocols.
  • the methods include oral administration of TAF/TDF/tenofovir/salt to a subject having AMD, or at risk of developing AMD.
  • the methods include oral co-administration of FTC, TAF/TDF/tenofovir/salt to a subject having AMD, or at risk of developing AMD.
  • FTC, TAF/TDF/tenofovir/salt, and EVG are administered to the subject having AMD, or at risk of developing AMD.
  • the method includes oral co-administration of FTC, TAF/TDF/tenofovir/salt, optionally EVG and optionally COBI to the subject.
  • FTC, TAF/TDF/tenofovir/salt, EVG, and COBI are administered to the subject.
  • TAF is used in any of the above combinations.
  • TDF is used in any of the above combinations.
  • an oral dose of TAF can be in the range from about 0.0001 to about 100 mg/kg body weight per day, for example, from about 0.01 to about 10 mg/kg body weight per day, from about 0.01 to about 5 mg/kg body weight per day, from about 0.5 to about 50 mg/kg body weight per day, from about 1 to about 30 mg/kg body weight per day, from about 1.5 to about 10 mg/kg body weight per day, or from about 0.05 to about 0.5 mg/kg body weight per day.
  • the daily dose for an adult human of about 70 kg body weight will range from about 0.1 mg to about 1000 mg, or from about 1 mg to about 1000 mg, or from about 5 mg to about 500 mg, or from about 1 mg to about 150 mg, or from about 5 mg to about 150 mg, or from about 5 mg to about 100 mg, or about 10 mg, and may take the form of single or multiple doses.
  • the oral dose of TAF may be in the form of a combination of agents (e.g., TAF/FTC/EVG/COBI).
  • COBI or a pharmaceutically acceptable salt thereof is combined with certain specific solid carrier particles (e.g., silica derivatives), the resulting combination possesses improved physical properties.
  • the resulting combination has low hygroscopicity as compared to COBI alone.
  • the resulting combination is a free-flowing powder, with high loading values for COBI, acceptable physical and chemical stability, rapid drug release properties, and excellent compressibility.
  • the resulting combination can readily be processed into solid dosage forms (e.g., tablets).
  • COBI can be used with any suitable solid carrier, provided the resulting combination has physical properties that allow it to be more easily formulated than the parent compound.
  • suitable solid carriers include kaolin, bentonite, hectorite, colloidal magnesium-aluminum silicate, silicon dioxide, magnesium trisilicate, aluminum hydroxide, magnesium hydroxide, magnesium oxide and talc.
  • the solid carrier can comprise calcium silicate or magnesium aluminometasilicate.
  • COBI can be coated in the pores and on the surface of a solid carrier.
  • Suitable silica derivatives of use are disclosed in PCT Publication WO 03/037379.
  • Exemplary oral dosages of use in the disclosed methods 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 can be used in amounts of less than 300 mg, 200 mg or less and 100 mg or less.
  • COBI can be used in amounts of 50-500 mg, 100-400 mg, 100-300 mg, and 150 mg.
  • Tenofovir (or TDF or TAF or another salt form) and COBI or pharmaceutically acceptable salt(s) 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.
  • the amount administered can be adjusted relative to the weight of the component added to produce the salt or complex.
  • the method can include co-administering 200 mg of FTC and 150 mg of EVG.
  • the method can include co-administering 150 mg COBI, 100 mg or tenofovir, 150 mg EVG, and 200 mg FTC.
  • the method can include co-administering 150 mg COBI, 200 mg or less tenofovir, 150 mg EVG, and 200 mg FTC.
  • the method can include co-administering 150 mg COBI, less than 300 mg tenofovir, 150 mg EVG, and 200 mg FTC.
  • the method can include co-administering 150 mg COBI, 50 mg tenofovir, 150 mg EVG, and 200 mg FTC.
  • the method can include co-administering 150 mg EVG, 150 mg COB, 200 mg FTC, and 10 mg TAF.
  • These compositions can be administered orally. See U.S. Published Patent Application No.2015/0105350, incorporated herein by reference for additional dosing information.
  • GENVOYA® is administered to the subject.
  • administration is oral.
  • An effective amount of one or more additional agents can be administered to a subject.
  • agents include, but are not limited to, and effective amount of L-745,870 trihydrochloride and an effective amount of metformin or metformin hydrochloride.
  • the active agents may be administered to the subject in any conventional manner. Local modes of administration include, by way of example, intraocular, intraorbital, subconjunctival, sub-Tenon’s, subretinal or transscleral routes. In an aspect, significantly smaller amounts of the components (compared with systemic approaches) may exert an effect when administered locally (for example, intravitreally) compared to when administered systemically (for example, orally).
  • the system disclosed herein is delivered by intravitreal injection. Intravitreal injection has a relatively low risk of retinal detachment.
  • administration is from an internal reservoir (for example, from an implant disposed at an intra- or extra-ocular location (see, U.S. Pat. Nos.5,443,505 and 5,766,242)) or from an external reservoir (for example, from an intravenous bag).
  • Components can be administered by continuous release for a particular period from a sustained release drug delivery device immobilized to an inner wall of the eye or via targeted transscleral controlled release into the choroid (see, for example, PCT/US00/00207, PCT/US02/14279, Ambati et al., Invest. Opthalmol. Vis.
  • the salt, carrier, or diluent should be acceptable in the sense of being compatible with the other ingredients and not deleterious to the recipient thereof.
  • carriers or diluents for oral administration include cornstarch, lactose, magnesium stearate, talc, microcrystalline cellulose, stearic acid, povidone, crospovidone, dibasic calcium phosphate, sodium starch glycolate, hydroxypropyl cellulose (e.g., low substituted hydroxypropyl cellulose), hydroxypropylmethyl cellulose (e.g., hydroxypropylmethyl cellulose 2910), and sodium lauryl sulfate.
  • compositions can be prepared by any suitable such as those methods well known in the art of pharmacy, for example, methods such as those in Gennaro et al., Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., 1990), especially Part 8: Pharmaceutical Preparations and their Manufacture.
  • the active agents are formulated for oral administration.
  • the active agents are formulated for local administration to the eye. Such methods include the step of bringing into association the active agents with the carrier or diluent and optionally one or more accessory ingredients.
  • Such accessory ingredients include those conventional in the art, such as, fillers, binders, excipients, disintegrants, lubricants, colorants, flavoring agents, sweeteners, preservatives (e.g., antimicrobial preservatives), suspending agents, thickening agents, emulsifying agents, and/or wetting agents.
  • preservatives e.g., antimicrobial preservatives
  • suspending agents thickening agents
  • emulsifying agents emulsifying agents
  • wetting agents e.g., wetting agents.
  • the pharmaceutical compositions of use in the methods disclosed herein can provide controlled, slow release or sustained release of the active agents over a period of time.
  • the controlled, slow release or sustained release of the agents can maintain the agents in the bloodstream of the human for a longer period of time than with conventional formulations.
  • compositions include, but are not limited to, coated tablets, pellets, solutions (such as eye drops), powders, capsules, and dispersions in a medium that is insoluble in physiologic fluids, or where the release of the therapeutic compound follows degradation of the pharmaceutical composition due to mechanical, chemical, or enzymatic activity.
  • fine powders or granules may contain diluting, dispersing, and or surface-active agents and may be present, for example, in water or in a syrup, in capsules or sachets in the dry state, or in a non- aqueous solution or suspension wherein suspending agents may be included, or in tablets wherein binders and lubricants may be included.
  • the formulation 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 the liquid solution or suspension include suitable sweeteners, flavoring agents, preservatives (e.g., antimicrobial preservatives), buffering agents, solvents, and mixtures thereof.
  • a component of the formulation may serve more than one function.
  • a suitable buffering agent also may act as a flavoring agent as well as a sweetener.
  • Suitable sweeteners include, for example, saccharin sodium, sucrose, and mannitol. A mixture of two or more sweeteners may be used.
  • the sweetener or mixtures thereof are typically present in an amount of from 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 to make the pharmaceutical composition easier for a human to ingest.
  • the flavoring agent or mixtures thereof are typically present in an amount of about 0.0001% to about 5% by weight of the total composition.
  • Preservatives can also be present in the composition.
  • Suitable preservatives include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. A mixture of two or more preservatives may be used.
  • the preservative or mixtures thereof are 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 compositions.
  • Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. A mixture 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.
  • a solvent can be used when a liquid suspension is desirable. Suitable solvents for a liquid solution or suspension include, for example, sorbitol, glycerin, propylene glycol, and water. A mixture of two or more solvents may be used. The solvent or solvent system can 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.
  • active agents such as TAF/TDF/tenofovir/salt, FTC, EVG, darunavir, and optionally COBI
  • active agents are co-administered to a subject at specific time points.
  • the present methods utilize doses of agents.
  • each dose can include oral co-administration of FTC, TAF/TDF/tenofovir/salt, and EVG.
  • each dose can include oral co-administration of FTC, TAF/TDF, EVG and COBI.
  • TAF is included in the dose(s).
  • ANG activity is also disclosed herein that one or more agents that increass ANG activity, such as increasing ability of ANG to generate tRNA fragments that are inhibitory to HERV-K, and reduce expression of HERV-K, are of use for treating AMD.
  • ANG itself, or a nucleic acid molecule encoding ANG, can be used treat AMD in a subject, or can be used to decrease the risk of AMD.
  • An increase in ANG activity generates tRNA fragments that are complementary to an HERV-K coding sequence. Without being bound by theory, downregulation of ANG expression is associated with an increase in HERV-K mRNA.
  • ANG a ribonuclease (cleaves double stranded RNA) uses a tRNA fragment complementary to HERV-K. This tRNA fragment forms a double strand to HERV-K allowing ANG to degrade this double stranded RNA.
  • An increase in ANG expression (with or without the tRNA fragment) downregulates HERV-K expression, and can be used to treat AMD, or reduce the risk of developing AMD.
  • agents that increase ANG activity are of use to treat AMD. These agents can increase ANG activity, for example, by about 1 to about 10 fold, such as about 2 to about 10 fold, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold.
  • the methods can include, without limitation, the administration of ANG, a nucleic acid molecule encoding ANG, tRNA fragment, or a nucleic acid molecule encoding a tRNA fragment. Combinations of these agents are also of use.
  • ANG Proteins An exemplary protein sequence for ANG is disclosed in GENBANK® Accession No.AAA51678.1, October 30, 1994, incorporated herein by reference.
  • An exemplary amino acid sequence for ANG also is provided below: MVMGLGVLLLSH VFVLGLGLTP PTLAQDNSRY THFLTQHYDA KPQGRDDRYC ESIMRRRGLT SPCKDINTFI HGNKRSIKAI CENKNGNPHR ENLRISKSSF QVTTCKLHGG SPWPPCQYRA TAGFRNVVVA CENGLPVHLD QSIFRRP 11).
  • the amino acid sequence for an angiogenin precursor protein is provided in GENBANK® No. NP_001091046, August 16, 2022, incorporated herein by reference.
  • An exemplary nucleic acid sequence encoding ANG is provided in GENBANK Accession No. NM_001145.4, August 15, 2022, incorporated herein by reference.
  • a variant of ANG of use in the methods disclosed herein includes 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 a nucleic acid encoding such).
  • the variant of ANG can include at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative substitutions in SEQ ID NO: 11.
  • the protein can be naturally occurring or recombinant.
  • a variant of ANG of use in the disclosed methods, comprises, consists essentially of, or consists of an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 11 (or a nucleic acid encoding such).
  • ANG comprises, consists essentially of, or consists of, SEQ ID NO: 11 (or a nucleic acid encoding such).
  • a fragment of ANG is of use in the disclosed methods. The use of ANG, and variants thereof, is disclosed, for example, in U.S. Published Patent Application No.2013/0136727 A1.
  • ANG variants and fragments thereof, 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 cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are known (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4 th 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). Hosts can include microbial, yeast, insect and mammalian organisms.
  • DNA sequences having eukaryotic or viral sequences can be expressed in prokaryotes or eukaryotes.
  • suitable host cells include bacteria, archea, insect, fungi (for example, yeast), plant, and animal cells (for example, mammalian cells, such as human).
  • Exemplary cells of use include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, HEK 293 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines.
  • mammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although cell lines may be used, such as cells designed to provide higher expression, desirable glycosylation patterns, or other features.
  • the host cells include HEK 293 cells or derivatives thereof, such as GnTI -/- cells (ATCC® No. CRL-3022), or HEK-293F cells.
  • Transformation of a host cell with recombinant DNA can be carried out by conventional techniques.
  • the host is prokaryotic, such as, but not limited to, E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method.
  • heat shock MgCl 2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.
  • Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding a disclosed antigen, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus a eukaryotic viral vector
  • Appropriate expression systems such as plasmids and vectors of use in producing proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.
  • ANG also can be produced using chemical synthesis. 2. Nucleic acid molecules encoding ANG and tRNA In some aspects, nucleic acid molecules ANG, a precursor, variant or fragment are also of use in the disclosed methods.
  • a tRNA complementary to HERV-K, or a fragment thereof is also of use in the disclosed methods, and can be encoded by a DNA molecule (see Gene ID: 100189107).
  • Two tRNA fragments (encoded by a DNA) that are complementary to HERV-K are LYS CTT (GCCCCACGTTGGGCGCCA, SEQ ID NO: 12) and LYS TTT (GTCCCTGTTCGGGCGCCA, SEQ ID NO: 13), see, for example, Tao et al., supra, 2020.
  • SEQ ID NO: 12 and SEQ ID NO: 13 are the sequences from the genomic DNA of tRNA- CTT (Gene ID: 100189107, May 13, 2022 from the National Library of Medicine, incorporated herein by reference) and tRNA-TTT (Gene ID: 7206, May 13, 2022, for the National Library of Medicine, incorporated herein by reference). These tRFs or tiRNA are complementary to HERV-K. Three nucleotides, CCA, added at the end of a tRNA and may not be complementary to HERV-K. These are all of use in the methods disclosed herein.
  • the tRNA fragment is 16 to 22 nucleotides in length, such as 17 to 21 nucleotides in length, for example, 16, 17, 18, 19.20, 21 or 22 nucleotides in length. In one aspect, the tRNA fragment is 18 nucleotides in length. In more aspects, the tRNA is encoded by SEQ ID NO: 12 or SEQ ID NO: 13. In further aspects, the tRNA includes CCA. One or both of these tRNA fragments can be expressed to increase ANG activity and downregulate HERV-K expression. tRFs are generated by ANG, but AGO proteins can also be involved in using these tRFs to target HERV-K coding sequence.
  • increasing ANG activity can, in some aspects, block HERV-K translation into GAG, POL, ENV proteins and induce degradation of the entire mRNA due to double-stand loops between tRFS and the HERV-K coding RNA.
  • a nucleic acid molecule encoding one or both of these tRNAs is of use in the disclosed methods.
  • tRNA can be used in the presently disclosed methods, with, or without, ANG or a nucleic molecule encoding exogenous ANG.
  • Nucleic acid molecules can be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill. RNA molecules are also of use.
  • the polynucleotides encoding ANG (ora precursor, variant, or fragment thereof), or encoding a tRNA, can include a recombinant DNA which is incorporated into a vector (such as an expression vector) into an autonomously replicating plasmid or virus or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (such as a cDNA) independent of other sequences.
  • the nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double forms of DNA.
  • Polynucleotides encoding ANG, a precursor, variant or fragment thereof, or encoding a tRNA fragment are of use in the disclosed methods include DNA, cDNA, tRNA sequences themselves, and an RNA sequences that encode ANG, a precursor, variant, or fragment thereof. Silent mutations in the coding sequence result from the degeneracy (i.e., redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue.
  • leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC, or ATA.
  • Tables showing the standard genetic code can be found in various sources (e.g., L. Stryer, 1988, Biochemistry, 3.sup.rd Edition, W.H.5 Freeman and Co., NY). Degenerate variants are also of use in the methods disclosed herein. Additional nucleic acid molecules encoding ANG, a precursor, variant, or fragment thereof, or encoding a tRNA fragment, can readily be produced using the amino acid sequences provided herein and the genetic code.
  • Nucleic acid sequences encoding ANG, a precursor, variant or fragment thereof, or encoding a tRNA fragment can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as 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; the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra.
  • Exemplary nucleic acids that include sequences encoding ANG, a precursor, variant, or fragment thereof, or encoding a tRNA fragment can be prepared by cloning.
  • a nucleic acid molecule encoding ANG, a precursor, variant, or fragment thereof, or encoding a tRNA fragment can be cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self- sustained sequence replication system (3SR), and the Q ⁇ replicase amplification system (QB).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • 3SR self- sustained sequence replication system
  • QB Q ⁇ replicase amplification system
  • a polynucleotide encoding ANG can be isolated by a polymerase chain reaction of cDNA using primers based on the DNA sequence of the molecule.
  • a wide variety of cloning and in vitro amplification methodologies can be used. PCR methods are described in, for example, 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 also can be isolated by screening genomic or cDNA libraries with probes selected from the sequences of the desired polynucleotide under stringent hybridization
  • a polynucleotide sequence encoding ANG, a precursor, variant, or fragment thereof, or encoding a tRNA fragment is operably linked to transcriptional control sequences including, for example a promoter and a polyadenylation signal.
  • Any promoter can be used that is a polynucleotide sequence recognized by the transcriptional machinery of the host cell (or introduced synthetic machinery) that is involved in the initiation of transcription.
  • a polyadenylation signal is a polynucleotide sequence that directs the addition of a series of nucleotides on the end of the mRNA transcript for proper processing and trafficking of the transcript out of the nucleus into the cytoplasm for translation.
  • exemplary promoters include viral promoters, such as cytomegalovirus immediate early gene promoter (“CMV”), herpes simplex virus thymidine kinase (“tk”), SV40 early transcription unit, polyoma, retroviruses, papilloma virus, hepatitis B virus, and human and simian immunodeficiency viruses.
  • CMV cytomegalovirus immediate early gene promoter
  • tk herpes simplex virus thymidine kinase
  • SV40 early transcription unit polyoma
  • retroviruses papilloma virus
  • hepatitis B virus hepatitis B virus
  • promoters include promoters isolated from mammalian genes, such as the immunoglobulin heavy chain, immunoglobulin light chain, T cell receptor, HLA DQ ⁇ and DQ ⁇ , ⁇ -interferon, interleukin-2, interleukin-2 receptor, MHC class II, HLA-DR ⁇ , ⁇ -actin, muscle creatine kinase, prealbumin (transthyretin), elastase I, metallothionein, collagenase, albumin, fetoprotein, ⁇ -globin, c-fos, c-HA-ras, neural cell adhesion molecule (NCAM), ⁇ 1-antitrypsin, H2B (TH2B) histone, type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TNI), platelet-derived growth factor, and dystrophin, as well as promoters specific for retinal pigment epithelial
  • the promoter can be either inducible or constitutive.
  • An inducible promoter is a promoter that is inactive or exhibits low activity except in the presence of an inducer substance.
  • promoters include, but are not limited to, MT II, MMTV, collagenase, stromelysin, SV40, murine MX gene, ⁇ -2-macroglobulin, MHC class I gene h-2kb, HSP70, proliferin, tetracycline inducible, tumor necrosis factor, or thyroid stimulating hormone gene promoter.
  • an inducible promoter is the interferon inducible ISG54 promoter.
  • the promoter is a constitutive promoter that results in high levels of transcription upon introduction into a host cell in the absence of additional factors.
  • the constitutive promoter is a human ⁇ -actin, human elongation factor-1 ⁇ , chicken ⁇ -actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, or a herpes simplex virus thymidine kinase promoter (see Damdindorj et al., PLOS One 9(8): e106472, 2014).
  • the promoter can be a human ⁇ -actin promoter, human elongation factor-1 ⁇ promoter, ⁇ -actin promoter, simian virus 40 promoter, or a herpes simplex virus thymidine kinase promoter.
  • transcription control include one or more enhancer elements, which are binding recognition sites for one or more factors that increase transcription above that observed for the minimal promoter alone, and also be operably linked to the polynucleotide encoding the nucleic acid molecule encoding ANG, a precursor, variant, or fragment, or encoding a tRNA fragment.
  • Introns can also be included that help stabilize mRNA and increase expression.
  • a polyadenylation signal can be included to effect proper termination and polyadenylation of the transcript. Exemplary polyadenylation signals have been isolated from beta globin, bovine growth hormone, SV40, and the herpes simplex virus thymidine kinase genes.
  • a nucleic acid molecule encoding ANG, a precursor, variant, or fragment thereof, or encoding a tRNA fragment can be included in a viral vector, for example for expression of the protomer to produce the corresponding protein, variant or fragment thereof, or encoding a tRNA fragment in a host cell, or for administration to a subject as disclosed herein.
  • viral vectors include a nucleic acid molecule encoding ANG, a precursor, variant or fragment thereof, or encoding a tRNA fragment.
  • the viral vector encoding ANG, a precursor, variant, or fragment, or encoding a tRNA fragment can be replication-competent.
