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WO2018118567A1 - Administration de thérapies antivirales - Google Patents

Administration de thérapies antivirales Download PDF

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Publication number
WO2018118567A1
WO2018118567A1 PCT/US2017/066049 US2017066049W WO2018118567A1 WO 2018118567 A1 WO2018118567 A1 WO 2018118567A1 US 2017066049 W US2017066049 W US 2017066049W WO 2018118567 A1 WO2018118567 A1 WO 2018118567A1
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composition
agent
virus
nanoparticle
programmable nuclease
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Inventor
Derek D. Sloan
Xin Cindy XIONG
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Agenovir Corp
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Agenovir Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5184—Virus capsids or envelopes enclosing drugs
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention generally relates to targeted delivery of antiviral therapies from a site of topical application to an infected cell.
  • Viruses are a significant medical problem. Viral infections can cause physical discomfort or significant pain, cancer, immune deficiency, and death. During an active infection, a virus replicates, makes new proteins, and releases new viral particles. The active infection can cause the death of the host cell. Some viruses have the ability to go into a latent stage or a persistent stage of infection, in which the virus does not replicate itself as it does in the active stage. For some viruses, in the latent phase, the viral genome is maintained in the host cell as an episome, such as a closed circular DNA molecule that replicates independently of the host chromosomes.
  • compositions for the topical delivery of a programmable nuclease for antiviral therapy use agents that promote topical penetration of the nuclease.
  • compositions of the invention are useful for transdermal, transmucosal, transneural, and translingual delivery. Delivery agents may also be used to target the cells to be treated with compositions of the invention.
  • a programmable nuclease for use in compositions of the invention is applied topically (e.g., to the skin, a mucosal region, the eye, the tongue, etc.) and, once it penetrates the area of topical application, is delivered to an infected cell population.
  • the delivery agent may include proteases to help permeate points of cellular adhesion; cell- penetrating peptides to promote uptake of the therapeutic across the membrane of an infected cell; or a ligand that binds to a cell-surface receptor and triggers uptake by receptor-mediated endocytosis.
  • the programmable nuclease is a Cas endonuclease, or a messenger RNA (mRNA) encoding the Cas endonuclease, and is delivered along with a guide RNA that includes a recognition sequence substantially complementary to a target within the genome of the infecting virus.
  • mRNA messenger RNA
  • compositions include a Cas endonuclease and a guide RNA that targets the HPV genome.
  • the nuclease and guide RNA may be together as an active RNP or the nuclease may be provided as mRNA to be translated within the infected cell.
  • the RNP or mRNA is preferably packaged in a nanoparticle delivery vector, such as a liposome or lipid nanoparticle, that promote penetration through tissue and, optionally, that target infected cells.
  • any suitable agents may be included to promote penetration from the site of delivery.
  • glycoconjugates of the RNP, mRNA, or a nanoparticle may be used to mediate cellular uptake.
  • a ligand specific for a cell-surface receptor such as survivin, may be conjugated to the RNP, mRNA or the nanoparticle to promote uptake by infected cells in the basal epithelium.
  • the RNP, mRNA or nanoparticle is conjugated to HPV LI or L2 capsid proteins to promote receptor-mediated endocytosis to deliver the Cas endonuclease into the infected cells.
  • the antiviral nuclease may be delivered as RNP or mRNA along with one or more proteases that cleave cell adhesion proteins to allow the nuclease to penetrate, e.g., anal or vaginal epithelium to treat infections such as HPV infections, which are known to be a dominant cause of cervical cancer.
  • compositions of the disclosure include agents that promote tissue penetration and optionally cellular uptake, the compositions are suited for topical delivery of antiviral therapeutic. Since the antiviral therapeutic can be delivered topically, difficulties such as dosing or off-target activity associated with systemic delivery can be avoided. Because systemic delivery is avoided, the delivery site and dosages can be selected for effective elimination of the viral infection.
  • compositions may be used to treat viral infections including both active as well as latent or persistent infections. Because the included agents promote penetration and uptake from the site of delivery to, and into, the infected cells, the viral infection may be fully cleared. Thus, where infections such as high grade, pre-cancerous HPV lesions are targeted, compositions may be used to avoid such unfortunate outcomes as anal or cervical cancer. Thus compositions of the invention may be used to treat and clear viral infections including latent or persistent viral infections and successfully prevent ongoing infections and associated symptoms like lesions, cold sores, or lymphomas such as Burkitt's lymphomas
  • compositions that include
  • compositions herein enhance transport of compositions into tissue and through individual cellular membranes to allow digestion of viral genetic material in target infected cells within tissue.
  • Tissue uptake compounds and methods can include protease combination therapies targeting proteins involved in cell-to-cell adhesion as well transcytosis enabling proteins.
  • Cellular uptake methods may invoke receptor-mediated endocytosis or enhanced basal epithelial cell uptake through the use of glycoconjugates or keratin 5 (K5) and keratin 14 (K14) ligands.
  • the programmable nuclease is a Cas endonuclease with a guide RNA (gRNA) in which the targeting sequence is substantially complementary to a target sequence in a viral genome.
  • Uptake enhancing methods and agents may be specific to the target tissue and may bind different receptors or target different paracellular adhesion proteins based on the anatomical site of topical application (e.g., anal or vaginal epithelium).
  • compositions for treating a viral infection include a programmable nuclease or a nucleic acid encoding the programmable nuclease and a delivery agent that promotes delivery of the composition through a site of topical application and optionally into an infected cell.
  • the delivery agent may be one that promotes transport through epithelial tissue.
  • the programmable nuclease can be a Cas endonuclease.
  • the Cas endonuclease may be complexed with a guide RNA (gRNA) as an active ribonucleoprotein (RNP).
  • gRNA guide RNA
  • RNP active ribonucleoprotein
  • the viral genome may be that of a hepatitis virus such as hepatitis B virus (HBV), an Epstein-Barr virus, a Kaposi's sarcoma-associated herpesvirus (KSHV), a herpes-simplex virus (HSV), a cytomegalovirus (CMV), human papilloma virus (HPV), or Merkel cell polyomavirus.
  • HBV hepatitis B virus
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • HSV herpes-simplex virus
  • CMV cytomegalovirus
  • HPV human papilloma virus
  • Merkel cell polyomavirus Merkel cell polyomavirus.
  • the viral genetic material may be associated with a latent viral infection and compositions of the disclosure, because the targeting sequence guides the programmable nuclease to digest the DNA genome at the site of infection, may be used to treat latent or persistent infections.
  • the RNP is encapsulated in, or delivered via, a non-viral vector or nanoparticle.
  • a non-viral vector is a lipid nanoparticle (LNP).
  • the agent may be disposed on an outer surface of the LNP.
  • the delivery agent may be one that promotes transcytosis, such as one or a combination of transferrin, insulin, or IgA.
  • the agent may be a protein that binds gp340.
  • the agent may comprise a human papillomavirus (HPV) capsid protein.
  • the agent can comprise a sugar such as ⁇ -acetylgalactosamine (GalNAc), fucose, or galactose.
  • GalNAc ⁇ -acetylgalactosamine
  • the delivery agent may be a protein that binds keratin 5 (K5) or keratin 14 (K14).
  • the agent can be a monoclonal antibody that binds K5 or K14.
  • the agent may comprise survivin.
  • the infected cell may be a human papilloma virus -infected dysplastic cell.
  • the delivery agent includes a protease.
  • the protease can target F11R/JAM-A, E-cadherin, Claudin-1, TJP1 (ZO-1), or Occludin.
  • the protease may target keratin, desmosomes, collagen, or integrins.
  • the protease may include one or more of subtilisin, trypsin, and dispase.
  • the Cas endonuclease and the delivery agent may be together as part of a fusion protein.
  • the Cas endonuclease may further be fucosylated or galactosylated.
  • the nucleic acid encoding the Cas endonuclease may be an mRNA that is co-delivered with a guide RNA (gRNA).
  • the nucleic acid encoding the Cas endonuclease may alternatively be DNA provided in a plasmid that also encodes a guide RNA (gRNA).
  • the invention provides a method for cleaving viral genetic material within a target cell. Steps of the method can include administering a protease and a
  • the target tissue may comprise vaginal epithelium.
  • the protease may target Fl 1R/JAM-A, E- cadherin, Claudin-1, TJP1 (ZO-1), or Occludin.
  • the protease may target keratin, desmosomes, collagen, or integrins.
  • the protease may include subtilisin, trypsin, or dispase.