  • the viral vector can have a mutation (e.g., insertion of nucleic acid encoding the protomer) in the viral genome that attenuates, but does not completely block viral replication in host cells.
  • Various viral vectors which can be utilized for nucleic acid based therapy as taught herein include adenovirus or adeno-associated virus (AAV), herpes virus, vaccinia, or an RNA virus such as a retrovirus (including HVJ, see Kotani et al., Curr. Gene Ther.4:183-194, 2004).
  • the retroviral vector is a derivative of a murine or avian retrovirus, or a human or primate lentivirus.
  • retroviral vectors in which a foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • a vector such as the gibbon ape leukemia virus (GaLV) can be utilized.
  • GaLV gibbon ape leukemia virus
  • a pseudotyped retroviral vector can be utilized that includes a heterologous envelope gene.
  • the viral vector is AAV.
  • retroviral vectors can incorporate multiple genes. These vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • Retroviral vectors can be made target specific by modifications of the envelope protein by attaching, for example, a sugar, a glycolipid, or a protein. In one specific, non-limiting example, targeting is accomplished by using an antibody to target the retroviral vector. Since recombinant retroviruses are non-replicating by design, they require assistance in order to produce infectious vector particles.
  • helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the long terminal repeat (LTR). These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an transcript for encapsidation.
  • Helper cell lines which have deletions of the packaging signal include, but are not limited to ⁇ 2, PA317, and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
  • NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional transfection methods. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • the adenovirus vectors include replication competent, replication deficient, gutless forms thereof. Defective viruses, such as adenovirus vectors or adeno-associated virus (AAV) vectors, that entirely or almost entirely lack viral genes, can be used. Use of defective viral vectors allows for administration to specific cells without concern that the vector can infect other cells.
  • the AAV vectors of use are replication deficient.
  • a vector of use is an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest., 90:626-6301992; La Salle et al., Science 259:988-990, 1993); or a defective AAV vector (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).
  • Recombinant AAV vectorsey are capable of directing the expression and the production of the selected transgenic products in targeted cells.
  • the recombinant vectors can include at least all of the sequences of AAV essential for encapsidation and the physical structures for infection of target cells.
  • 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 sense and antisense strands of AAV DNA are packaged into AAV capsids with equal frequency.
  • the AAV DNA includes a nucleic acid including a promoter operably linked to a nucleic acid molecule encoding ANG, a precursor, variant, or fragment thereof, or encoding a tRNA fragment.
  • recombinant vectors such as recombinant adenovirus vectors and recombinant adeno-associated virus (rAAV) vectors comprising a nucleic acid molecule(s) disclosed herein.
  • the AAV is rAAV8, and/or AAV2.
  • the AAV serotype can be any other suitable AAV serotype, such as 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 No. WO 2014/127196.
  • Gene therapy vectors that utilize AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell.
  • the present disclosure contemplates the use of an rAAV for the methods disclosed herein.
  • AAV including has the ability to bind and enter target cells, enter the nucleus, the ability to be expressed in the nucleus for a prolonged period of time, and low toxicity.
  • AAV can be used to transfect cells, and suitable vector are known i, see for example, U.S. Published Patent Application No. 2014/0037585, incorporated herein by reference.
  • Methods for producing rAAV suitable for gene therapy are known (see, for example, U.S. Published Patent Nos.2012/0100606; 2012/0135515; 2011/0229971; and 2013/0072548; and Ghosh et al., Gene Ther 13(4):321-329, 2006), and can be utilized with the methods disclosed herein.
  • the vector is a rAAV8 vector, a rAAV2 vector, a rAAV9 vector.
  • the vector is an AAV8 vector.
  • AAV8 vectors are disclosed, for example, in U.S. Patent No.8,692,332, which is incorporated by reference herein.
  • the location and sequence 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 location and hypervariable regions of AAV8 are also provided.
  • the vector is an AAV2 variant vector, such as AAV7m8.
  • vectors of use in the methods disclosed herein can contain nucleic acid sequences encoding an intact AAV capsid which may be from a single AAV serotype (e.g., AAV2, AAV6, AAV8 or AAV9).
  • vectors of use can also be recombinant, and thus can contain sequences encoding artificial capsids which contain one or more fragments of the AAV8 capsid fused to heterologous AAV or non-AAV capsid proteins (or fragments thereof).
  • These artificial capsid proteins are selected from non-contiguous portions of the AAV2, AAV6, AAV8 or AAV9 capsid or from capsids of other AAV serotypes.
  • a AAV vector may have a capsid protein comprising one or more of the AAV8 capsid regions selected from the VP2 and/or VP3, or from VP1, or fragments thereof selected from amino acids 1 to 184, amino acids 199 to 259; amino acids 274 to 446; amino acids 603 to 659; amino acids 670 to 706; amino acids 724 to 738 of the AAV8 capsid, which is presented as SEQ ID NO: 2 in U.S. Patent No.8,692,332.
  • the AAV may contain one or more of the AAV serotype 8 capsid protein hypervariable regions, for example aa 185- 198; aa 260-273; aa447-477; aa495-602; aa660-669; and aa707-723 of the AAV8 capsid which is presented as SEQ ID NO: 2 in U.S. Patent No.8,692,332.
  • Additional viral vectors that can be used for expression of ANG, a precursor, variant, or fragment, or encoding a tRNA fragment include polyoma, i.e., SV40 (Madzak et al., 1992, J. Gen.
  • HSV herpes viruses
  • EBV and CMV herpes viruses
  • 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), Sindbis viruses (H. Herweijer et al., 1995, Human Gene Therapy 6:1161-1167; U.S. Pat. Nos.
  • Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • One colloidal dispersion system is a liposome.
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from about 0.2-4 microns, can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules.
  • LUV large unilamellar vesicles
  • RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et al., Trends Biochem. Sci.6:77, 1981).
  • liposomes In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells.
  • a liposome In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the nucleic acid of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino et al., Biotechniques 6:682, 1988).
  • the composition of the liposome is usually a combination of phospholipids, particularly high-phase- transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
  • Particularly useful are diacylphosphatidyl-glycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
  • Illustrative phospholipids include, for example, phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
  • the targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) in organs which contain sinusoidal capillaries.
  • RES reticuloendothelial system
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein
  • Another targeting delivery system is the use of biodegradable and biocompatible polymer scaffolds (see Jang et al., Expert Rev. Medical Devices 1:127-138, 2004) for use in the bone.
  • These scaffolds usually contain a mixtures of one or more biodegradable polymers, for example and without limitation, saturated aliphatic polyesters, such as poly(lactic acid) (glycolic acid), or poly(lactic-co-glycolide) (PLGA) copolymers, unsaturated linear as polypropylene fumarate (PPF), or microorganism produced aliphatic polyesters, such as polyhydroxyalkanoates (PHA), (see Rezwan et al., Biomaterials 27:3413-3431, 2006; Laurencin et al., Clin. Orthopaed. Rel. Res.447:221-236).
  • PHA polyhydroxyalkanoates
  • An exemplary scaffold contains a ratio of PLA to PGA is 75:25, but this ratio may change depending upon the specific application.
  • Other exemplary scaffolds include surface bioeroding polymers, such as poly(anhydrides), such as trimellitylimidoglycine (TMA-gly) or pyromellitylimidoalanine (PMA-ala), or poly(phosphazenes), such as high molecular weight poly(organophasphazenes) (P[PHOS]), and bioactive ceramics.
  • TMA-gly trimellitylimidoglycine
  • PMA-ala pyromellitylimidoalanine
  • P[PHOS] high molecular weight poly(organophasphazenes)
  • a nanodispersion system includes a biologically active agent and a dispersing agent (such as a polymer, copolymer, or low molecular weight surfactant).
  • Exemplary polymers or copolymers include polyvinylpyrrolidone (PVP), poly(D,L-lactic acid) (PLA), poly(D,L-lactic-co-glycolic acid (PLGA), poly(ethylene glycol).
  • Exemplary low molecular weight surfactants include sodium dodecyl sulfate, hexadecyl pyridinium chloride, polysorbates, sorbitans, poly(oxyethylene) alkyl ethers, poly(oxyethylene) alkyl esters, and combinations thereof.
  • the nanodispersion system includes PVP and ODP or a variant thereof (such as 80/20 w/w).
  • the nanodispersion is prepared using the solvent evaporation method, 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.
  • Dendrimers are synthetic three-dimensional macromolecules that are prepared in a step-wise fashion from simple branched monomer units, the nature and functionality of which can be easily controlled and varied. Dendrimers consist of an initiator core, surrounded by a layer of a selected polymer that is grafted to the core, forming a branched macromolecular complex.
  • Dendrimers are typically produced using polymers such as poly(amidoamine) or poly(L-lysine).
  • a dendrimer can be synthesized from the repeated addition of building blocks to a multifunctional core (divergent approach to synthesis), or towards a multifunctional core (convergent approach to synthesis) and each addition of a three-dimensional shell of building blocks leads to the formation of a higher generation of the dendrimers.
  • Polypropylenimine dendrimers contain 100% protonable nitrogens and up to 64 terminal amino groups. Protonable groups are usually amine groups which are able to accept protons at neutral pH.
  • dendrimers can be formed from polyamidoamine and phosphorous containing compounds with a mixture of amine/ amide or N- P(O2)S as the conjugating units. Dendrimers of use for delivery of nucleic acid molecules is disclosed, for example, in PCT Publication No.2003/033027, incorporated herein by reference.
  • the surface of the targeted delivery be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
  • Various linking groups can be used for joining the lipid chains to the targeting ligand.
  • an mRNA can be used to deliver a nucleic acid encoding ANG, a precursor, variant, or fragment thereof, directly into cells.
  • a tRNA fragment is delivered.
  • nucleic acid-based vaccines based on mRNA may provide a potent alternative to the previously mentioned approaches. mRNA delivery precludes safety concerns about DNA integration into the host genome and can be directly translated in the host cell cytoplasm. Moreover, the simple cell-free, in vitro synthesis of RNA avoids the manufacturing complications associated with viral vectors.
  • RNA Two exemplary forms of RNA that can be used to deliver a nucleic acid include 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 conserveed 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., 89:179
  • CRISR/Cas9 Included are in the present disclosure are methods for site-specific modification of a nucleic acid molecule in a cell to introduce ANG or another molecule that increases ANG activity, such as one or more tRNA fragments. These modifications can include, but are not limited to, site-specific insertions, and replacements of nucleotides (a “knock-in”), such that an increase in ANG activity is produced. These modifications can be made anywhere within the genome, for example, in genomic elements, including, among others, coding sequences, regulatory elements, and non-coding DNA sequences.
  • an insertion is made into a safe harbor locus (see Pavani and Amendola, Front Genome Ed., https://doi.org/10.3389/fgeed.2020.609650, 20 January 2021). Any number of such insertions can be made, in any order or combination.
  • Such methods may be used to modify expression of a gene, such as to increase expression of ANG or a tRNA fragment. These modifications include a “knock-in” of a nucleic acid molecule encoding ANG and one or more tRNA fragments.
  • Techniques for making such modifications by genome editing include, for example, use of CRISPR-Cas systems, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), among others. These modifications can be used to introduce (knock-in) a nucleic acid molecule encoding ANG, a precursor, variant, or fragment into a safe harbor locus in a genome. These modifications can be used to introduce (knock-in) a nucleic acid molecule encoding a tRNA fragment into a safe harbor locus in the genome. Cas9 mediated integration of genes is disclosed, for example, in Li et al., G3 10(2):467-473.
  • a typical set of CRISPR system is two components, a CRISPR-associated nuclease 9 (Cas9) and one or more guide RNAs (gRNAs), each of which contains a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA).
  • Cas9 CRISPR-associated nuclease 9
  • gRNAs guide RNAs
  • crRNA CRISPR RNA
  • tracrRNA trans-activating CRISPR RNA
  • Simple gene disruptions can be generated by cleavage of the target site, followed by alteration of nucleic acids, such as a deletion, and repair by the non-homologous- end-joining pathway (NHEJ).
  • Target recognition by crRNAs occurs through complementary base pairing with target DNA, which directs cleavage of foreign sequences by means of Cas proteins.
  • DNA recognition by guide RNA and consequent cleavage by the endonuclease requires complementary base-pairing with a protospacer adjacent motif (PAM) (e.g., 5’-NGG-3’) and with a protospacer region in the target.
  • PAM protospacer adjacent motif
  • the PAM motif recognized by a Cas9 varies for different Cas9 proteins.
  • any Cas9 protein can be used in the systems and methods disclosed herein, including mutant Cas9 proteins (such as those having an R691A, D10A, H840A, or combination of such substitutions).
  • a promoter is operably linked to the nucleic acid encoding Cas9.
  • a retinal pigment epithelial cell promoter is utilized, such as, but not limited to, RPE65, BEST1, DCT, TYR, or a TYRP1 promoter.
  • the Cas9 RNA guide system includes a mature crRNA that is base-paired to trans- activating crRNA (tracrRNA), forming a two-RNA structure that directs Cas9 to the locus of a desired double-stranded (ds) break in target DNA.
  • tracrRNA trans- activating crRNA
  • base-paired tracrRNA:crRNA combination is engineered as a single RNA chimera to produce a guide sequence (e.g., gRNA) which preserves the ability to direct sequence-specific Cas9 dsDNA cleavage.
  • the Cas9-guide sequence complex results in cleavage of one or both strands at a target sequence within a safe harbor locus, which allows a knock in.
  • the Cas9 endonuclease and the gRNA molecules are used sequence-specific target recognition, cleavage, and genome editing of the safe harbor locus.
  • the cleavage site is at a specific nucleotide, such as, but not limited to the 16, 17, or 18 th nucleotide (nt) of a 20-nt target.
  • the cleavage site is at the 17 th nucleotide of a 20-nt target sequence.
  • the cleavage can be a double stranded cleavage.
  • the gRNA molecule is selected so that the target genomic targets bear a protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • DNA recognition by guide RNA and consequent cleavage by the endonuclease requires the presence of a protospacer adjacent motif (PAM) (e.g., 5’-NGG-3’) in immediately after the target.
  • PAM protospacer adjacent motif
  • the PAM is present in the targeted nucleic acid sequence but not in the crRNA that is produced to target it.
  • the proto-spacer adjacent motif (PAM) corresponds to 2 to 5 nucleotides starting immediately or in the vicinity of the proto-spacer at the leader distal end.
  • the PAM motif also can be NNAGAA, NAG, NGGNG, AWG, CC, CC, CCN, TCN, or TTC.
  • cleavage occurs at a site about 3 base-pairs upstream from the PAM.
  • the Cas9 nuclease cleaves a double stranded nucleic acid sequence.
  • the guide sequence is selected to reduce the degree of secondary structure within the sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold (Zuker and Stiegler, Nucleic Acids , 133-148).
  • RNAfold Another example folding algorithm is the online webserver RNAfold, which uses the centroid 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 and 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.
  • the crRNA can be 18-48 nucleotides in length.
  • the crRNA can be 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In one example, the crRNA is 20 nucleotides in length.
  • the system disclosed herein introduces double stranded DNA breaks, such that the target, e.g., the safe harbor locus, is cleaved by Cas9. This allows insertion of a nucleic acid sequence encoding ANG, a precursor, variant, or fragment, and/or encoding a tRNA fragment.
  • more than one DNA break can be introduced by using more than one gRNA.
  • two gRNAs can be utilized, such that two breaks are achieved.
  • the two or more cleavage events may be made by the same or different Cas9 proteins.
  • a single Cas9 nuclease may be used to create both double strand breaks.
  • the disclosed methods include the use of one or more vectors comprising: a) a retinal specific promoter operably linked to a nucleotide sequence encoding a Type II Cas9 nuclease, b) a promoter, such as a U6 promoter, operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas guide RNAs that hybridize with a safe harbor locus in a target cell, such as a human cell; and c) a nucleic acid molecule encoding ANG, a precursor, variant, or fragment, and/or encoding a tRNA fragment that is introduced into the target cell.
  • the one or more guide RNAs target the noncoding region, such that the Cas9 protein cleaves the DNA and the nucleic acid molecule encoding ANG, a precursor, variant, or fragment, or encoding a tRNA fragment, is introduced.
  • the one or more vectors are viral vectors such as lentiviral vectors.
  • the viral vectors are adenovirus vectors, adeno-associated virus vectors, or retroviral vectors.
  • Cas9 and gRNAs can be delivered to the using AAV, a lentivirus, piggybac, an episomal constructs, or injected as purified nanoparticles constituted by pure Cas9 protein and pure guides RNAs (see, for example, Steyer et al., Drug Discov Today Technol 28: 3-12, 2018).
  • Chemical Compounds Agents 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.
  • Agents that increase ANG activity include molecules that are identified from large libraries of both natural product or (or semi-synthetic) extracts or chemical libraries. Screening methods that detect increases in ANG activity are useful for identifying compounds from a variety of sources for activity. The initial screens may be performed using a diverse library of compounds, a variety of other compounds and compound libraries. Thus, molecules that increase the activity of ANG 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 commercial sources, as well as commercially available analogs of identified agonists.
  • test extracts or compounds are not critical to the identification of agents that increase ANG activity. Accordingly, 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 modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds.
  • Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.).
  • ANG protein agonists can be identified from synthetic compound libraries that are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N. J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.).
  • La protein agonists can be identified from a rare chemical library, such as the library that is available from Aldrich (Milwaukee, Wis.).
  • La protein agonists can be identified in libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means. Useful compounds may be found within numerous chemical classes, though typically they are organic compounds, including small organic compounds. Small organic compounds have a molecular weight of more than 50 yet less than about 2,500 daltons, such as less than about 750 or less than about 350 daltons can be utilized in the methods disclosed herein.
  • Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like.
  • the compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like.
  • III. Methods of Treatment and Pharmaceutical Compositions Methods are disclosed herein for treating, or reducing the risk of developing, AMD.
  • the methods inhibit drusen formation.
  • the disclosed agents can administered systemically (for example by peripheral vein infusion or orally) and may be administered locally or regionally (intraocular administration).
  • the agent can be an agent that inhibits HERV-K.
  • the agent can be an agent that increases ANG activity.
  • the subject can be at the early, or advanced stages of AMD. In some examples the subject is a mammalian subject, such as a human or veterinary subject.
  • the subject has wet AMD. In other aspects, the subject has dry AMD. In further aspects, the subject is at risk of developing AMD. In some aspects, the disclosed methods reduce or arrest loss of vision.
  • Anti-retroviral agents can be administered according to approved dosing methods, see above. Chemical compounds that increase ANG activity can be administered to the subject. In some aspects, administration is systemic, such as oral or intravenous. However, other routes of administration are contemplated, such as inhalation, vaginal, rectal and intranasal are of use. In some aspects, an anti-retroviral agent is formulated for local administration, such as to the eye.
  • Therapeutic compounds can be prepared for storage by mixing a polypeptide(s) having the desired degree of purity, such as ANG, a precursor, variant, or fragment thereof, an antibody, or another agent, such as an inhibitory nucleic acid molecule or a CRISPR/Cas13 system, a nucleic acid molecule encoding ANG, a precursor, variant, or fragment thereof, or a CRISPR/Cas9 system, with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]), in the form of lyophilized formulations or aqueous solutions.
  • a polypeptide(s) having the desired degree of purity such as ANG, a precursor, variant, or fragment thereof, an antibody, or another agent, such as an inhibitory nucleic acid molecule or a CRISPR/Cas13 system, a nucleic acid molecule encoding ANG, a precursor, variant, or fragment thereof, or a CRIS
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • the pharmaceutical composition can be formulated for local delivery, such as to the eye.
  • the pharmaceutical composition is formulated as an eye drop.
  • the active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • the formulations to be used for in vivo administration can be sterile.
  • a pharmaceutical composition can be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • the relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition can vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may include 0.1% to 100% (w/w) active ingredient.
  • the vehicles and compositions can include various formulatory ingredients, such as anti-microbial preservatives and tonicity agents.
  • antimicrobial preservatives include: benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, EDTA, sorbic acid, POLYQUAD® or other agents.