  • the protease may be co-administered with the programmable nuclease or may be administered before the programmable nuclease.
  • the protease and the programmable nuclease may be administered topically to the target tissue.
  • Methods of the invention may include preparing compositions described herein by providing a recombinant gene that encodes the programmable nuclease along with a delivery agent such as an agent that promote transcytosis, paracellular transport, endocytosis, or cell penetration, or nuclear localization (e.g., transferrin, a protease, a capsid protein fragment, a cell penetrating peptide, or a nuclear localization sequence), as well as optionally a linker.
  • the gene may be provided as part of a vector such as a plasmid or an adeno-associated virus and the protein may be expressed in culture, e.g., within E. coli, a Lactobacillus, yeast, or other such organism.
  • compositions and methods may include linking the programmable nuclease to a number of proteins or other molecules discussed below to enhance tissue or cellular uptake through, for example, receptor mediated endocytosis or transcytosis.
  • the linking may include the use of chemical linkers and reagents, click chemistry, or other chemical methods.
  • the uptake facilitating molecule may be attached to a side chain through a linker that may include one or more of a disulfide bond; a thioether; an amine bond; a hydrazine linkage; an amide bond; an imidoester; a peptide bond; maleimide; a click reaction product; one or more five-membered heterocycles; polyethylene glycol (PEG); BM(PEG)n with 1 ⁇ n ⁇ 9; poly lactic-co-glycolic acid (PLGA)-b- PEG; and bio tin.
  • a linker may include one or more of a disulfide bond; a thioether; an amine bond; a hydrazine linkage; an amide bond; an imidoester; a peptide bond; maleimide; a click reaction product; one or more five-membered heterocycles; polyethylene glycol (PEG); BM(PEG)n with 1 ⁇ n
  • FIG. 1 shows a composition that includes a programmable nuclease and an agent that promotes penetration of the nuclease from a site of topical delivery to, and into, cells infected by a virus.
  • FIG. 2 shows a composition that includes a messenger RNA (mRNA) encoding a programmable nuclease, and in which the mRNA is packaged in a nanoparticle that has a delivery agent on a surface thereof.
  • mRNA messenger RNA
  • FIG. 3 illustrates a composition in which a nanoparticle encapsulates a plasmid encoding a programmable nuclease.
  • FIG. 4 shows a composition that includes a delivery agent that promotes transcytosis across epithelial tissue.
  • FIG. 5 shows a composition that includes an agent that promotes paracellular transport.
  • FIG. 6 shows a nanoparticle that encapsulates an antiviral programmable nuclease and is coated with an agent to promotes transcytosis.
  • FIG. 7 shows a composition that includes a programmable nuclease and transferrin as an agent to promote transport across epithelial cells via receptor- mediated endocytosis.
  • FIG. 8 shows a composition that includes a nanoparticle with insulin disposed on its outer surface.
  • FIG. 9 shows a composition that includes a nanoparticle with IgA on its surface.
  • FIG. 10 shows a composition in which a nanoparticle has a gp340-binding protein on its outer surface.
  • FIG. 11 shows a composition that includes a programmable nuclease encapsulated in an nanoparticle along with an agent that promotes delivery into infected cells by promoting receptor- mediated endocytosis.
  • FIG. 12 shows a composition that includes a nanoparticle with a sugar on its surface.
  • FIG. 13 shows a composition that includes a nanoparticle conjugated to GalNAc.
  • FIG. 14 shows a composition that includes a nanoparticle with fucose on its surface.
  • FIG. 15 shows a composition with galactose on a nanoparticle.
  • FIG. 16 shows a a keratin-binding protein on a nanoparticle.
  • FIG. 17 shows an antibody on a nanoparticle encapsulating an antiviral programmable nuclease.
  • FIG. 18 shows a composition that includes nanoparticle with survivin on its surface.
  • FIG. 19 shows a composition with a keratin-binding protein being transported through epithelial tissue from a site of topical application to an HPV-infected dysplastic cell.
  • FIG. 20 shows a composition with an agent is linked to an RNP comprising a
  • programmable nuclease complexed with a guide RNA as part of a fusion protein is programmable nuclease complexed with a guide RNA as part of a fusion protein.
  • FIG. 21 shows a composition in which a programmable nuclease, or nucleic acid encoding a programmable nuclease, is contained in nanoparticle coupled to, coated with, comprising, or otherwise associated with one or more delivery agents.
  • FIG. 22 shows steps of a method of preparing antiviral compositions.
  • FIG. 23 shows steps of a method of treating a viral infection.
  • compositions and methods of the invention relate to targeted delivery of programmable nucleases to infected cells to cleave viral genetic material.
  • the invention provides practical methods and compositions for treatment of viral infections including latent phase viral infections.
  • the invention relates to co-delivery of programmable nucleases with a delivery agent that increases penetration into or uptake by a target tissue or cell.
  • the programmable nuclease may be linked to a transport-promoting agent as a fusion protein or through encapsulation in a non- viral vector having the agent disposed on its outer surface.
  • Programmable nuclease may alternatively not be chemically linked to the uptake enhancing agent but may be administered at approximately the same time to enhance uptake.
  • Programmable nucleases that may be delivered include, for example, Cas9, ZFNs, TALENs, Cpfl, NgAgo, or a modified programmable nuclease having an amino acid sequence substantially similar to the unmodified version, for example, a programmable nuclease having an amino acid sequence at least 90% similar to one of Cas9, ZFNs, TALENs, Cpfl, or NgAgo, or any other programmable nuclease.
  • Programmable nuclease generally refers to an enzyme that cleaves nucleic acid that can be or has been designed or engineered by human contribution so that the enzyme targets or cleaves the nucleic acid in a sequence-specific manner.
  • Programmable nucleases include zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA- guided nucleases such as the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) nucleases or Cpfl.
  • Programmable nucleases also include PfAgo and NgAgo.
  • ZFNs cut genetic material in a sequence- specific matter and can be designed, or programmed, to target specific viral targets.
  • a ZFN is composed of two domains: a DNA- binding zinc-finger protein linked to the Fokl nuclease domain.
  • the DNA-binding zinc-finger protein is fused with the non-specific Fokl cleave domain to create ZFNs.
  • the protein will typically dimerize for activity.
  • Two ZFN monomers form an active nuclease; each monomer binds to adjacent half- sites on the target.
  • the sequence specificity of ZFNs is determined by ZFPs.
  • Each zinc-finger recognizes a 3-bp DNA sequence, and 3-6 zinc-fingers are used to generate a single ZFN subunit that binds to DNA sequences of 9-18 bp.
  • the DNA-binding specificities of zinc-fingers is altered by mutagenesis.
  • New ZFPs are programmed by modular assembly of pre-characterized zinc fingers.
  • Transcription activator-like effector nucleases cut genetic material in a sequence-specific matter and can be designed, or programmed, to target specific viral targets.
  • TALENs contain the Fokl nuclease domain at their carboxyl termini and a class of DNA binding domains known as transcription activator- like effectors (TALEs).
  • TALEs are composed of tandem arrays of 33-35 amino acid repeats, each of which recognizes a single base-pair in the major groove of target viral DNA.
  • nucleotide specificity of a domain comes from the two amino acids at positions 12 and 13 where Asn-Asn, Asn-Ile, His-Asp and Asn-Gly recognize guanine, adenine, cytosine and thymine, respectively. That pattern allows one to program
  • TALENs to target viral nucleic acid.
  • RNA-guided nucleases were first found as part of bacterial immune systems.
  • the host bacteria capture small DNA fragments (-20 bp) from invading viruses and insert those sequences (termed protospacers) into their own genome to form a CRISPR.
  • Those CRISPR regions are transcribed as pre-CRISPR RNA(pre-crRNA) and processed to give rise to target- specific crRNA.
  • Invariable target-independent trans-activating crRNA (tracrRNA) is also transcribed from the locus and contributes to the processing of precrRNA.
  • the crRNA and tracrRNA have been shown to be combinable into a single guide RNA.
  • guide RNA or gRNA refers to either format.
  • the gRNA forms a RNP with Cas9, and the RNP cleaves a target that includes a portion complementary to the guide sequence in the gRNA and a sequence known as proto spacer adjacent motif (PAM).
  • the RNA-guided nucleases are programmed to target a specific viral nucleic acid by providing a gRNA that includes a ⁇ 20-bp guide sequences that is substantially complementary to a target in viral nucleic acid.