  • Such preservatives can be used in an amount from about 0.0001 wt. % to 1.0 wt. %.
  • Suitable agents which may be used to adjust tonicity or osmolality of the compositions include: mannitol, dextrose, glycerine and propylene glycol. If used, such agents will be employed in an amount of about 0.1 wt. % to 10.0 wt. %.
  • the composition does not include preservatives or tonicity agents which adversely affect or irritate the eye.
  • Ophthalmic compositions are provided herein, such as eye drops, that include, in addition to the active ingredient(s), a preservative and optionally a viscosifier.
  • the preservative can be, for example, benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, benzyl alcohol, sodium dehydroacetate, paraoxybenzote esters, sodium edetate, boric acid and the like.
  • the viscosifier can be, for example, hydroxypropyl methyl cellulose (HPMC), or water-soluble cellulose derivatives such as methylcellulose, hydroxyethylcellulose, carboxymethylcellulose and the like.
  • additives include, but are not limited to, e.g., isotonicity agents such as sodium chloride, potassium chloride, glycerol, mannitol, sorbitol, boric acid, glucose, propylene glycol and the like; buffering agents such as phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, tris buffer, glutamic acid, ⁇ -aminocaproic acid and the like; stabilizers such as e.g., sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, dibutylhydroxytoluene and the like, thickeners such as sodium chondroitin sulfate, sodium hyaluronate, corboxyvinyl polymer, polyvinyl alcohol, polyvinylpyrrolidone, macrogol and the like, pH adjusters such as hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, and the like
  • the disclosed eye drops may further include one or more other ingredients or additives, which can be contained in artificial tears, i.e., aminoethylsulfonic acid, sodium chondroitin sulfate, potassium L-aspartate, magnesium L-aspartate, potassium magnesium L-aspartate (equimolar mixture), sodium hydrogen carbonate, sodium carbonate, chloride, calcium chloride, sodium chloride, sodium hydrogen phosphate, sodium dihydrogen potassium dihydrogen phosphate, exsiccated sodium carbonate, magnesium sulfate, polyvinylalcohol, polyvinylpyrrolidone, hydroxyethylcellulose, glucose and methylcellulose.
  • other ingredients or additives which can be contained in artificial tears, i.e., aminoethylsulfonic acid, sodium chondroitin sulfate, potassium L-aspartate, magnesium L-aspartate, potassium magnesium L-aspartate (equimolar mixture), sodium hydrogen carbonate, sodium carbonate, chloride
  • eye drops are provided that include agent can be an agent that inhibits HERV-K and/or an agent that increases ANG activity.
  • the pharmaceutical composition can include a preservative and/or a viscosifier.
  • other ingredients can be included.
  • concentrations of other ingredients or additives contained in a pharmaceutical composition formulated for ocular administration, such as eye drops are generally, but without limitation, not less than 0.01% w/v, not less than 0.1% w/v, not less than 0.5% w/v, and not more than 20% w/v, such not more than 10% w/v, or not more than 7% w/v, or not more than 5% w/v.
  • the ophthalmic compositions may be made as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably as solutions in isotonic, pH adjusted sterile saline.
  • the pH of the compositions provided herein can vary across a range acceptable for the eye, as known to a person of ordinary skill in the art. Preferably, the pH of the formulations is within the range of approximately 4-8.
  • the ophthalmic compositions can be formulated in an ointment such as petrolatum.
  • the ophthalmic formulations include lipophiliccally modified compositions and transplantable carriers.
  • Ophthalmic compositions, formulated for topical ocular administration can be eye drops, such as aqueous eye drops, for example, monophasic aqueous eye drops.
  • Ophthalmic compositions also be formulated and used in the form of a mist, a frost, a foam, a cream, an ointment or an emulsion for direct application to the eye, or used in ocular implants or injectable ocular therapies or else administered to the eyelid or sclera.
  • An ophthalmic composition can also include a compound such as a Nox4 inhibitor, a compound that modulates NF-kB, mTOR, or one or more Rho GTPases such as CDC42 and/or RACI; a compound that modulates AMPK or a compound that regulates RPE epithelial to mesenchymal transition or RPE dedifferentiation, cholesterol pathway modulators, complement inhibitors, epigenetic modifiers, mitochondrial activity inducers, oxidative stress inhibitors, and metabolic stress inhibitors.
  • a compound such as a Nox4 inhibitor, a compound that modulates NF-kB, mTOR, or one or more Rho GTPases such as CDC42 and/or RACI
  • a compound that modulates AMPK or a compound that regulates RPE epithelial to mesenchymal transition or RPE dedifferentiation cholesterol pathway modulators, complement inhibitors, epigenetic modifiers, mitochondrial activity inducers, oxidative stress inhibitors, and metabolic stress inhibitors
  • Examples of such compounds include, but are not limited to, aminocapropic acid, L-701,324, Vas2870, L-745,870 hydrochloride, Me3,4-dephostatin, 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, Telenzepine dihydrochloride, NO-711 hydrochloride, U-99194A maleate, S(+)-Rac10pride L-tartrate, Pirenzepine dihydrochloride, Captopril, thioperamide maleate, alpre
  • compositions and methods can be used in combination with a compound, or a pharmaceutically acceptable salt thereof, which inhibits Nox4 or reactive oxygen species formation, or modulates serine protease, a dopamine receptor, NF-kB, mTOR, AMPK, RPE epithelial to mesenchymal transition, RPE dedifferentiation, or one or more Rho GTPases.
  • the compound is a Nox4 inhibitor or an inhibitor of reactive oxygen species formation.
  • the compound modulates NF-kB, mTOR, or one or more Rho GTPases.
  • the compound modulates one or more Rho GTPases, wherein the Rho GTPase is CDC42 and/or RACl.
  • the compound inhibits serine protease.
  • the compound regulates AMPK.
  • the compound modulates a dopamine receptor.
  • the compound can be a dopamine receptor D4 antagonist.
  • the compound is the compound is Aminocapropic acid, L-701,324, Vas2870, L-745,870 hydrochloride, Me-3,4-dephostatin, N-Methyl-l-deoxynojirimycin, L- 750,667 trihydrochloride, (+)-MK-801 hydrogen maleate, Pempidine 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, Telenzepine dihydrochloride, NO-711 hydrochloride, U- 99194A maleate, S(+)-Raclopride L-tartrate, Pirenzepine dihydrochloride, Captopril, Thioperamide maleate,
  • the compound is Aminocaproic Acid; Vas2870, L-745,870; Riluzole; Acadenisine; Metformin or a pharmaceutically acceptable salt thereof.
  • Suitable compounds are disclosed, for example, in PCT Publication No. WO2021/050980 A1, incorporated herein by reference.
  • Dosage treatment may be a single dose schedule or a multiple dose schedule to ultimately deliver the amount specified above.
  • the doses can be intermittent.
  • the subject may be administered as many doses as appropriate.
  • Individual doses are typically not less than an amount required to produce a measurable effect on the subject, and may be determined based on the pharmacokinetics and pharmacology for absorption, distribution, metabolism, and excretion ("ADME") of the subject composition or its by- products, and thus based on the disposition of the composition within the subject. This includes consideration of the route of administration as well as dosage amount, which can be adjusted for local applications. Effective amounts of dose and/or dose regimen can readily be determined empirically from preclinical assays, from safety and escalation and dose range trials, individual clinician-patient relationships, as well as in vitro and in vivo assays. Administration may be provided as a a periodic bolus or as continuous infusion (for example, from an implant disposed at an location.
  • Intraocular injection of the nucleic acid molecules, antibodies, and CRISPR/Cas13 systems disclosed herein can be performed once, or can be performed repeatedly, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more times. Administration can be performed biweekly, weekly, every other week, monthly, or every 2, 3, 4, 5, or 6 months. Local modes of administration include intraocular routes, as discussed below. In an aspect, significantly smaller amounts (compared with systemic approaches) may exert an effect when administered locally (for example, intraocularly) compared to when administered systemically (for example, intravenously). Local modes of administration can reduce or eliminate the incidence of potential side effects.
  • the method includes administraton of an agent, such as, but not lmited to, an anti- retroviral agent, inhibitory RNA, or an agent that increases ANG activity, to the eye.
  • an agent such as, but not lmited to, an anti- retroviral agent, inhibitory RNA, or an agent that increases ANG activity
  • the amount of the ophthalmic formulation to be administered to the eye can depend on the individual to be subjected to the treatment, and is preferably an amount optimized to achieve the desired treatment without accompanying marked side effects.
  • Dosage can include administration to the eye, such as, but not limited to, in the form of an eye drop. These drops can be administered, for example, 1, 2, 3, 4, 5, 6, or more times a day (such as every hour, every 2 hours, every 4 hours, every 6 hours, every 12 hours, or every 24 hours).
  • Intraocular administration can be by subretinal, direct retinal, suprachoroidal or intravitreal injection.
  • the volume of the medicament composition injected may, for example, be about 10-500 ⁇ L, for example about 50-500, 100-500, 200-500, 300-500, 400-500, 50-250, 100-250, 200-250 or 50-150 ⁇ L.
  • the volume may, for example, be about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 ⁇ L.
  • the volume of the pharmaceutical composition injected is 100 ⁇ L.
  • Subretinal injections are injections into the subretinal space, i.e., underneath the neurosensory retina.
  • the injected material is directed into, and creates a space between, the photoreceptor cell and retinal pigment epithelial (RPE) layers.
  • RPE retinal pigment epithelial
  • a retinal detachment may be created.
  • the detached, raised layer of the retina that is generated by the injected material is referred to as a “bleb.”
  • the hole created by the subretinal injection can be sufficiently small that the injected solution does not significantly reflux back into the vitreous cavity after administration. Such reflux can be particularly problematic when a medicament is injected, if effects of the medicament are directed away from the target zone.
  • the injection creates a self-sealing entry point in the neurosensory retina, i.e. once the injection needle is removed, the hole created by the needle reseals such that very little or substantially no injected material is released through the hole.
  • Subretinal injection needles are available (e.g., DORC 41 G Teflon subretinal injection needle, Dutch Ophthalmic Research BV, Zuidland, The Netherlands). Unless damage to the retina occurs during the injection, and as long as a sufficiently small needle is used, substantially all injected material remains localized between the detached neurosensory retina and the RPE at the site of the localized retinal detachment (i.e., does not reflux into the vitreous cavity).
  • the typical persistence of the bleb over a short time frame indicates that there is usually little escape of the injected material into the vitreous.
  • the bleb may dissipate over a longer time frame as the injected material is absorbed.
  • Visualizations of the eye, in particular the retina for example using optical coherence tomography can be performed. Under certain circumstances, for example during end-stage retinal degenerations, identifying the retina is difficult because it is thin, transparent and difficult to see against the disrupted and heavily pigmented epithelium on which it sits.
  • a blue vital dye e.g., BRILLIANT PEEL®, Geuder; MEMBRANEBLUE-DUAL®, Dore
  • BRILLIANT PEEL® Geuder
  • MEMBRANEBLUE-DUAL® Dore
  • the use of the blue vital dye also identifies any regions of the retina where there is a thickened internal limiting membrane or epiretinal membrane, as injection through either of these structures would hinder clean access into the subretinal space.
  • Suprachoridal injection can be utilized.
  • the agent be delivered to the suprachoroidal space using an ab extemo approach that utilizes an microcatheter (see, for example, Peden et al. (2011) PLoS One 6(2): e17140).
  • a limbal conjunctival peritomy is performed to expose bare sclera, followed by sclerotomy to expose bare choroid.
  • a microcatheter such as the iTrack 250A from iScience Interventional, optionally connected to an illumination system such as the iLumin laser-diode based micro- illumination system (iScience Interventional) is introduced into the suprachoroidal space and advanced posteriorly towards the optic disc. Following manipulation of the microcatheter tip into the desired position, injection of the product, polynucleotide or vector forms a bleb within the retina and choroid.
  • a therapeutically effective amount of an agent disclosed herein is administered by intraocular, for example intravitreal, injection.
  • a general method for intravitreal injection may be illustrated by the following brief outline. This example is merely meant to illustrate certain features of the method, and is in no way meant to be limiting.
  • 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.
  • a subject for intravitreal injection may be prepared for the procedure by pupillary dilation, sterilization of the eye, and administration of anesthetic. Any suitable mydriatic agent known in the art may be used for pupillary dilation. Adequate pupillary dilation may be confirmed before treatment.
  • Sterilization may be achieved by applying a sterilizing eye treatment, e.g., an iodide-containing solution such as povidone-iodine (BETADINE®).
  • a sterilizing eye treatment e.g., an iodide-containing solution such as povidone-iodine (BETADINE®).
  • BETADINE® povidone-iodine
  • a similar solution may also be used to clean the eyelid, eyelashes, and any other nearby tissues (e.g., skin).
  • Any suitable anesthetic may be used, such as lidocaine or proparacaine, at any suitable concentration.
  • Anesthetic may be administered by any method known in the art, including without limitation topical drops, gels or jellies, and subconjuctival application of anesthetic.
  • a sterilized eyelid speculum may be used to clear the eyelashes from the area. The site of the injection may be marked with a syringe.
  • the site of the injection may be chosen based on the lens of the patient.
  • the injection site may be 3-3.5 mm from the limus in pseudophakic or aphakic patients, and 3.5-4 mm from the limbus in phakic patients.
  • the patient may look in a direction opposite the injection site.
  • the needle can be inserted perpendicular to the sclera and pointed to the center of the eye.
  • the needle can be inserted such that the tip ends in the vitreous, rather than the subretinal space. Any suitable volume known in the art for injection may be used.
  • the eye can be treated with a sterilizing agent such as an antibiotic.
  • the eye can also be rinsed to remove excess sterilizing agent.
  • Intravitreal injection of an agent as disclosed herein can be performed once, or can be performed repeatedly, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. Administration can be performed biweekly, weekly, every other week, monthly, or every 2, 3, 4, 5, or 6 months.
  • the method can include administering a therapeutically effective amount of an agent to inhibit unwanted angiogenesis, for example, to counteract the choroidal new vessel (CNV) growth under the macula in AMD patients.
  • An exemplary therapeutic agent can reduce activity of vascular endothelial growth factor (VEGF), for example, by binding to the receptor site of active forms of VEGF and preventing interaction of VEGF with its receptors.
  • VEGF vascular endothelial growth factor
  • Other drugs can prevent atrophy of RPE cells by targeting complement pathway, autophagy, or NF-kB pathways.
  • Treatments of use for AMD include medications directed to stopping the growth of new blood vessels, such as bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), and aflibercept (EYLEA®); photodynamic therapy; photocoagulation; and low vision rehabilitation
  • the method includes administering to the 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, for example, to promote development or function of neurons such as photoreceptor cells.
  • CNTF Ciliary Neurotrophic Factor
  • BDNF Brain-Derived Neurotrophic Factor
  • PEDF Pigment Epithelial Derived Factor
  • exemplary, non-limiting aspects include administering to the subject a therapeutically effective amount of thrombospondin 1, an anti-inflammatory cytokine (for example, interleukin (IL)-lra, IL-6, Fas ligand or tumor factor (TGF)-beta, a neurotrophic/neuroprotective growth factor such as, but not limited to, glial cell derived growth factor, brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3, - 4/5, -6, and vitamin E.
  • IL interleukin
  • TGF tumor factor
  • a neurotrophic/neuroprotective growth factor such as, but not limited to, glial cell derived growth factor, brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3, - 4/5, -6, and vitamin E.
  • Such agents may be provided singly or in combination.
  • Implants are also of use in the methods disclosed herein.
  • the implants can be inserted into the eye by a variety of methods, including placement by forceps or by trocar following making an incision in the sclera (for example, a 2-3 mm incision) or other suitable sites.
  • the implant can be placed by trocar without making a separate incision, but instead by forming a hole directly into the eye with the trocar.
  • the method of placement can influence the release kinetics. For example, implanting the device into the vitreous or the posterior chamber with a trocar may result in placement of the device deeper within the vitreous than placement by forceps, which may result in the implant being closer to the edge of the vitreous.
  • an implant is formulated with a bioerodible polymer matrix.
  • the therapeutic agent is homogeneously distributed through the polymeric matrix, such that it is distributed evenly enough that no detrimental fluctuations in rate of release occur because of uneven distribution of the immunosuppressive agent in the polymer matrix.
  • the selection of the polymeric composition to be employed varies with the desired release kinetics, the location of the implant, patient tolerance, and the nature of the implant procedure.
  • the polymer can 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 aspect, the implant comprises more than one polymer. These factors are described in detail in U.S. Patent No.6,699,493. Characteristics of the polymers generally include biodegradability at the site of implantation, compatibility with the agent of interest, ease of encapsulation, and water insolubility, amongst others. Generally, the polymeric matrix is not fully degraded until the drug load has been released.
  • Topical administration to the eye is also of use with the disclosed methods.
  • Topical preparations can include eye drops, ointments, sprays and the like. Eye drops or sprays can be provided in unit dose dispensers (such as eye drop bottles that dispense a metered unit dose. These can include, for example, wetting agents and an inert matrix.
  • liposomes may be prepared from dipalmitoyl phosphatidylcholine (DPPC), such as egg phosphatidylcholine (PC).
  • DPPC dipalmitoyl phosphatidylcholine
  • PC egg phosphatidylcholine
  • Liposomes can be applied topically, either in the form of drops or as an aqueous based cream, or can be injected intraocularly.
  • the active agent is slowly released over time as the liposome capsule degrades due to wear and tear from the eye surface.
  • the liposome capsule degrades due to cellular digestion. Both of these formulations provide advantages of a slow release drug delivery system, allowing the subject to be exposed to a substantially constant concentration of the active agent over time.
  • the active agent can be dissolved in an organic solvent such as DMSO or alcohol as previously described and contain a polyanhydride, poly(glycolic) acid, poly(lactic) acid, or polycaprolactone polymer.
  • compositions including Nucleic Acid Molecules can be formulated and administered in a variety of ways (see, e.g., U.S. Published Application No.2005/0054567, which discloses pharmaceutical compositions as well as administration of such compositions and is incorporated herein by reference).
  • the pharmaceutical compositions can include a nanoparticle or dendrimer. These pharmaceutical compositions are of use in the methods disclosed herein.
  • Pharmaceutical compositions including a nucleic acid molecule are provided that are formulated for local delivery to the eye. These include nucleic acid molecules encoding antibodies and antigen binding fragments thereof, inhibitory RNA molecules, Cas13 proteins, and gRNAs.
  • the nucleic acid molecule can be administered in vivo to the subject, such as, but not limited to, oral, intravenous, or intraoclar (such as instravitreous) administration.
  • methods for making a pharmaceutical composition containing the nucleic acid molecules, or vectors described above, are included herein.
  • preparation of a pharmaceutical composition entails preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurities that could be harmful to humans or animals.
  • the pharmaceutical composition contains appropriate salts and buffers to render the composition stable and allow for uptake of nucleic acids or virus by target cells.
  • compositions including nucleic acid molecules can be formulated for injection, such as for introcular or intravenous administration. Such compositions are formulated generally by mixing a disclosed nucleic acid molecule at the desired degree of purity in a unit dosage injectable form (solution, suspension, or emulsion) with a pharmaceutically acceptable carrier, for example, one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • a pharmaceutically acceptable carrier for example, one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • Pharmaceutical compositions can include an effective amount of the nucleic acid molecule dispersed (for example, 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 E. W.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.
  • a disclosed nucleic acid molecule can be suspended in an aqueous carrier, for example, in an isotonic or hypotonic buffer solution at a pH of about 3.0 to about 8.5, such as about 4.0 to about 8.0, about 6.5 to about 8.5, or about 7.4.
  • Useful buffers buffered phosphate or an ionic boric acid buffer can also be in the form of a lyophilisate and can be made into a solution prior to administration by the addition of suitable solvents.
  • the pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well-known in the art.
  • compositions can include the vectors or viruses in water, mixed with a suitable surfactant, such as hydroxy-propylcellulose.
  • Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • the excipients confer a protective effect to a virus including the nucleic acid molecules, such as AAV virion or lentivirus virion, such that loss of AAV virions or lentivirus virions, as well as transduceability resulting from formulation procedures, packaging, storage, transport, and the like, is minimized.
  • These excipient compositions are therefore considered "virion-stabilizing" in the sense that they provide higher virion titers and higher transduceability levels than their non-protected counterparts, as measured using standard assays, see, for example, Published U.S.