  • the targetable sequences include, among others, 5 ' -X 20NGG-3 ' or 5 ' -X 20NAG-3 ' ; where X 20 corresponds to the 20-bp crRNA sequence and NGG and NAG are PAMs. It will be appreciated that recognition sequences with lengths other than 20 bp and PAMs other than NGG and NAG are known and are included within the scope of the invention.
  • FIG. 1 shows a composition 101 that includes a programmable nuclease and an agent 175 that promotes penetration of the nuclease from a site of topical delivery to, and into, cells infected by a virus.
  • the programmable nuclease is a Cas endonuclease 151 complexed with a guide RNA 115 to form an active ribonucleoprotein (RNP) 153.
  • the RNP 153 includes the Cas endonuclease 151 complexed with a gRNA 115 comprising a targeting sequence 401 at least substantially complementary to a target in a viral genome.
  • compositions of the invention include a Cas endonuclease (e.g., as an active RNP 153) or an mRNA encoding the Cas endonuclease.
  • compositions of the invention include a DNA-guided programmable nuclease such as an argonaute.
  • Argonaute proteins are a family of proteins that play a role in RNA silencing as a component of the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the Argonaute of the archaeon Pyrococcus furiosus (PfAgo) uses small 5'-phosphorylated DNA guides to cleave both single stranded and double stranded DNA targets, and does not utilize RNA as guide or target.
  • NgAgo uses 5' phosphorylated DNA guides (so called "gDNAs") and appear to exhibit little preference for any certain guide sequences and thus may offer a general-purpose DNA- guided programmable nuclease.
  • NgAgo does not require a PAM sequence, which contributes to flexibility in choosing a genomic target.
  • NgAgo also appears to outperform Cas9 in GC-rich regions.
  • NgAgo is only 887 amino acids in length.
  • NgAgo randomly removes 1-20 nucleotides from the cleavage site specified by the gDNA.
  • PfAgo and NgAgo represent potential DNA-guided programmable nucleases that may be modified for use as a composition of the invention.
  • the programmable nuclease may be an RNA-guided nuclease such as Cas9 or Cpfl; a modified RNA-guided nuclease, e.g., with an amino acid sequence at least 90% similar to Cas9 or Cpfl; a DNA-guided nuclease such as NgAgo; a modified DNA-guided nuclease, e.g., with an amino acid sequence at least 90% similar to NgAgo; or a TALEN protein engineered to recognize a target in a viral genome but not recognize the patient's genome.
  • RNA-guided nuclease such as Cas9 or Cpfl
  • a modified RNA-guided nuclease e.g., with an amino acid sequence at least 90% similar to Cas9 or Cpfl
  • a DNA-guided nuclease such as NgAgo
  • a modified DNA-guided nuclease e.g., with an amino acid sequence at least 90% similar to
  • Programmable nucleases may be prepared and delivered into cells and tissue as mRNA to be translated into active proteins within the target host cells.
  • the programmable nuclease may be an RNA-guided nuclease complexed with a guide RNA as an active ribonucleoprotein (RNP), wherein the guide RNA is complementary to a target within viral genetic material and is not complementary to any target within a human genome.
  • RNP active ribonucleoprotein
  • the RNP, plasmid DNA, or mRNA is encapsulated in a nanoparticle, which may include, for example, lipids.
  • a nanoparticle which may include, for example, lipids.
  • the RNA-guided nuclease is Cas9.
  • FIG. 2 shows a composition 201 that includes a messenger RNA (mRNA) 2701 encoding a programmable nuclease, and in which the mRNA 2701 is packaged in a nanoparticle 203 that has a delivery agent 111 on a surface thereof.
  • the composition 201 includes a non- viral vector 203 such as an LNP having an agent 111 on its outer surface and containing gRNA 115 and mRNA 2701 encoding a Cas endonuclease.
  • FIG. 3 illustrates a composition 301 in which a nanoparticle 203 encapsulates a plasmid 2801 encoding the programmable nuclease.
  • the nanoparticle 203 has a delivery agent 111 on its outer surface and contains the plasmid 2801 encoding, for example, a Cas endonuclease and a gRNA.
  • compositions include nanoparticles useful in topical delivery such as liposomes, albumin-based particles, PEGylated proteins, biodegradable polymer-drug composites, polymeric micelles, dendrimers, among others. See Davis et al., 2008,
  • Nanotherapeutic particles an emerging treatment modality for cancer, Nat Rev Drug Discov. 7(9):771-782, incorporated by reference.
  • FIG. 4 shows a composition 401 that includes a delivery agent that promotes transcytosis 125 of the composition 401 across epithelial tissue 213 from a site of topical application 133 to an infected cell 135.
  • Transcytosis is a type of transcellular transport that involves transporting a composition into a tissue by passing it through the cells themselves.
  • the composition 401 preferably includes a Cas endonuclease— or an mRNA encoding the Cas endonuclease— with a guide RNA which has a targeting sequence complementary to a target within a viral genome.
  • the Cas endonuclease or the mRNA are packaged in a nanoparticle such as a lipid nanoparticle that is coated with transcytosis enabling proteins such as gp340 antibody, insulin, transferrin, or IgA.
  • gp340 is a human scavenger receptor that mediates transcytosis through vaginal epithelium and thus may be preferred for delivery of an anti-HPV therapeutic.
  • Insulin, transferrin, and IgA are also molecules that promote transport across epithelial cells. Additionally or alternatively, delivery to infected cells may be accomplished after topical delivery via a paracellular route.
  • FIG. 5 shows a composition 501 that includes an agent that promotes paracellular transport and an antiviral programmable nuclease (for example, a protease 1427 and a Cas endonuclease with a guide RNA that targets HPV).
  • the composition 501 including the Cas endonuclease and a protease 1427 is passed in between the cells of a tissue 213 from a site of topical application 133 to an infected cell 135.
  • the protease 1427 can break down paracellular adhesion proteins that may be general to living tissue or specific to the tissue to which the composition is topically applied. As discussed, delivery may be accomplished by paracellular mechanisms, trans-cellular mechanisms, or a combination thereof.
  • FIG. 6 shows a nanoparticle 203 that encapsulates an antiviral programmable nuclease and is coated with an agent 111 to promotes transcytosis.
  • the nanoparticle 202 is preferably a lipid nanoparticle (LNP) and preferably contains a programmable nuclease 107 as described above.
  • the nanoparticle 203 is coated with a protein or other agent 111 having affinity for a cell surface receptor expressed in the cells of epithelial tissue 213. Coatings may be dependent on the target cell or tissue.
  • gp340 is a human scavenger receptor that mediates HIV transcytosis through vaginal epithelium.
  • the non- viral vector 203 or other molecule may be coated 205 with an agent 111 such as gp340 antibody or other gp340-binding proteins to form a coated delivery molecule 207.
  • the coated delivery molecule 207 may then be delivered 209 to the target tissue such as epithelium 213.
  • the gp340 or other surface receptor then facilitates transcytosis 211 of the coated molecule 207 through individual epithelial cells, in turn facilitating passage 213 through the tissue 213.
  • the LNP or other delivery molecules may be coated with, for example, insulin, transferrin, or IgA, each of which are transported across epithelial cells in a receptor- mediated process.
  • FIG. 7 shows a composition 701 that includes a programmable nuclease 151 and transferrin 1401 as an agent to promote transport across epithelial cells via receptor-mediated endocytosis.
  • the composition preferably includes a lipid nanoparticle (LNP) 415 with the transferrin 1401 disposed on its outer surface, the nanoparticle 415 containing a Cas endonuclease 151 complexed with a gRNA 115.
  • LNP lipid nanoparticle
  • FIG. 8 shows a composition 801 that includes an nanoparticle 415 with insulin 1403 disposed on its outer surface, the nanoparticle 415 containing a Cas endonuclease 151 complexed with a gRNA 115.
  • FIG. 9 shows a composition 901 that includes an nanoparticle 415 with IgA 1405 on its outer surface.
  • the nanoparticle 415 contains a Cas endonuclease 151 complexed with a gRNA 115. It may be found that insulin or IgA promote receptor-mediated endocytosis.
  • the programmable nuclease may be delivered as a plasmid or mRNA encoding the nuclease or as an RNP that is in turn delivered in a coated molecule.
  • the delivered agent may be a fusion protein comprising a programmable nuclease RNP or protein coupled to a target-specific transcytosis enhancing agent such as those discussed above (e.g., gp340 antibody or binding protein, insulin, transferrin, or IgA).
  • a target-specific transcytosis enhancing agent such as those discussed above (e.g., gp340 antibody or binding protein, insulin, transferrin, or IgA).