  • excipients that can used to protect a virion from activity degradative 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, such as but not limited to polyethylene glycols (PEG) of varying molecular weights, such as PEG-200, PEG-400, PEG-600, PEG-1000, PEG-1450, PEG-3350, PEG-6000, PEG-8000 and any molecular weights in between these values, with molecular weights of 1500 to 6000 preferred, propylene glycols (PG), sugar alcohols, such as a carbohydrate, preferably, sorbitol.
  • PEG polyethylene glycols
  • PG propylene glycols
  • sugar alcohols such as a carbohydrate, preferably, sorbitol.
  • the detergent when present, can be an anionic, a cationic, a zwitterionic or a nonionic detergent.
  • An exemplary detergent is a nonionic detergent.
  • One suitable type of nonionic detergent is a sorbitan ester, e.g., polyoxyethylenesorbitan monolaurate (TWEEN®-20) polyoxyethylenesorbitan monopalmitate (TWEEN®- 40), polyoxyethylenesorbitan monostearate (TWEEN®-60), polyoxyethylenesorbitan tristearate (TWEEN®- 65), polyoxyethylenesorbitan monooleate (TWEEN®-80), polyoxyethylenesorbitan trioleate (TWEEN®- 85), such as TWEEN®-20 and/or TWEEN®-80.
  • a sorbitan ester e.g., polyoxyethylenesorbitan monolaurate (TWEEN®-20) polyoxyethylenesorbitan monopalmitate (TWEEN®- 40), polyoxyethylenesorbitan monostearate
  • excipients are commercially available from a number of vendors, such as Sigma, St. Louis, Mo.
  • the amount of the various excipients in any of the disclosed compositions including virus, such as AAV or a lentivirus, varies and is readily determined by one of skill in the art.
  • a protein excipient such as BSA, if present, will can be present at a concentration of between 1.0 weight (wt.) % to about 20 wt. %, such as 10 wt. %.
  • an amino acid such as glycine is used in the formulations, it can be present at a concentration of about 1 wt. % to wt. %.
  • a carbohydrate, such as sorbitol, if present, can be present at a concentration of about 0.1 wt % to about 10 wt. %, such as between about 0.5 wt. % to about 15 wt. %, or about 1 wt. % to about 5 wt. %.
  • polyethylene glycol it can generally be present on the order of about 2 wt. % to about 40 wt. %, such as about 10 wt. % top about 25 wt. %.
  • propylene glycol is used in the subject formulations, it will typically be present at a concentration of about 2 wt. % to about 60 wt. %, such as about 5 wt.
  • an aqueous virion-stabilizing formulation comprises a carbohydrate, such as sorbitol, at a concentration of between 0.1 wt. % to about 10 wt. %, such as between about 1 wt. % to about 5 wt.
  • Virions are generally present in the composition in an amount sufficient to provide a therapeutic effect when given in one or more doses, as defined above.
  • a compositon can be formulated in unit dosage form, suitable for individual administration of precise dosages. The amount of active compound(s) administered will depend on the subject being treated, the severity of the affliction, and the manner of administration and is best left to the judgment of the prescribing clinician.
  • the formulation to be administered will contain a quantity of the active compound(s) in amounts effective to achieve the desired effect in the subject being treated.
  • Nucleic acid molecules can be incporated into an inert matrix.
  • liposomes may be prepared from dipalmitoyl phosphatidylcholine (DPPC), such as egg phosphatidylcholine (PC). Liposomes, including cationic and anionic liposomes, can be prepared and used in the persent methods. In a formulation for intrahepatic injection, the liposome capsule degrades due to cellular digestion.
  • DPPC dipalmitoyl phosphatidylcholine
  • PC egg phosphatidylcholine
  • nucleic acid molecule can be dissolved in an organic solvent, such as DMSO or alcohol, as previously described, and contain a polyanhydride, poly(glycolic) acid, poly(lactic) acid, or polycaprolactone polymer.
  • the nucleic acid molecule may be formulated to permit release over a specific period of time.
  • a release system can include a matrix of a biodegradable material or a material which releases the incorporated nucleic acid molecule by diffusion.
  • the nucleic acid molecule can be homogeneously or heterogeneously distributed within the release system.
  • release systems may be useful; however, the choice of the appropriate system will depend upon rate of release required by a particular application. Both non- degradable and degradable release systems can be used. Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents such as, but not limited to, calcium carbonate and sugar (for trehalose). Release systems may be natural or synthetic. However, synthetic release systems are because generally they are more reliable, more reproducible and produce more defined release profiles. The release system material can be selected so that active ingredients having different molecular weights are released by diffusion through or degradation of the material.
  • Representative synthetic, biodegradable polymers include, for example: polyamides such as poly(amino acids) and poly(peptides); polyesters such as poly(lactic acid), poly(glycolic acid), poly(lactic- co-glycolic acid), and poly(caprolactone); poly(anhydrides); polyorthoesters; polycarbonates; and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof.
  • polyamides such as poly(amino acids) and poly(peptides)
  • polyesters such as poly(lactic acid), poly(glycolic acid), poly(lactic- co-glycolic acid), and poly(caprolactone)
  • poly(anhydrides) polyorthoesters
  • polycarbonates and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylation
  • Representative synthetic, non-degradable polymers include, for example: polyethers such as poly(ethylene oxide), poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymers-polyacrylates and polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and methacrylic acids, and others such as poly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate); poly(urethanes); cellulose and its derivatives such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various cellulose acetates; polysiloxanes; and any chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers, and mixtures thereof.
  • polyethers such as poly(ethylene oxide), poly(ethylene glycol), and poly
  • Poly(lactide-co-glycolide) microspheres can also be used for intrahepatic injection.
  • the microspheres are composed of a polymer of lactic acid and glycolic acid, which are structured to form hollow spheres.
  • the spheres can be approximately 15-30 microns in diameter and can be loaded with the biologicla molecules described herein.
  • An implant can be uised, that can be inserted into the eye by a variety of methods, which can influence the release kinetics.
  • the location of the implanted device may influence the concentration gradients of the nucleic acid molecule surrounding the device and, thus, influence the release rates.
  • the nucleic acid molecule is homogeneously distributed through the polymeric matrix, such that it is distributed evenly enough that no detrimental fluctuations in rate of release occur due to uneven distribution in the polymer matrix.
  • the selection of the polymeric composition to be employed varies with the desired release kinetics, the location of the implant, patient tolerance, and the nature of the implant procedure.
  • the polymer can 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 aspect, the implant comprises more than one polymer.
  • Characteristics of the polymers can include biodegradability at the site of implantation, compatibility with the agent of interest, ease of encapsulation, and water insolubility, among others. Generally, the polymeric matrix is not fully degraded until the drug load has been released.
  • the chemical composition of suitable polymers is known in the art (for example, see U.S. Patent No.6,699,493).
  • the nucleic acid molecule can be formulated in an implantable form with other carriers and solvents.
  • buffering agents and preservatives can be The implant sizes and shape can also be varied for use in particular regions of the liver Patent No.5,869,079).
  • a nanoparticle or dendrimer is used.
  • Nucleic acid molecules can be delivered by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, nanoparticle mediated delivery (such as lipid or polymeric nanoparticle mediate delivery), dendrimer mediated delivery, as a conjugate to GalNAc, as an mRNA modified by base linker sugars, in association with a degradable polymer, as an mRNA-Lipoplex, as mRNA cargo of PEG-10, or other methods known in the art.
  • the mRNA An appropriate dose depends on the subject being treated (e.g., human or nonhuman primate or other mammal), age and general condition of the subject to be treated, the severity of the condition being treated, the mode of administration, among other factors.
  • a “therapeutically effective amount” will fall in a relatively broad range that can be determined through clinical trials.
  • a viral vector is utilzed.
  • a therapeutically effective dose will be on the order of from about 10 5 to 10 16 of virions (such as AAV virions, lentivirus or baculovirs), such as 10 8 to 10 14 virions. The dose can depend on the efficiency of transduction, promoter strength, the stability of the message and the protein encoded thereby, and clinical factors. Effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.
  • an effective amount will be about 1 X10 8 vector genomes or more, in some cases about 1 X 10 9 , about 1 X 10 10 , about 1 X 10 11 , about 1 X 10 12 , or about 1 X 10 13 vector genomes or more, in certain instances, about 1 X 10 14 vector genomes or more, and usually no more than about 1 X 10 15 vector genomes administered to the recipient.
  • the amount of vector that is delivered is about 1 X 10 14 vectors or less, for example about 1 X 10 13 , about 1 X 10 12 , about 1 X 10 11 , about 1 X 10 10 , or about 1 X 10 9 vectors or less, in certain instances about 1 X 10 8 vectors, and typically no less than 1 X 10 8 vectors administered to the recipient.
  • the amount of vector genomes that is delivered is about 1 X 10 10 to about 1 X 10 11 vectors.
  • the amount of vector that is delivered is about 1 X 10 10 to about 1 X 10 12 vector genomes.
  • the amount of pharmaceutical composition to be administered may be measured using multiplicity of infection (MOI).
  • MOI refers to the ratio, or multiple of vector or viral genomes to the cells to which the nucleic may be delivered.
  • the MOI may be about 1 X 10 6 .
  • the MOI can be about 1 X 10 5 to about 1 X 10 7 .
  • the MOI may be about 1 X 10 4 to about 1 X 10 8 .
  • recombinant viruses of the disclosure are at least about 1 X 10 1 , about 1 X 10 2 , about 1 X 10 3 , about 1 X 10 4 , about 1 X 10 5 , about 1 X 10 6 , about 1 X 10 7 , about 1 X 10 8 , about 1 X 10 9 , about 1 X 10 10 , about 1 X 10 11 , about 1 X 10 12 , about 1 X 10 13 , about 1 X 10 14 , about 1 X 10 15 , about 1 X 10 16 , about 1 X 10 17 , and about 1 X 10 18 MOI. In some cases, recombinant viruses of this disclosure are about 1 X 10 8 to 1 X 10 14 MOI.
  • the amount of pharmaceutical composition delivered comprises about 1 X 10 8 to about 1 X 10 15 of recombinant viruses, about 1 X 10 9 to about 1 X 10 14 particles of recombinant viruses, about 1 X 10 10 to 1 X 10 13 particles of recombinant viruses, or about 1 X 10 11 to about 1 X 10s 12 particles of recombinant viruses.
  • Dosage treatment may be a single dose schedule or a multiple dose schedule to ultimately deliver the amount specified above.
  • the subject may be administered as many doses as appropriate.
  • the recipient may be given, e.g., 10 5 to 10 16 AAV virions in a single dose, or two, four, five, six or more doses that collectively result in delivery of, e.g., 10 5 to 10 16 AAV virions.
  • an AAV is administered to the recipient and/or to the donor liver (such as in an ex vivo perfusion system) at a dose of about 1 x 10 11 to about 1 x 10 14 viral particles (vp)/kg.
  • the AAV is administered to the recipient at a dose of about 1 x 10 12 to about 8 x 10 13 vp/kg.
  • the AAV is administered to the recipient liver at a dose of about 1 x 10 13 to about 6 x 10 13 vp/kg.
  • the AAV is administered to the recipient at a dose of at least about 1 x 10 11 , at least about 5 x 10 11 , at least about 1 x 10 12 , at least about 5 x 10 12 , at least about 1 x 10 13 , at least about 5 x 10 13 , or at least about 1 x 10 14 vp/kg.
  • the AAV is administered to the recipient at a dose of no more than about 5 x 10 11 , no more than about 1 x 10 12 , no more than about 5 x 10 12 , no more than about 1 x 10 13 , no more than about 5 x 10 13 , or no more than about 1 x 10 14 vp/kg. In one non-limiting example, the AAV is administered to the recipient at a dose of about 1 x 10 12 vp/kg.
  • the AAV can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more doses) as needed for the desired therapeutic results.
  • a lentivirus is administered at a dose of about 1 x 10 11 to about 1 x 10 14 viral particles (vp)/kg. In some examples, the lentivirus is administered to the recipient at a dose of about 1 x 10 12 to about 8 x 10 13 vp/kg. In other examples, the lentivirus is administered to the recipient at a dose of about 1 x 10 13 to about 6 x 10 13 vp/kg.
  • the lentivirus is administered to the recipient at a dose of at least about 1 x 10 11 , at least about 5 x 10 11 , at least about 1 x 10 12 , at least about 5 x 10 12 , at least about 1 x 10 13 , at least about 5 x 10 13 , or at least about 1 x 10 14 vp/kg.
  • the lentivirus is administered to the recipient at a dose of no more than about 5 x 10 11 , no more than about 1 x 10 12 , no more than about 5 x 10 12 , no more than about 1 x 10 13 , no more than about 5 x 10 13 , or no more than about 1 x 10 14 vp/kg.
  • the lentivirus is administered to the recipient at a dose of about 1 x 10 12 vp/kg.
  • the lentivirus can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more doses) as needed for the desired therapeutic results.
  • iPSC induced pluripotent stem
  • CC-HS activated complement
  • BODIPY® fluorophore staining revealed higher staining for the dye in CC-HS treated cells. Drusen are not formed if cells are treated with inactive complement (CI-HS). Furthermore, treatment with CC-HS led to degeneration of the RPE monolayer as confirmed by reduced F-ACTIN staining that marks cell borders (FIG.1) and loss of trans-epithelial resistance (TER) of the monolayer. TER of the monolayer is formed by functional tight junctions between neighboring RPE cells. As cells degenerate in a diseased state, the tight junctions fall apart dropping the TER of cells, as seen in CC-HS treatment conditions (FIG. 1B). Activation of endogenous retroviruses can cause degeneration of cells.
  • Endogenous retroviral RNA (FIG.2) and the three translated proteins – gag, pol, and env may induce degeneration of cells.
  • Comparative RNAseq analysis was used to identify that the expression of several members of the HERV-K family of endogenous retroviruses was significantly increased in CC-HS treated cells as compared to CI-HS treated cells (FIGS.3A-3B).
  • Approximately, 80 different loci were discovered that showed differential expression of HERV-K retroviruses (FIG.3A).
  • CC-HS treatment showed that the expression of different components of HERV-K family of endogenous retroviruses (ENV and GAG) was increased in CC-HS treated cells, as compared to CI-HS treated cells (FIG.4A, 4B and 5). Expression of other retroviruses like HERV-R were not increased in this context (FIG.4A, 4B). Similar increase in expression of HERV-K RNA was noted in cadaveric AMD eyes, confirming physiological relevance (FIG.6).
  • CC-HS treatment increases the expression of inflammatory cytokines (IL 6 and IL 8) secreted by RPE cells towards apical and basal sides (FIGS.7A, 7B). It was determined whether this increase in expression of inflammatory cytokines was mediated by increased HERV-K expression in CC-HS treated cells.
  • HERV-K To suppress the expression of HERV-K we used anti-retroviral drug Tenofovir that block transcription of HERV-K mRNA. Co-treatment of iRPE cells with CC-HS and Tenofovir decreased apical and basal secretions of both IL6 and IL8 (FIGS.7A, 7B). HERV-K ENV protein and RNA are thought to trigger inflammatory changes inside cells by activating receptors TLR3 and TLR4. Hence, it was determined if blocking the activity of both these receptors would reduce the expression of IL6 and IL8.
  • HERV-K ENV protein was overexpressed in healthy cells (FIGS.9A-9C).
  • the ENV protein of HERV-K that is thought to drive HERV-K pathology in cells was fused to a V5 tag allowing detection of the overexpressed protein separate from the endogenously make ENV protein.
  • Red fluorescent protein mcherry was driven from the same expression cassette driving ENV protein expression. However, once transcribed the two proteins were made separately because of the presence of an internal ribosome entry site (IRES) in between ENV and mcherry. Expression of the entire cassette was driven by a CMV promoter allowing high expression.
  • IVS internal ribosome entry site
  • This construct was packaged into a lentivirus and iRPE cells were transduced with it.
  • Western blot performed on cell lysates from iRPE transduced with ENV-expressing lentivirus compared to non- transduced cells showed the construct was able to derive the expression of ENV protein of HERV-K.
  • Immunostaining for the ENV protein in cells transduced at different multiplicities of infection (MOI) of 0.5 and 3.0 showed a lentivirus dose dependent increase in expression of the ENV protein (FIG.9A-9C).
  • ENV protein in wildtype iRPE was sufficient to trigger the AMD phenotype without CC- HS treatment, measured as loss of cellular barrier resistance (TER) and increased BODIPY® staining (FIGS.10A-10C).
  • the decrease in monolayer TER was dose dependent for the ENV protein. For instance, higher MOI led to higher amount of ENV protein (FIG.10C) and led to higher decrease in monolayer TER (FIG.10A) but the increase in subRPE lipid as stained by BODIPY® was not a dose dependent phenomenon (FIG.10B). Overall, this indicates ENV protein of HERV-K was sufficient to trigger AMD phenotype in iRPE cells.
  • ANG angiogenin
  • Lys-specific tRNA is used by ANG as a template to degrade HERV-K RNA.
  • qRT-PCR confirmed that fragments of Lys-specific tRNA (LysCTT, LysTTT, pro-AGG) were all decreased within 24 hours of CC-HS treatment of iRPE cells, whereas the expression of another tRNA fragment for a leucine-specific tRNA (LeuTAA) that doesn’t act as a template for HERV-K degradation by ANG, did not decrease in CC-HS treated iRPE cells (FIGS.13A, 13B).
  • HERV-K levels increase in CC-HS treated iRPE via downregulation of ANG expression and downregulation of tRNA fragment required for HERV-K degradation.
  • a gene therapy strategy was developed to reduce HERV-K levels in CC-HS treated iRPE cells.
  • HERV-K is made from multiple loci within the genome and these loci have slight sequence variations between them. Therefore, to develop a gene therapy for HERV-K, RNA made from most of these different loci of HERV-K was targeted.
  • CAS13 is a nuclease that can target RNA using a complementary guide sequence and degrade it.
  • HERV-K Using bioinformatics analysis, three guide sequences were identified in HERV-K that are used to target HERV-K expression in RPE cells. These sequences were designed against the GAG part of HERV-K mRNA, which is the start of the RNA. Degradation of the start of RNA leads to degradation of the entire RNA.
  • bioinformatics analysis it was discovered three guide sequences in HERV-K that are used to target HERV-K expression in RPE cells (Fig.14A-14B). The sequences selected showed complementarity across multiple HERV-K loci and also showed good consensus for CAS13 based targeting (FIGS.15A-15B).
  • FIGS.17A-17E show Cas13Rx mediated HERV-K knock down reverses complement-induced lipid accumulation.
  • iRPE cells were transduced with lentivirus expressing a HA-tagged CAS13Rx under the control of a doxycycline inducible promoter. Seven days of doxycycline treatment induced the expression of CAS13Rx (FIG.16). At this stage, iRPE cells were transduced with another lentiviral construct that expresses one of the three guide RNAs along or together and the control guide RNA. Another 7 days was allowed for guide RNAs to be expressed at high levels and then treated cells with CC-HS.
  • FIG.17B shows a TER graph of CIHS (grey bars) and CCHS (black bars) treated samples that were tranduced with a scrambled guide RNA (NEG) or no guide RNA (Cas13Rx) or guide RNA #2 (FIG.15). Only guide RNA#2 is able to rescue TER downregulated by CCHS treatment.
  • the top panel shows digital images of CIHS or CCHS treated iRPE, transduced either with a scrambled control (NEG) or guide RNA#2.
  • CCHS-induced increase in HERV-K expression is downregulated by guide RNA 2 against HERV-K.
  • the bottom panel shows digital images of CIHS or CCHS treated iRPE, transduced either with a scrambled control (NEG) or guideRNA#2.
  • CCHS-induced increase in lipid deposits is downregulated by guide RNA 2 against HERV-K.
  • FIG.17D shows quantification of HERV-K levels seen in FIG.17C
  • the top panel shows guide RNA#2 is able to downregulate HERV-K levels in CCHS treated iRPE cells.
  • FIG.17E shows quantification of BODIPY® levels seen in FIG.17C
  • bottom panel shows guide RNA#2 is able to downregulate BODIPY in CCHS treated iRPE cells.
  • FIG.18 shows Cas13Rx mediated HERV K knock down reverses complement-induced lipid accumulation by all three guides selected in FIG.15.
  • CCHS induced increase in lipid deposits (measured as BODIPY® signal) in CCHS is downregulated by all three guides against HERV-K.
  • Left graph shows quantification of digital imaging data. Additional results are provided in FIG.19.
  • Immunofluorescent or BODIPY®-fluorescent staining 6–8 week old fully differentiated iRPE monolayers, grown on transwells, were used for all experiments. iRPE cells were treated for 48 h 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 change. For chronic treatment, 0.1% CC-HS (or CI- HS) were added to both apical and basal media of iRPE for up to 6 days. Medium was changed daily.
  • iRPE either prepared as cross-sections or a monolayer on transwell membranes (TWM), were fixed in 4% paraformaldehyde and blocked 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), 0.25% TRITON® X-100 (9002-64-5, Sigma)) for 1 h at RT.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • TWEEN®20 900-64-5, Affymetrix
  • TRITON® X-100 9002-64-5, Sigma
  • APOE ⁇ 250 ng/mL; AB947, Millipore, FIG.1
  • HERV-K ⁇ 1 ⁇ g/mL, HERM-1811-5
  • Austral Biologicals Angiogenin (1ug/ml, AF265, R&D systems, FIGS.13, 17
  • HA tag 2 ⁇ g/mL, SAB2702217, Sigma Aldrich,
  • ZO-1 5 ⁇ g/mL; MA3-39100-A488, ThermoFisher).
  • Secondary antibodies include the following: ALEXA FLUOR® 555 goat anti-rabbit (1:200)(5 ⁇ g/mL; A21429, ThermoFisher), ALEXA FLUOR® 488 goat anti-rabbit (1:1000) (5 ⁇ g/mL; A11078, ThermoFisher), ALEXA FLUOR® 555 goat anti-mouse (1:500) (5 ⁇ g/mL; A-21422, ThermoFisher), ALEXA FLUOR® 488 goat anti-mouse (1:200) (5 ⁇ g/mL; A32723, ThermoFisher), Hoechst-33542 (1:1000) (H3570, ThermoFisher), and Phalloidin (1:300) (ALEXA FLUOR® 488, A12379, ThermoFisher
  • the cells were washed with ICC buffer and mounted on a glass slide with Fluoromount-G aqueous mounting medium (0100-01; Southern tech) and a glass coverslip.
  • Samples were imaged using Zeiss 880 confocal microscopes (Carl Zeiss). Images were processed and exported as tiff files using Zenblue3.2 software (Carl Zeiss). Lipid deposits were stained using BODIPY® dye (D3922, or D3835, ThermoFisher, Figure 1 bottom, 9a, 11b bottom, 17C bottom).
  • the cells were fixed in 4% paraformaldehyde for 20 m at RT and washed with 1 ⁇ PBS.
  • the BODIPY® stock solution was prepared in a concentration of 1 mg/mL (3.8 mM) in DMSO then diluted 1:1000 in 1 ⁇ PBS to generate the working solution. Bring both the stock and working solutions to RT, then filtering with a 0.22- ⁇ m filter before use. Cells were incubated in the working solution for 30 minutes at RT, washed with 1 ⁇ PBS, mounted, and imaged using the procedure described above. Quantification of lipid droplets (BODIPY® dye) was performed in ImageJ (v1.8.0, Bethesda, USA). For HERV-K, particles were counted with maxima prominence 8000, and for BODIPY® dye, maxima prominence 2900.
  • RNAscope to localize mRNA of HERV-K Probes and reagents were obtained from Advanced Cell Diagnostics (acdbio.com) except as otherwise stated. Fully matured confluent monolayer of iRPE grown on transwells were treated with CI-HS or CC-HS (5%) for 48 hours. Cells were fixed in 10% formalin. The transwell membrane was punched out and embedded in paraffin and sectioned transversely into 5 uM cross sections in RNA free environment. The assays were performed using RNAscope 2.5 HD Chromogenic Detection Kit (PN332360) according to the manufacturer’s protocol.
  • the slides were then treated with H 2 O 2 for 10 minutes at room temperature (RT), washed in distilled water 3 X and continued with target retrieval in1X retrieval buffer in Black and Decker Steamer HS3000, at 99C for 15 minutes, the rinsed thoroughly with distilled water for 15 minutes.
  • the slides were transferred to 100% ETOH for 3 minutes and dry at RT. Slides were then treated with protease plus in the 40°C prewarmed humidity control tray, incubated inside the 40 °C oven for 15 minutes, immediately followed by washing in distilled water for 5X.
  • Slides were air-dried and incubated with the probes in hybEZ rack located in the humidity control tray inside the oven for 2 h at 40°C, followed by wash 2 minutes at room temperature (RT) with 1X wash buffer for 3 times. Slides were stored in 5 X SSC at RT overnight. The next day, the slides were washed with 1X Wash Buffer for 2 minutes at RT for 2X, then incubated with AMP 1 for 30 minutes at 40°C; AMP 2: for 15 minutes at 40°C, AMP 3, 30 minutes at 40°C; AMP 4:15 minutes at 40°C, Amp 5: 30 minutes at RT; Amp 6, 15 minutes at RT, added the same wash step after each AMP.
  • the signals were detected by incubated 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) 10 minutes at RT, then washed with tap water. Slides were then counterstained in 50% Hematoxylin staining solution (Gill’s Hematoxylin I, HXGHE1LT, American MasterTech Scientific (1:4)) for 20” at RT, washed with tap water for 3X, dipped quickly in 0.02% ammonia water, then washed with tap water 5X and dried in a 60°C dry oven for 15.
  • 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
  • Slides were then counterstained in 50% Hematoxylin staining solution (Gill’s Hematoxylin I, HXGHE1LT, American MasterTech Scientific (1:4)) for 20” at RT
  • the slides were dehydrated in pure xylene (not pure ETOH) and immediately mounted with EcoMount media (EM897L, Biocare medical) on the slide before the xylene dried. Images were collected with a Zeiss Axio Imager M2 microscope fitted with a 40 ⁇ oil-immersion objective lens and a AxioCamIcc1 digital camera and Zen software (Carl Zeiss AG, Oberkochen, Germany, zeiss.com). The probes included human Positive Control probe Hs PPIB C1 (PN 321641) or RNASCOPE® Probe V-HERV-K-pol (469831). The slides of human cadaver eyes were obtained through the Advancing Sight Network (Alabama Eye Bank).
  • TER measurement Transepithelial resistance (TER) in iRPE was measured using commercially available Epithelial Volt/Ohm Meter (EVOM2, WPI). The electrodes in the form of chopsticks (STX2, WPI) were positioned into apical and basal media of iRPE simultaneously. Resistance values (Ohms) were noted. Actual resistance (Ohms ⁇ cm 2 ) is calculated by multiplying the raw TER value by the area of measurement (12 mm diameter transwell).
  • RNAseq analysis to identify CC-HS-induced loci specific expression of HERV-K:
  • the data set was first analyzed for CCHS induced Transposon Element (TE) expression by employing TEToolkit GTF (github.com/mhammell-laboratory/tetoolkit) to build a custom index for RNA quantification of established the RNAseq data (nature.com/articles/s41467-021-27488-x) in following ways: 1) Assign user provided transposable element (TE) info file.
  • the set of coordinates was used to direct/count mapping events at the positions in the genome.
  • loci specific HERV-K expression was performed as follows: according to the annotated loci of HERV-K in 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 loci annotation in GENBANK® of version hg38 human genome.
  • a comprehensive dataset of ⁇ 80 HERV-K loci in the human genome was composed which contained either gag (full or almost full loci) and partial loci (loci without gag and or also missing other portions of HERV-K) and the sequence identifiers for each loci.
  • RNA seq data Site-specific expression data at all of the HERV-K loci were narrowed down to the 20-most differentially expressed (CI- HS & CC- HS). Data is represented as scaled depth of each loci, which is a calculation that takes the total reads of a region and divides it by the number of base pairs in that region.
  • sample Bam files were created which permit the data to be viewed by the Integrative Genomics Viewer (IGV), (ncbi.nlm.nih.gov/pmc/articles/PMC3346182/) that enables real-time examining the RNAseq datasets in whole genome.
  • IGV Integrative Genomics Viewer
  • RNAseq data set CI-HS& CC-HS was queried via customized RNA sequencing (RNA-seq) transcriptome database of healthy human eye tissues (pubmed.ncbi.nlm.nih.gov/31343654, eyeIntegration.nei.nih.gov) to retrieve differential expression data for a set of RNASEs (including Angiogenin, Dicer, RNases I,2,3,4). The data was plotted as Box plot.
  • cDNA was prepared using the ISCRIPTTM cDNA Synthesis Kit (1708891, Bio-Rad) according to manufacturer’s protocol. PCR reaction was performed in a 10ul reaction with 5ng cDNA and 5 ⁇ L of SSOADVANCEDTM Universal SYBR® Green Supermix (1725274, Bio-Rad), amplified in triplicates in a Viia7 Real-Time PCR System (ThermoFisher Scientific). Sequences of HERV-K primers were synthesized according to the published data (Li et al., 2015 Sep 30;7(307):307ra153. doi: 10.1126/scitranslmed.aac8201.
  • RNA quantification Fully matured iRPE grown on transwell were treated with CI-HS or CC- HS (5%) for 3, 24 or 48h. Total RNA was pretreated to remove post-transcriptional modifications based on an established protocol (RTSTARTM tRF&tiRNA pretreatment Kit (Cat#: AS-FS-005). Briefly, 3’-terminal Deacylation of 1ug of total RNA was carried out in a 15 ul reaction mix at 37°C for 40 minutes and the reaction was stopped by the incubation with 19 ⁇ L Deacylation Stop Buffer, at RT for 5 minutes.
  • the reaction was continued by addition of a terminal treatment mix to remove 3’-phosphate and add a 5’- phosphate to the RNA in a reaction mix of 50ul at 37°C for 40 minutes.
  • the Terminal Enzyme was inactivated by incubating at 70°C for 5 minutes.
  • the RNA was purified by magnetic beads-based RNA purification methods (SEQ-STARTM RNACLEAN® and SMALLENRICH® Beads, AS-MB-009, Arraystar, Inc). See below).
  • the purified RNA samples were treated in a demethylation reaction performed by incubation the adaptor ligated RNA with demethylation mix at 37°C for 2 h, followed by adding stop Buffer to terminate the reaction.
  • RNA was purified again by the beads-based RNA purification methods described above. RNA purification was carried out with SEQ-STARTM RNACLEAN® and SMALLENRICH® Beads assay.
  • the pretreated RNA product was mixed thoroughly with 1.8X (1.8:1 v:v ratio) beads suspension and 4.2X 100% isopropanol (collected RNA>17bp) by vortexing for 30 minutes, and then the mixture was incubated at RT for 10 minutes to allow the RNA to bind to the beads.
  • the mix tube was placed on a magnetic stand (DYNAMAG TM ,12321D, Invitrogen) for 3 minutes until the supernatant became completely clear. The supernatant was carefully aspirated and discarded.
  • Reverse transcription was performed with RTSTARTM First-Strand cDNA Synthesis Kit for tiRNA Detection (AS-FS-003, ARRAYSTAR).
  • the 3’ adaptor ligated product was then incubated with a 5’ adaptor ligation mixture at 25°C for 1hr.
  • the adaptor-ligated RNA was then incubated with a reverse transcription mixture at 45°C for 1h.
  • PCR for the was carried out following the same protocol as above (cDNA synthesis from mRNA) using predesigned tiRNA primers from ARRASTAR Inc (RTSTARTM 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) Relative quantification was calculated using the 2 - ⁇ CT method (Schmittgen and Livak Nat Protoc.
  • SDS-PAGE for Western blot analysis were performed using 4–16% pre-made gels (AnykDTM CRITERIONTM TGXTM Precast Midi Protein Gel, 12 + 2 well, 45 ⁇ l, catalog #5671123, Bio-Rad) with equal protein amount (30 ⁇ g/well) loaded.
  • a semi-dry transfer apparatus (Trans-Blot Turbo transfer system, 1704150, Bio-Rad) was used to transfer proteins to the polyvinylidene fluoride (PVDF) membrane.
  • the blots were blocked in buffer containing 5% BSA in PBST (1 ⁇ PBS and 1% Tween20) for 1 h at RT and then incubated with primary antibody overnight at 4 °C on a shaker.
  • the blots were washed with 1 X phosphate buffered saline-1% TWEEN® (PBST), then incubated with secondary antibodies for 1 h at RT.
  • the blots were washed again and developed in horse radish peroxidase (HRP) substrate (Bio-rad Clarity Western ECL Substrate) and imaged using a Bio-Rad imaging machine (ChemiDoc MP Imaging System).
  • HRP horse radish peroxidase
  • Bio-Rad imaging machine Bio-Rad imaging machine
  • HERV-K (1:500) (HERM-1811-5, Austral Biologicals, Figure 10B), Angiogenin (1:500) (AF265, R&D systems, Figure 13A), HA tag (1:1000) (SAB2702217, Sigma Aldrich, FIG.16a), and Beta-tubulin (1:500) (ab6046, Abcam) to normalize the loading control.
  • the secondary antibodies included goat anti-rabbit HRP- conjugate (1705046, Bio-Rad) and goat anti-mouse HRP-conjugate (1705047, Bio-Rad) diluted (1:10,000) in PBST and 10% SDS (1:500).
  • Tenofovir or inhibitors to TLR3, TLR4 The antiviral drug Tenofovir (a nucleotide analogue reverse transcriptase , which causes premature termination of DNA transcription (20uM, SML1795, Sigma-Aldrich)) or TLR3/dsRNA complex inhibitor (10uM, 614310, Sigma-Aldrich) or TLR4 inhibitor (5uM, CLI-095 or TAK-242, Invivogen) was used. Each inhibitor was added to both apical and basal media of RPE transwell of fully matured iRPE 16 hours prior to the addition of CIHS (5%) or CCHS (5%), which lasted for 48h.
  • Luminex discovery assay human premixed multi-Analyte Kit Cat#: LXSAHM-02, R& D systems Inc. to include interleukin (IL)-8, IL-6). Cytokine standard cocktails were prepared as 3-fold serial dilution with calibration diluent RD6-52. iRPE media samples were diluted 2X in RD6-52.
  • Magnetic microparticles cocktails were spined briefly and vortexed for 30”, were then incubated for 2 hours at room temperature (RT) with samples or standards in a horizontal orbital plate shaker at 800rpm (IKA R MS 3 digital). Washing was carried out using a hand-held magnet (Clarion Safety Systems Inc., catalog # C7002-01) that accommodates the microplate, by filling the wells with washing buffer and applying the magnet to the bottom of the assay plate for 1 minute before removing the liquid. for total 3 washes. This was followed by the incubation with biotinylated antibody cocktails for an hour in RT on the shaker set on 800rpm. The wash was repeated, then incubated with streptavidin-PE for 30mins, with shaking (on the same setting).
  • ANG ELISA was carried out with media collected from 0.1% CC-HS CI-HS treated iRPE with QUANTIKINE® ELISA (human Angiogenin, DAN00, R&D systems) according to standard manufacturer’s protocol.
  • lentivirus to express HERV-K ENV and lentiviral transduction Coding sequences of HERV-K ENV (2.1kb) was cloned into modified pLx304 vector with CMV promoter, C-V5 tag, IRES2 – mCherry, blasticidin resistance and packaged into lentivirus (Genecopoeia , LPP-CS-CC1718L-Lx304-01- 050). The virus was transduced into iRPE in two multiplicity of infection MOI (0.5, 3) in a polybrene (5ug/ml) containing 5%RPE media for 16 hours. The media was replaced with fresh media and cells were continuously cultured regularly for additional 14 days.
  • the Mcherry signal was imaged live with Epi- fluorescent microscope (Zeiss AXIO, Vert.A1 with was equipped with AxioCam MRm digital camera) prior to experiment end point ( Figure 10B). Images were processed and exported as tiff files using Zenblue3.2 software (Carl Zeiss). Production and expression of Cas13Rx compatible HERV-K giRNA and induction of Cas13Rx expression: Comprehensive dataset of HERV-K for the ⁇ 80 loci (see “RNAseq analysis to identify CC-HS- induced loci specific expression of HERV-K” were aligned and searched via NCBI blast search to compile HERV-K loci with intact 5’LTR and gag protein.
  • gRNAs were selected (SEQ ID NOS; 1, 2 and 3) using the following criteria: 1) Target multiple loci; 2) Cover majority of the selected expression HERV-K loci (among 20 selected HERV-K loci, 16 contains 5’-LTR and conserved gag sequence in HERV-K GAG region. This led to 16 loci that were used to evaluate the gRNAs.
  • guide RNA The sequences of guide RNA are as followings: guide 1: TTAGAACGACATTGACTAGCCCA (SEQ ID NO: 1), guide 2: AGCTGCTTTAATAATGGCCCAAT (SEQ ID NO: 2); guide 3: TTGTACCCCATCAATCCACCAAG (SEQ ID NO: 3); negative guide: TCAGGGCAAACAGAACTTTGACTCCCAT (SEQ ID NO: 4).
  • the established negative guide RNA was included as control. Genomic sequences of the 16 HERV-K loci were aligned by (MegaAlignPro, DNAstar software) and gRNA1 or -2 or-3 was located within the aligned region (highlighted in blue) in a reverse and complement manner.
  • the gRNA information are listed as followings: g1: LPP-CS-CC1540L-01, titer: 3.51 X 10 8 TU/ml; g2: LPP- CS-CC1541L-01, titer 3.50 X 10 8 TU/ml; g3: LPP-CS-CC1542L-01, titer, 3.82 X 10 8 TU/ml; neg: LPP-CS- CC1544L-01, titer 3.23 X 10 8 TU/ml.
  • Cas13RX-expression lentivector (Addgene: 138149), a TET-ON expression vector, was packaged similarly (LPP-CS-PK1403L, titer: 7.82 X 10 8 TU/ml).
  • LPP-CS-PK1403L titer: 7.82 X 10 8 TU/ml.
  • Transduction iRPE with Cas13Rx and the guides via lentiviral transduction was sued.
  • Fully matured iRPE grown on transwell was transduced with Cas13Rx expressing lentivirus (MOI 1) in the presence of polybrene (5ug/ml) following similar procure as described previously (Miyagishima et al., Commun. Biol.4, 1360, doi.org/10.1038/s42003-021-02872-x, 2021). The following day, the viral containing media was removed and replaced with fresh media containing doxycycline (5ug/ml), which was refreshed every other day to induce Cas13Rx expression.
  • MOI 1 Cas13Rx expressing lentivirus
  • Example 4 ANG Overexpression Reduces Expression of HERV-K A schematic diagram of methods performed to determine the effects of ANG expression is provided in FIG.20.
  • Fully differentiated iRPE (D3C) cells grown on in a transwell plate were transduced with lentivirus that encodes human angiogenin (co-expression of mcherry reporter) at a multiplicity of infection (MOI) of 0.5, a MOI of 1.5, or with addition of polybrene reagent (PB) are as a control.
  • MOI multiplicity of infection
  • PB polybrene reagent
  • the virus containing media were removed the next day.
  • the cells were continually cultured in regular RPE media for 2 days, followed by the incubation with 0.1% CCHS RPE media for 6 days, which was refreshed daily. Cells were analyzed on day 6 for ANG expression (FIG.21) HERVK expression (FIG.22), BODIP® accumulates (FIG.23), and TER (FIG.24).
  • FIG.21 shows lentiviral overexpression of ANG.
  • Secreted ANG was measured in both apical and basal media by ELISA. The data are the ratio of secreted ANG at the day 6 end point as compared to secreted ANG at day 0. There was a virus dose-dependent increase in ANG secretion by RPE cells.
  • FIG.22 shows that ANG prevented the CCHS-induced HERVK increase.
  • iRPE were fixed and processed to show cell borders with phalloidin (magenta), transduced cells with mcherry (red), HERV-K env protein (green), and nuclei (blue).
  • FIG.23 shows that ANG reduces the CCHS-induced lipid accumulation.
  • iRPE were fixed and processed to show cell borders with phalloidin (magenta), transduced cells with mcherry (red), lipid deposits as stained with BODIPY® dye (green), and nuclei (blue).
  • ANG expression prevents CCHS-induced transepithelial resistance (TER) decrease in the iRPE.
  • VMD2-HERV-K-ENV transgenic mice To investigate the impact of HERV-K ENV in RPE, a classic transgenic mouse model was generated in which HERV-K ENV expression is controlled by a custom-made RPE specific VMD2 promoter (Iacovelli J, et. Al., Invest Ophthalmol Vis Sci.2011 Mar 14;52(3):1378-83.
  • FIGS.26A-26B show the HERV-K ENV protein was specifically detected in 1–2-month-old HERV-K ENV tg/tg mouse RPE, approximately 9X above that in the control RPE.
  • the expression was detected similarly in 7–8-month-old HERV-K ENV transgenic RPE (FIGS.26C-26D).
  • the overexpression increased lipid accumulation, detected significantly at 1–2-month-old RPE (FIG.26E), roughly 2X higher compared to wildtype control (FIG.26F).
  • the HERV-K ENV caused increases of lipid accumulation were continually detected in the 7-8 month old RPE (FIG.26G), 5X greater than the control (FIG.26H), also higher than the degree of increase detected at 1-2 month old RPE.
  • These overexpressing HERV-K ENV were patchy (FIGS.27A, 27B).
  • the HERV-K level was higher in a group of RPE than their neighboring cells and were co-stained with Bodiby fluorescent signals in these cells and localized at basolateral side of RPE monolayer (FIG.27B), indicating lipid accumulation occurred in HERVK-ENV tg/tg cells, consistent with HERV-K ENV being causative to the lipid accumulation in these cells.
  • Microscopy showed age-related histopathological changes in the HERV-K ENV tg+ or tg/tg eye relative to WT control (FIG.28B). Histological sections are compared between WT (left) and transgenic mice in 7-month-old (7 mo), mis-localized cells appeared in the subretinal space (SRS) (arrows, middle, right panel), associated with disruption of the interphotoreceptor matrix (IPM).
  • SRS subretinal space
  • IPM interphotoreceptor matrix
  • Retrovirology 9, 6 (2012); Joel Gruchot, et.al., Brain, Behavior, and Immunity 107, 2023, pp.242-252, ISSN 0889-1591, scielo.br/j/rsbmt/a/qrzhZzKqZNzmKkV5dv53LVR/?lang en). Therefore, evaluations of additional therapeutic strategies are needed for HIV-AMD patients, and HIV-infected people in general. A study was performed (FIGS.30A, 30B) with matured iRPE.
  • a combination of antiretroviral drugs (tenofovir, darunavir) was more effective than a single drug against HERV-K mediated cell degeneration, evaluated as lipid accumulation (FIG.30C) morphological alterations (cell area, aspect ratio and hexagonality, FIG.30D),increased IL-8 and IL-6 secretion (FIGS.30E, 30F), through a systematic multimodalities examination. Additionally, combining retroviral drugs (tenofovir, darunavir) with non- retroviral drugs (metformin hydrochloride, L-745,870 trihydrochloride) was more effective than each drug separately. For therapeutics, these drugs can be delivered as a pill, eye drop, or slow-release particles.
  • Example 7 shRNA Young mice (3-4 weeks old) are subjected to subretinal or intravitreal injection of AAV HERV-K ENV or control AAV (1-2ul at titer ⁇ 5X1012tu/ml).
  • the RPE degeneration is monitored monthly via live imaging with fundus fluorescent imager or Heidelberg SPECTRALIS HRA + OCT system, to detect hyperfluorescent spots.
  • ERG is performed at experimental endpoint when RPE tissues are collected for determining lipid accumulations and morphological changes.
  • HERV-K serves as a common hub/passage to pathogenesis during tissue degeneration. Targeting HERV-K via AAV delivery strategy will provide a therapeutic for treatment of AMD.
  • the F0 breeder mice were genotyped by the automated genotyping system (Transnetyx Cordova, TNI) using the primers: Forward Primer: CGACGGAGACTACAAGGATCATGATA (SEQ ID NO: 15; Reverse Primer: GGGTTTAAACGGGCCCTCTAG (SEQ ID NO: 16), Reporter 1: CCGCCTACTACTTATCG (SEQ ID NO: 17).
  • the HERV-k ENV positive founder mice were each bred to C57Bl/J mice to maintain a line in F1 generation in tg+. Mice were housed and cared for according to an animal study protocol.
  • mice All mice were screened to be negative for the spontaneous rd8 mutation, which leads to retinal degeneration (Mattapallil , et.al., Invest. Ophthalmol. Vis. Sci., 53, 2921–2927).
  • F1 heterozygous HERV-K ENV mice tg+ were crossed to obtain homozygous HERVK-ENV tgtg mice.
  • Mouse RPE flat-mount tissues were prepared as previously described (Schneider et al., PLoS ONE 13(11): e0207222).
  • HERV-K ENV transgene expression in mouse RPE was determined by qRT-PCR using the primer set that recognizes HERV-K ENV.
  • FAF Fundus Autofluorescence
  • mice aged 7-8 months, n>6 each for wildtype+/+ and HERV-K ENV tg/+ or HERV-K ENV tgtg were placed under anesthesia via a subcutaneous injection of a ketamine/xylazine mixture (i.p.100 mg/kg and 6 mg/kg, respectively).
  • Pupils were dilated with phenylephrine (2.5%) and tropicamide (1%).
  • a drop of GENTEAL® Gel (Novartis) was placed on each eye to maintain moistness.
  • a 30-degree lens was used for the autofluorescence images (AF).
  • Eye motion artifacts were eliminated by in- system eye tracking. Histopathology-H&E staining: Eyes were enucleated and fixed with 4% glutaraldehyde for 1h, followed by 10% formalin for 24h at room temperature (RT). The whole eyes were embedded in methacrylate and serially sectioned vertically through cornea -optic nerve plane. Four sections were collected per slide and stained with hematoxylin and eosin. The images of whole samples were collected by scanning in ZEISS AxioScan 7 scanner. Representative images were sampled, see FIG.26B. n>6.
  • Electron microscopy Mouse eyes were fixed in 4% glutaraldehyde at RT for 24 h and were processed according to a routine TEM protocol to the RPE micrographs.
  • Immunostaining and signal quantification Mouse RPE flat-mount tissues were prepared according to a protocol described previously, (Schneider et al., PLoS ONE 13(11): e0207222), washed in 1X phosphate buffered saline (PBS) on ice and fixed in 4% paraformaldehyde/1 ⁇ PBS (20 min) at RT.
  • PBS 1X phosphate buffered saline
  • Lipid deposits were stained using BODIPY® dye (D3922, or D3835, ThermoFisher) in the absence of any detergent, similarly as described above for staining iRPE. Briefly, the tissues were incubated with 10 ⁇ M BODIPY® dye overnight at 4C, washed with 1 ⁇ phosphate buffer saline (PBS), and immunostaining with ZO-1 Monoclonal Antibody ALEXA FLUORTM 594 (ZO1-1A12, ThermoFisher) was carried out as described previously (see Sharma et al., Nature Comm.12: 7293, doi.org/10.1038/s41467-021-27488-x, 2021).
  • the tissues were permeabilized with immunocytochemistry (ICC) blocking buffer (1 ⁇ PBS, 0.5% normal goat serum, 0.3% Triton X-100, 0.3% tweent-20) for 1 hour (h) at RT, then, incubated with the conjugated antibody diluted 1:200 in ICC buffer for 1hr at RT.
  • ICC immunocytochemistry
  • the images were captured using Zeiss LSM 980 confocal microscopes (Carl Zeiss), and then processed and exported as tiff files using Zenblue3.2 software (Carl Zeiss).
  • IPSC- RPE treatments with combination drugs including Metformin, antiviral drugs, and DrD4 blocker: IPSC- RPE were treated with antiviral drug tenofovir (a nucleotide analogue reverse transcriptase inhibitor, nRTI), which causes premature termination of DNA transcription (20uM, SML1795, Sigma-Aldrich)), darunavir, a protease inhibitor which inhibits cleavage of gag-pol polyproteins (50 uM, Sigma L0937), Metformin hydrochloride (3 mM , Tocris Bioscience 2864), and L-745,870 trihydrochloride (6 uM, Tocris Bioscience,10-021-0).
  • antiviral drug tenofovir a nucleotide analogue reverse transcriptase inhibitor, nRTI
  • darunavir a protease inhibitor which inhibits cleavage of gag-pol polyproteins (50 uM, Sigma L0937)
  • Metformin hydrochloride

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Virology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Mycology (AREA)
  • Neurology (AREA)
  • AIDS & HIV (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Neurosurgery (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Vascular Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)

Abstract

L'invention divulgue des méthodes de traitement ou de réduction du risque de développer une dégénérescence maculaire liée à l'âge (DMLA) chez un sujet. Ces méthodes peuvent comprendre la sélection d'un sujet ayant, ou risquant de développer, une DMLA et l'administration au sujet d'une quantité efficace d'un agent qui inhibe HERV-K. Un agent qui inhibe HERV-K comprend, entre autres, un anticorps, une molécule d'ARN inhibiteur, un agent anti-rétroviral, un système CRISPR/Cas13 ciblant HERV-K, ainsi que l'angiogénine. L'invention concerne également un système CRISPR/Cas13 ciblant HERV-K. Ces méthodes peuvent également comprendre la sélection d'un sujet ayant, ou risquant de développer, une DMLA et l'administration au sujet d'une quantité efficace d'un agent qui augmente l'activité de l'ANG.
PCT/US2024/020294 2023-03-17 2024-03-15 Méthodes de traitement de la dégénérescence maculaire liée à l'âge Pending WO2024196814A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2024240009A AU2024240009A1 (en) 2023-03-17 2024-03-15 Methods for treatment of age-related macular degeneration

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363453006P 2023-03-17 2023-03-17
US63/453,006 2023-03-17

Publications (1)

Publication Number Publication Date
WO2024196814A1 true WO2024196814A1 (fr) 2024-09-26

Family

ID=90810892

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/020294 Pending WO2024196814A1 (fr) 2023-03-17 2024-03-15 Méthodes de traitement de la dégénérescence maculaire liée à l'âge

Country Status (2)

Country Link
AU (1) AU2024240009A1 (fr)
WO (1) WO2024196814A1 (fr)

Citations (55)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4036945A (en) 1976-05-03 1977-07-19 The Massachusetts General Hospital Composition and method for determining the size and location of myocardial infarcts
US4331647A (en) 1980-03-03 1982-05-25 Goldenberg Milton David Tumor localization and therapy with labeled antibody fragments specific to tumor-associated markers
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
WO1990002809A1 (fr) 1988-09-02 1990-03-22 Protein Engineering Corporation Production et selection de proteines de liaison diversifiees de recombinaison
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1991017271A1 (fr) 1990-05-01 1991-11-14 Affymax Technologies N.V. Procedes de triage de banques d'adn recombine
WO1992001047A1 (fr) 1990-07-10 1992-01-23 Cambridge Antibody Technology Limited Procede de production de chainon de paires a liaison specifique
US5091309A (en) 1986-01-16 1992-02-25 Washington University Sindbis virus vectors
WO1992009690A2 (fr) 1990-12-03 1992-06-11 Genentech, Inc. Methode d'enrichissement pour des variantes de l'hormone de croissance avec des proprietes de liaison modifiees
WO1992015679A1 (fr) 1991-03-01 1992-09-17 Protein Engineering Corporation Phage de visualisation d'un determinant antigenique ameliore
WO1992018619A1 (fr) 1991-04-10 1992-10-29 The Scripps Research Institute Banques de recepteurs heterodimeres utilisant des phagemides
WO1992020791A1 (fr) 1990-07-10 1992-11-26 Cambridge Antibody Technology Limited Methode de production de chainons de paires de liaison specifique
WO1993001288A1 (fr) 1991-07-08 1993-01-21 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Phagemide utile pour trier des anticorps
US5217879A (en) 1989-01-12 1993-06-08 Washington University Infectious Sindbis virus vectors
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5443505A (en) 1993-11-15 1995-08-22 Oculex Pharmaceuticals, Inc. Biocompatible ocular implants
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5858988A (en) 1993-02-24 1999-01-12 Wang; Jui H. Poly-substituted-phenyl-oligoribo nucleotides having enhanced stability and membrane permeability and methods of use
US5869079A (en) 1995-06-02 1999-02-09 Oculex Pharmaceuticals, Inc. Formulation for controlled release of drugs by combining hydrophilic and hydrophobic agents
US5935946A (en) 1997-07-25 1999-08-10 Gilead Sciences, Inc. Nucleotide analog composition and synthesis method
US6251090B1 (en) 1994-12-12 2001-06-26 Robert Logan Avery Intravitreal medicine delivery
US6291438B1 (en) 1993-02-24 2001-09-18 Jui H. Wang Antiviral anticancer poly-substituted phenyl derivatized oligoribonucleotides and methods for their use
US6299895B1 (en) 1997-03-24 2001-10-09 Neurotech S.A. Device and method for treating ophthalmic diseases
US6413540B1 (en) 1999-10-21 2002-07-02 Alcon Universal Ltd. Drug delivery device
US6416777B1 (en) 1999-10-21 2002-07-09 Alcon Universal Ltd. Ophthalmic drug delivery device
WO2003033027A2 (fr) 2001-10-19 2003-04-24 University Of Strathclyde Dendrimeres utilises pour une administration ciblee
WO2003037379A1 (fr) 2001-10-30 2003-05-08 Degussa Ag Utlisation de materiaux granuleux a base de dioxyde de silicium produit de façon pyrogenique dans des compositions pharmaceutiques
US6699493B2 (en) 2000-11-29 2004-03-02 Oculex Pharmaceuticals, Inc. Method for reducing or preventing transplant rejection in the eye and intraocular implants for use therefor
US6780324B2 (en) 2002-03-18 2004-08-24 Labopharm, Inc. Preparation of sterile stabilized nanodispersions
US20050054567A1 (en) 2001-11-06 2005-03-10 Rowlinson Scott William Use of il-19,il-22 and il-24 to treat hematopoietic disorders
US20080019979A1 (en) 2006-05-22 2008-01-24 Feng Wang-Johanning HERV-K Antigens, Antibodies, and Methods
US7390791B2 (en) 2000-07-21 2008-06-24 Gilead Sciences, Inc. Prodrugs of phosphonate nucleotide analogues
US20090175953A1 (en) 2006-07-13 2009-07-09 Doris Angus Nanodispersions
US20110229971A1 (en) 2008-01-29 2011-09-22 Applied Genetic Technologies Corporation Recombinant virus production using mammalian cells in suspension
US20120100606A1 (en) 2009-04-02 2012-04-26 Sergei Zolotukhin Inducible System for Highly Efficient Production of Recombinant Adeno-Associated Virus (rAAV) Vectors
US20120135515A1 (en) 2003-05-21 2012-05-31 Guang Qu Methods for producing preparations of recombinant aav virions substantially free of empty capsids
US20120219528A1 (en) 1998-12-03 2012-08-30 Sista Hema S Excipients for use in adeno-associated virus pharmaceutical formulations, and pharmaceutical formulations made therewith
US20130072548A1 (en) 2010-01-28 2013-03-21 John Fraser Wright Scalable Manufacturing Platform for Viral Vector Purification and Viral Vectors So Purified for Use in Gene Therapy
US20130136727A1 (en) 2009-11-19 2013-05-30 President And Fellows Of Harvard College Angiogenin and Variants Thereof for Treatment of Neurodegenerative Diseases
US20140037585A1 (en) 2011-02-14 2014-02-06 The Children's Hospital Of Philadelphia AAV8 Vector with Enhanced Functional Activity and Methods of Use Thereof
US8692332B2 (en) 2010-01-14 2014-04-08 United Microelectronics Corp. Strained-silicon transistor and method of making the same
WO2014127196A1 (fr) 2013-02-15 2014-08-21 The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services Vecteur d'expression de rétinoschisine aav8 pour le traitement de la rétinoschisite liée à l'x
US20150105350A1 (en) 2012-02-03 2015-04-16 Gilead Sciences, Inc. Combination therapy comprising tenofovir alafenamide hemifumarate and cobicistat for use in the treatment of viral infections
US20150246067A1 (en) * 2009-05-21 2015-09-03 The Regents Of The University Of Michigan Antiviral treatment of lymphoma and cancer
WO2017059122A1 (fr) 2015-09-29 2017-04-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Méthodes de traitement et de prévention d'une sclérose latérale amyotrophique
WO2019040664A1 (fr) 2017-08-22 2019-02-28 Salk Institute For Biological Studies Méthodes et compositions de ciblage d'arn
US10392616B2 (en) 2017-06-30 2019-08-27 Arbor Biotechnologies, Inc. CRISPR RNA targeting enzymes and systems and uses thereof
WO2020142629A1 (fr) * 2019-01-02 2020-07-09 The General Hospital Corporation Agents de blocage de la transcriptase inverse et leurs procédés d'utilisation
US10723787B2 (en) 2017-01-20 2020-07-28 Geneuro Sa Methods for alleviating symptoms of sporadic amyotrophic lateral sclerosis by neutralizing a HERV-K envelope protein using an anti-HERV-K envelope protein antibody
WO2021050980A1 (fr) 2019-09-13 2021-03-18 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Cible à potentiel pharmacologique pour traiter la dégénérescence rétinienne
US10981976B2 (en) 2016-08-31 2021-04-20 University Of Rochester Human monoclonal antibodies to human endogenous retrovirus K envelope (HERV-K) and use thereof
US11293011B2 (en) 2017-03-28 2022-04-05 Locanabio, Inc. CRISPR-associated (CAS) protein
US20220280543A1 (en) * 2019-08-23 2022-09-08 University Of Virginia Patent Foundation Ddx17 and nlrc4 targeting for inflammatory diseases
US20220332799A1 (en) * 2019-09-04 2022-10-20 Deutsches Zentrum Für Neurodegenerative Erkrankungen E.V. (Dzne) Herv inhibitors for use in treating tauopathies

Patent Citations (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4036945A (en) 1976-05-03 1977-07-19 The Massachusetts General Hospital Composition and method for determining the size and location of myocardial infarcts
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4331647A (en) 1980-03-03 1982-05-25 Goldenberg Milton David Tumor localization and therapy with labeled antibody fragments specific to tumor-associated markers
US5091309A (en) 1986-01-16 1992-02-25 Washington University Sindbis virus vectors
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (fr) 1986-01-30 1990-11-27 Cetus Corp
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1990002809A1 (fr) 1988-09-02 1990-03-22 Protein Engineering Corporation Production et selection de proteines de liaison diversifiees de recombinaison
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5217879A (en) 1989-01-12 1993-06-08 Washington University Infectious Sindbis virus vectors
WO1991017271A1 (fr) 1990-05-01 1991-11-14 Affymax Technologies N.V. Procedes de triage de banques d'adn recombine
WO1992001047A1 (fr) 1990-07-10 1992-01-23 Cambridge Antibody Technology Limited Procede de production de chainon de paires a liaison specifique
WO1992020791A1 (fr) 1990-07-10 1992-11-26 Cambridge Antibody Technology Limited Methode de production de chainons de paires de liaison specifique
WO1992009690A2 (fr) 1990-12-03 1992-06-11 Genentech, Inc. Methode d'enrichissement pour des variantes de l'hormone de croissance avec des proprietes de liaison modifiees
WO1992015679A1 (fr) 1991-03-01 1992-09-17 Protein Engineering Corporation Phage de visualisation d'un determinant antigenique ameliore
WO1992018619A1 (fr) 1991-04-10 1992-10-29 The Scripps Research Institute Banques de recepteurs heterodimeres utilisant des phagemides
WO1993001288A1 (fr) 1991-07-08 1993-01-21 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Phagemide utile pour trier des anticorps
US5858988A (en) 1993-02-24 1999-01-12 Wang; Jui H. Poly-substituted-phenyl-oligoribo nucleotides having enhanced stability and membrane permeability and methods of use
US6291438B1 (en) 1993-02-24 2001-09-18 Jui H. Wang Antiviral anticancer poly-substituted phenyl derivatized oligoribonucleotides and methods for their use
US5443505A (en) 1993-11-15 1995-08-22 Oculex Pharmaceuticals, Inc. Biocompatible ocular implants
US5766242A (en) 1993-11-15 1998-06-16 Oculex Pharmaceuticals, Inc. Biocompatible ocular implants
US6251090B1 (en) 1994-12-12 2001-06-26 Robert Logan Avery Intravitreal medicine delivery
US5869079A (en) 1995-06-02 1999-02-09 Oculex Pharmaceuticals, Inc. Formulation for controlled release of drugs by combining hydrophilic and hydrophobic agents
US6299895B1 (en) 1997-03-24 2001-10-09 Neurotech S.A. Device and method for treating ophthalmic diseases
US5935946A (en) 1997-07-25 1999-08-10 Gilead Sciences, Inc. Nucleotide analog composition and synthesis method
US20120219528A1 (en) 1998-12-03 2012-08-30 Sista Hema S Excipients for use in adeno-associated virus pharmaceutical formulations, and pharmaceutical formulations made therewith
US6413540B1 (en) 1999-10-21 2002-07-02 Alcon Universal Ltd. Drug delivery device
US6416777B1 (en) 1999-10-21 2002-07-09 Alcon Universal Ltd. Ophthalmic drug delivery device
US7390791B2 (en) 2000-07-21 2008-06-24 Gilead Sciences, Inc. Prodrugs of phosphonate nucleotide analogues
US7803788B2 (en) 2000-07-21 2010-09-28 Gilead Sciences, Inc. Prodrugs of phosphonate nucoleotide analogues
US6699493B2 (en) 2000-11-29 2004-03-02 Oculex Pharmaceuticals, Inc. Method for reducing or preventing transplant rejection in the eye and intraocular implants for use therefor
WO2003033027A2 (fr) 2001-10-19 2003-04-24 University Of Strathclyde Dendrimeres utilises pour une administration ciblee
WO2003037379A1 (fr) 2001-10-30 2003-05-08 Degussa Ag Utlisation de materiaux granuleux a base de dioxyde de silicium produit de façon pyrogenique dans des compositions pharmaceutiques
US20050054567A1 (en) 2001-11-06 2005-03-10 Rowlinson Scott William Use of il-19,il-22 and il-24 to treat hematopoietic disorders
US6780324B2 (en) 2002-03-18 2004-08-24 Labopharm, Inc. Preparation of sterile stabilized nanodispersions
US20120135515A1 (en) 2003-05-21 2012-05-31 Guang Qu Methods for producing preparations of recombinant aav virions substantially free of empty capsids
US20080019979A1 (en) 2006-05-22 2008-01-24 Feng Wang-Johanning HERV-K Antigens, Antibodies, and Methods
US20090175953A1 (en) 2006-07-13 2009-07-09 Doris Angus Nanodispersions
US20110229971A1 (en) 2008-01-29 2011-09-22 Applied Genetic Technologies Corporation Recombinant virus production using mammalian cells in suspension
US20120100606A1 (en) 2009-04-02 2012-04-26 Sergei Zolotukhin Inducible System for Highly Efficient Production of Recombinant Adeno-Associated Virus (rAAV) Vectors
US20150246067A1 (en) * 2009-05-21 2015-09-03 The Regents Of The University Of Michigan Antiviral treatment of lymphoma and cancer
US20130136727A1 (en) 2009-11-19 2013-05-30 President And Fellows Of Harvard College Angiogenin and Variants Thereof for Treatment of Neurodegenerative Diseases
US8692332B2 (en) 2010-01-14 2014-04-08 United Microelectronics Corp. Strained-silicon transistor and method of making the same
US20130072548A1 (en) 2010-01-28 2013-03-21 John Fraser Wright Scalable Manufacturing Platform for Viral Vector Purification and Viral Vectors So Purified for Use in Gene Therapy
US20140037585A1 (en) 2011-02-14 2014-02-06 The Children's Hospital Of Philadelphia AAV8 Vector with Enhanced Functional Activity and Methods of Use Thereof
US20150105350A1 (en) 2012-02-03 2015-04-16 Gilead Sciences, Inc. Combination therapy comprising tenofovir alafenamide hemifumarate and cobicistat for use in the treatment of viral infections
WO2014127196A1 (fr) 2013-02-15 2014-08-21 The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services Vecteur d'expression de rétinoschisine aav8 pour le traitement de la rétinoschisite liée à l'x
WO2017059122A1 (fr) 2015-09-29 2017-04-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Méthodes de traitement et de prévention d'une sclérose latérale amyotrophique
US10981976B2 (en) 2016-08-31 2021-04-20 University Of Rochester Human monoclonal antibodies to human endogenous retrovirus K envelope (HERV-K) and use thereof
US10723787B2 (en) 2017-01-20 2020-07-28 Geneuro Sa Methods for alleviating symptoms of sporadic amyotrophic lateral sclerosis by neutralizing a HERV-K envelope protein using an anti-HERV-K envelope protein antibody
US11293011B2 (en) 2017-03-28 2022-04-05 Locanabio, Inc. CRISPR-associated (CAS) protein
US10392616B2 (en) 2017-06-30 2019-08-27 Arbor Biotechnologies, Inc. CRISPR RNA targeting enzymes and systems and uses thereof
WO2019040664A1 (fr) 2017-08-22 2019-02-28 Salk Institute For Biological Studies Méthodes et compositions de ciblage d'arn
WO2020142629A1 (fr) * 2019-01-02 2020-07-09 The General Hospital Corporation Agents de blocage de la transcriptase inverse et leurs procédés d'utilisation
US20220280543A1 (en) * 2019-08-23 2022-09-08 University Of Virginia Patent Foundation Ddx17 and nlrc4 targeting for inflammatory diseases
US20220332799A1 (en) * 2019-09-04 2022-10-20 Deutsches Zentrum Für Neurodegenerative Erkrankungen E.V. (Dzne) Herv inhibitors for use in treating tauopathies
WO2021050980A1 (fr) 2019-09-13 2021-03-18 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Cible à potentiel pharmacologique pour traiter la dégénérescence rétinienne
US20220339127A1 (en) 2019-09-13 2022-10-27 The United States Of America,As Represented By The Secretary,Department Of Health And Human Services Druggable target to treat retinal degeneration

Non-Patent Citations (136)

* Cited by examiner, † Cited by third party
Title
"Eukaryotic Viral Vectors", 1982, COLD SPRING HARBOR LABORATORY
"GENBANK", Database accession no. NP_001091046
"New Dimension In Biological Analyses", 1980, PLENUM PUBLISHING CORP.
"Remington's Pharmaceutical Sciences", 1995, MACK PUBLISHING CO.
ALTSCHUL ET AL., J MOL BIOL, vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL.: "Pierce Catalog and Handbook", vol. 6, 1994, PIERCE CHEMICAL CO., pages: 119 - 129
AMBATI ET AL., INVEST. OPTHALMOL. VIS. SCI, vol. 41, 2000, pages 1186 - 1191
BARANYMERRIFIELD, THE PEPTIDES: ANALYSIS, SYNTHESIS, BIOLOGY, vol. 2
BARBAS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 88, 1991, pages 7978 7982
BEAUCAGE ET AL., TETRA. LETT, vol. 22, 1981, pages 1859 - 1862
BEAUCAGECARUTHERS, TETRA. LETTS, vol. . 22, no. 20, 1981, pages 1859 - 1862
BEERMANN, CELL. MOL. BIOL, vol. 45, no. 7, 1999, pages 961 - 8
BERNSTEIN ET AL., NATURE, vol. 409, 2001, pages 363 - 366
BITTER ET AL., METHODS IN ENZYMOLOGY, vol. 153, 1987, pages 516 - 544
BRANDYOPADHYAY ET AL., MOL. CELL BIOL., vol. 4, 1984, pages 1730 - 1737
BREAKFIELD ET AL., MOL. NEUROBIOL., vol. 1, 1987, pages 337 - 371
BRITO ET AL.: "Self-amplifying mRNA vaccines", ADV GENET., vol. 89, 2015, pages 179 - 233, XP055301432, DOI: 10.1016/bs.adgen.2014.10.005
BROWN ET AL., J. IMMUNOL, vol. 127, 1981, pages 539 46
BROWN ET AL., MET . ENZYMOL, vol. 68, 1979, pages 109 - 151
BUCHNER ET AL., ANAL. BIOCHEM, vol. 205, 1992, pages 263 - 270
BUCHSCHALCHER ET AL., J. VIROL., vol. 66, 1992, pages 2731 - 2739
CANCHURCH, NATURE BIOTECHNOLOGY, vol. 27, no. 12, 2009, pages 1151 - 62
CARTER ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 89, 1992, pages 4285
CECH, J. AMER. MED. ASSN, vol. 260, 1988, pages 3030
CORPET ET AL., NUCLEIC ACIDS RESEARCH, vol. 16, 1988, pages 10881 - 10890
DAMDINDORJ ET AL., PLOS ONE, vol. 9, no. 8, 2014, pages e106472
EDELMAN ET AL.: "Methods in Enzymology", vol. 1, 1967, ACADEMIC PRESS, pages: 422
EGERER ET AL., MOL. THER, vol. 19, no. 7, 2011, pages 1236 - 1244
ESUMI ET AL., HUM. MOL. GEN., vol. 18, 2009, pages 128 - 141
FAGANAL-QURESHI, CLIN. EXPERIMENT, 2013
FINK ET AL., HUM. GENE THER, vol. 3, 1992, pages 11 - 19
FRALEY ET AL., TRENDS BIOCHEM. SCI, vol. 6, 1981, pages 77
FRESSE ET AL., BIOCHEM. PHARMACOL., vol. 40, 1990, pages 2189 - 2199
FROLOV ET AL., PROC. NATL. ACAD. SCI. USA, vol. 93, 1996, pages 11371 - 11377
GAPBESTFITFASTA: "Genetics Computer Group", vol. 575, SCIENCE DR., article "TFASTA in the Wisconsin Genetics Software Package"
GEALL ET AL.: "Nonviral delivery of self-amplifying RNA vaccines", PNAS, vol. 109, no. 36, 2012, pages 14604 - 14609
GEFTER, M. L ET AL., SOMATIC CELL GENET, vol. 3, 1977, pages 231 36
GHOSH ARKASUBHRA ET AL: "Elevated Angiogenin levels associated with both forms of age-related macular degeneration is regulated by hypoxia and telomerase", 1 June 2017 (2017-06-01), XP093171200, Retrieved from the Internet <URL:https://iovs.arvojournals.org/article.aspx?articleid=2639024> *
GHOSH ET AL., GENE THER, vol. 13, no. 4, 2006, pages 321 - 329
GRUBER ET AL., CELL, vol. 106, no. 1, 2008, pages 23 - 24
H. HERWEIJER ET AL., HUMAN GENE THERAPY, vol. 6, 1995, pages 1161 - 1167
HELENE, C., ANTICANCER DRUG DESIGN, vol. 6, no. 6, 1991, pages 569
HOFFMAN ET AL., J. VIROL, vol. 94, no. 23, 2000, pages e01682 - 20
HOOGENBOOM ET AL., NUCLEIC ACIDS RES, vol. 19, 1991, pages 4133 4137
HUSE ET AL., SCIENCE, vol. 246, 1989, pages 1275 - 153
IACOVELLI J, INVEST OPHTHALMOL VIS SCI, vol. 52, no. 3, 14 March 2011 (2011-03-14), pages 1378 - 83
JABS DA, AM J OPHTHALMOL, vol. 159, no. 6, June 2015 (2015-06-01), pages 1115 - 1122
JACINTO ET AL., J CELL MOL MED, vol. 24, pages 3766 - 3778
JANG ET AL., EXPERT REV. MEDICAL DEVICES, vol. 1, 2004, pages 127 - 138
JANJIC ET AL., BMC ORAL HEALTH, vol. 17, 2017, pages 87
JINEK, SCIENCE, vol. 337, 2012, pages 816 - 821
JOEL GRUCHOT, BRAIN, BEHAVIOR, AND IMMUNITY, vol. 107, 2023, pages 242 - 252
JOHNSON ET AL., J. VIROL.,, vol. 66, 1992, pages 29522965
JONES ET AL., NATURE, vol. 321, 1986, pages 522
KANAZE ET AL., DRUG DEV. INDUS. PHARM, vol. 36, 2010, pages 292 - 301
KANAZE ET AL., J. APPL. POLYMER SCI., vol. 102, 2006, pages 460 - 471
KOHLERMILSTEIN, NATURE, vol. 256, 1995, pages 495 49
KOTANI ET AL., CURR. GENE THER, vol. 4, 2004, pages 183 - 194
KOTLER ET AL., PNAS, vol. 85, 1988, pages 4185 - 4189
KOZBOR ET AL., IMMUNOL. TODAY, vol. 4, 1983, pages 72
KUBY, J.: "Immunology", 1997, W.H. FREEMAN & CO.
LATORRE ET AL., ANGEWANDTE CHEMIE, vol. 55, 2016, pages 3548 - 50
LAURENCIN ET AL., CLIN. ORTHOPAED. REL. RES, vol. 447, pages 221 - 236
LEBKOWSKI ET AL., MOL. CELL. BIOL., vol. 8, 1988, pages 3988 - 3996
LERNER, E. A, YALE J. BIOL. MED, vol. 54, 1981, pages 387 402
LI ET AL., G3, vol. 10, no. 2, 2020, pages 467 - 473
LI W, SCI TRANSLMED, vol. 7, no. 307, 30 September 2015 (2015-09-30)
LIM ET AL., THE LANCET, vol. 379, no. 9827, 2012, pages 1728 - 1738
MA ET AL., BIOMED RESEARCH INTERNATIONA, vol. 2013, 2013
MADZAK ET AL., J. GEN. VIROL., vol. 73, 1992, pages 15331536
MAGINI ET AL.: "Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge", PLOS ONE, vol. 11, no. 8, 2016, pages e0161193, XP055397457, DOI: 10.1371/journal.pone.0161193
MAHER ET AL., ANTISENSE RES. AND DEV, vol. 1, no. 3, 1991, pages 227
MANN ET AL., J. VIROL., vol. 54, 1985, pages 401 - 407
MANNINO ET AL., BIOTEC NIQUES, vol. 6, 1988, pages 682
MARCUS-SAKURA, ANAL. BIOCHEM., vol. 172, 1988, pages 289
MARGOLSKEE, CURR. TOP. MICROBIOL. IMMUNOL.,, vol. 158, 1992, pages 67 - 90
MATTAPALLIL, INVE T. OPHTHALMOL. VIS. SCI., vol. 53, pages 2921 - 2927
MERRIFIELD ET AL., J. AM. CHEM. SOC., vol. 85, 1963, pages 2149 - 2156
MILLER ET AL., MOL. CELL BIOL., vol. 5, 1985, pages 77 96 - 437
MILLER, CURR. TOP. MICROBIOL. IMMUNOL., vol. 158, 1992, pages 1 - 24
MITCHELL ET AL., THE LANCET, vol. 392, no. 10153, 2018, pages 1147 - 1159
MIYAGISHIMA ET AL., COMMUN. BIO, vol. 4, 2021, pages 1360
MURISIER ET AL., DEV. BIOL, vol. 303, 2007, pages 838 - 847
NAITO ET AL., BIOINFORMATICS, 20 November 2014 (2014-11-20)
NARANG ET AL., METH. ENZYMOL, vol. 68, 1979, pages 109 - 151
NARANG ET AL., METH. ENZYMOL., vol. 68, 1979, pages 90 - 99
NATALIA MAREK ET AL: "Decreased angiogenin concentration in vitreous and serum in proliferative diabetic retinopathy", MICROVASCULAR RESEARCH, ACADEMIC PRESS, US, vol. 82, no. 1, 18 April 2011 (2011-04-18), pages 1 - 5, XP028374273, ISSN: 0026-2862, [retrieved on 20110423], DOI: 10.1016/J.MVR.2011.04.006 *
NEEDHAM-VANDEVANTER ET AL., NUCL. ACIDS RES, vol. 12, 1984, pages 6159 - 6168
NICOLETTI ET AL., INVESTS OPHTHALMOL. VIS. SCI, vol. 39, no. 3, March 1998 (1998-03-01), pages 637 - 44
NISONHOFF ET AL., ARCH. BIOCHEM. BIOPHYS, vol. 89, 1960, pages 230
OPHTHALMOL, vol. 41, no. 5, pages 500 - 7
ORTOLAN, D, PNAS, vol. 119, no. 19, 6 May 2022 (2022-05-06), pages e2117553119
PACK ET AL., BIO/TECHNOLOGY, vol. 11, 1993, pages 1271
PAGE ET AL., J. VIROL., vol. 64, 1990, pages 5370 - 5276
PAVANIAMENDOLA, FRONT GENOME ED, 20 January 2021 (2021-01-20), Retrieved from the Internet <URL:https://doi.org/10.3389/fgeed.2020.609650>
PEARSONLIPMAN, PROC NATL ACAD SCI USA, vol. 85, 1988, pages 2444 - 2448
PEDEN ET AL., PLOS ONE, vol. 6, no. 2, 2011, pages e17140
PETSCH ET AL.: "Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection", NATURE BIOTECHNOLOGY, vol. 30, no. 12, 2012, pages 1210 - 6, XP055051005, DOI: 10.1038/nbt.2436
PEYMAN ET AL., RETINA, vol. 29, no. 7, 2009, pages 875 - 912
PLUCKTHUN, BIOTECHNOLOGY, vol. 9, 1991, pages 545
PORTER, BIOCHEM. J, vol. 73, no. 119, pages 1959
REZWAN ET AL., BIOMATERIALS, vol. 27, 2006, pages 3413 - 3431
RIECHMANN ET AL., NATURE, vol. 334, 1988, pages 585
S. SCHLESINGER, TRENDS BIOTECHNOL, vol. 11, 1993, pages 18 - 22
SALLE ET AL., SCIENCE, vol. 259, 1993, pages 988 - 990
SAMULSKI ET AL., J. VIROL., vol. 61, 1987, pages 3096 - 3101
SAMULSKI ET AL., J. VIROL., vol. 63, 1989, pages 3822 - 3828
SANDHU, CRIT. REV. BIOTECH, vol. 12, 1992, pages 437
SAXENA ET AL., BIOCHEMISTRY, vol. 48, 1970, pages 5015 - 5021
SCHMITTGENLIVAK, NAT PROTOC., vol. 3, no. 6, 2008, pages 1101 - 8
SCHNEIDER ET AL., PL S ONE, vol. 13, no. 11, pages e0207222
SCHNEIDER ET AL., PLOS ONE, vol. 13, no. 11, pages e0207222
SCHORNMAT-TICISSERT, TRENDS CELL BIOL, vol. 28, no. 10, 2018, pages 793 - 906
SHARMA ET AL., NATURE COMM, vol. 12, 2021, pages 7293
SHARMA ET AL., NATURE COMM., vol. 12, 2021, pages 7293
SHARMA R, NAT COMMUN, vol. 12, no. 1, 15 December 2021 (2021-12-15), pages 7293
SHORN ET AL., TRENDS CELL BIOL., vol. 28, no. 10, October 2018 (2018-10-01), pages 793 - 806
SINGER ET AL., J. IMMUNOL, vol. 150, 1993, pages 2844
SMITHASRINIVASACHARBADARINARAYAN, NUCLEIC ACIDS RESEARCH, vol. 48, no. 19, 4 November 2020 (2020-11-04), pages 10890 - 10908
SPECIAL METHODS IN PEPTIDE SYNTHESIS, PART A, pages 3 - 284
STEYER ET AL., DRUG DISCOV TODAY TECHNOL, vol. 28, 2018, pages 3 - 12
STRATFORD-PERRICAUDET ET AL., J. CLIN. INVEST., vol. 90, 1992, pages 626 - 630
TAKEDA ET AL., BIOCHEM. BIOPHYS. RES. COMM, vol. 300, 2003, pages 908 - 914
TAO ET AL., J CELL PHYSIOL., vol. 235, 2020, pages 683 - 690
TYAGI ET AL.: "Inhibition of human endogenous retrovirus-K by antiretroviral drugs", RETROVIROLOGY, vol. 14, 2017, pages 21
VAN DER KUYL, A.C, RETROVIROLOGY, vol. 9, 2012, pages 6
VERHOEYEN ET AL., SCIENCE, vol. 242, 1988, pages 1534
WARD ET AL., NATURE, vol. 341, 1989, pages 544
WEINTRAUB, SCIENTIFIC AMERICAN, vol. 262, 1990, pages 40
WHITLOW ET AL., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, vol. 2, 1991, pages 97
WITTGROVE CARLI ET AL: "AngiogAngiogenin is a potent modulator of neovascularization and inflammation that is upregulated in various human retinal diseases", 30 April 2014 (2014-04-30), XP093171218, Retrieved from the Internet <URL:https://iovs.arvojournals.org/article.aspx?articleid=2267566> *
WU ET AL., J. BIOL. CHEM, vol. 262, 1987, pages 4429
YEH ET AL., PROC. NATL. ACAD. SCI, vol. 76, 1976, pages 2927 31
ZAMORE, SCIENCE, vol. 296, 2002, pages 1265 - 1269
ZHOU ET AL., MOL. NEUROL, vol. 59, 9 July 2022 (2022-07-09), pages 5891 - 5901
ZUKERSTIEGLER, NUCLEIC ACIDS RES, vol. 9, 1981, pages 133 - 148

Also Published As

Publication number Publication date
AU2024240009A1 (en) 2025-09-18

Similar Documents

Publication Publication Date Title
Guziewicz et al. Recombinant AAV-mediated BEST1 transfer to the retinal pigment epithelium: analysis of serotype-dependent retinal effects
TWI698240B (zh) 使用腺相關病毒(aav)sflt-1治療老年性黃斑部退化(amd)
US20180325992A1 (en) Modulation of axon degeneration
Borrás et al. Inducible scAAV2. GRE. MMP1 lowers IOP long-term in a large animal model for steroid-induced glaucoma gene therapy
CN113966236A (zh) 眼睛病状的基因疗法
US20240384295A1 (en) Compositions and methods for inhibiting viral vector-induced inflammatory responses
US20210348193A1 (en) Compositions and methods for treating retinitis pigmentosa
Bogner et al. Capsid mutated adeno-associated virus delivered to the anterior chamber results in efficient transduction of trabecular meshwork in mouse and rat
Boyd et al. Reduced retinal transduction and enhanced transgene-directed immunogenicity with intravitreal delivery of rAAV following posterior vitrectomy in dogs
US20240100081A1 (en) Compositions and methods for treating age-related macular degeneration and geographic atrophy
US20220241316A1 (en) 25-hydroxycholesterol (25hc), cryab aggregation inhibitor, is a novel senolytic
Chambers et al. Lipid nanoparticle-mediated delivery of mRNA into the mouse and human retina and other ocular tissues
US20240050529A1 (en) Modulating lymphatic vessels in neurological disease
WO2024196814A1 (fr) Méthodes de traitement de la dégénérescence maculaire liée à l&#39;âge
US20250049815A1 (en) 25-hydroxycholesterol (25hc), cryab aggregation inhibitor to amelioriate vascular stiffness
CN116218985A (zh) Tbx家族转录因子作为靶点在制备用于治疗眼部疾病的药物中的应用
KR20180109278A (ko) 종간 교차활성을 지닌 mTOR를 표적으로 하는 siRNA를 함유하는 망막혈관질환 치료용 조성물
US20250186546A1 (en) Compositions and Methods to Prevent, Inhibit or Treat Autoimmune and Vascular Disease
CN112888440A (zh) Akt抑制剂在眼科中的用途
WO2025184515A1 (fr) Administration à base de microvésicules à médiation par arrdc1 d&#39;agents thérapeutiques à des cellules et des tissus de l&#39;œil
US20220401580A1 (en) Optidicer construct for age-related macular degeneration
Chern The Development of a Prostaglandin-Based Gene Therapy for the Treatment of Glaucoma
US20240301040A1 (en) Anti-herv-w envelope protein antibody for use in the treatment of psychotic diseases
JP2025532963A (ja) 眼の細胞および組織への治療用薬剤のａｒｒｄｃ１介在性の微小小胞体に基づく送達
HK40044304A (en) Use of akt inhibitors in ophthalmology

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24720357

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: AU2024240009

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2024240009

Country of ref document: AU

Date of ref document: 20240315

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2024720357

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2024720357

Country of ref document: EP

Effective date: 20251017