  • Lipid nanoparticles may include a cationic lipid to aid in tissue penetration.
  • FIG. 10 shows a composition 1001 in which an nanoparticle 415 has a gp340-binding protein 1407, such as a gp340 antibody, on its outer surface.
  • the nanoparticle 415 contains a Cas endonuclease 151 complexed with a gRNA 115.
  • the gp340-binding protein will bind to gp340 on epithelial cells and that binding promotes transcytosis of the composition 1001 through epithelial tissue (e.g., to cells of the basal epithelium).
  • the composition 1001 may be particularly effective for treating an HPV infection.
  • programmable nucleases of the invention may be co- delivered with various proteases to help break down paracellular adhesion in target tissue to enhance epithelial paracellular transport.
  • Target- specific proteases may be co-administered with any of the various programmable nucleases discussed herein.
  • Cellular adhesion proteins are in certain instances anatomically distinct, for example, vaginal epithelium includes cellular adhesion proteins such as FUR (JAM-A), E-cadherin, Claudin-1, TJP1 (ZO-1), and Occludin.
  • Programmable nucleases of the invention targeting infected cells within vaginal tissue may be topically delivered to vaginal epithelium along with proteases that target the cellular adhesion proteins listed above, thereby promoting paracellular transport of the programmable nuclease to infected cells from the site of topical application.
  • Topical proteases include, for example, subtilisin, trypsin, and dispase that target keratin, desomsomes, and type IV collagen respectively.
  • Targeting and disrupting proteins involved in cell-to-cell junctions e.g., cell adhesion molecules, integrins, and desmosomes
  • proteases may be co-administered to disrupt tissue- specific cell adhesion molecules (CAM) depending on the target tissue.
  • Targeted agents and methods for enhanced cellular uptake may take advantage of receptor-mediated endocytosis that may, in turn, be tailored to distinct receptors and surface proteins of the target tissue or cells.
  • Agents and methods may target cell surface proteins used for cell entry by various viruses such as human papillomavirus (HPV).
  • HPV genomic material enters a host cell through a multi-step process of viral LI and L2 capsid proteins binding heparin- sulfate proteoglycans, a6 integrin, annexin A2, tetraspanin (CD151, CD63), and
  • TRAPPC8 By associated programmable nucleases of the invention with HPV capsid proteins like LI and L2, compositions and methods of the invention may take advantage of this process to promote endocytosis of the programmable nuclease.
  • FIG. 11 shows a composition that includes a programmable nuclease 107 encapsulated in an nanoparticle 415 (e.g., preferably a lipid nanoparticle that includes cationic lipids), along with an agent that promotes delivery into infected cells by promoting receptor-mediated endocytosis.
  • the delivery agent is an HPV capsid protein 1409.
  • the nanoparticle 415 having a HPV capsid protein 1409 and containing a programmable nuclease 107 binds to the cell surface receptors and is transferred to the entry complex in tetraspanin enriched microdomains before CD- 151 and actin mediated endocytosis occurs, bringing the programmable nuclease 107 into the infected cell 135.
  • fusion proteins including programmable nucleases and HPV LI and L2 capsid proteins cellular uptake of the programmable nuclease may be facilitated via the same cell-mediated endocytotic pathway used by HPV.
  • programmable nucleases of the invention may be introduced in nucleic acid, RNP, or protein form and may be delivered within an HPV-derived capsid.
  • Glycoconjugates such as a programmable nuclease conjugated to beta-D-GLcNAc- substituted polylsine may be used to enhance topical delivery to the basal epithelium.
  • N- acetylgalactosamine (GalNAc) has been shown to mediate hepatocyte uptake allowing delivery of si RNA.
  • agents and methods of the invention relate to enhanced cellular uptake of programmable nucleases through the use of glycoconjugates.
  • Basal epithelial cells express high levels of a-L-fucose and a-D-galactose.
  • agents of the invention targeting basal epithelial cells may include a fucoslated or galactosylated programmable nuclease to facilitate epithelial uptake of the agent after topical delivery.
  • FIG. 12 shows a composition 1201 that includes an nanoparticle 415 with a sugar 1411 on its outer surface.
  • the nanoparticle 415 contains a Cas endonuclease 151 complexed with a gRNA 115. Glycoconjugates of programmable nucleases, nucleic acids encoding the
  • programmable nucleases or nanoparticles encapsulating those programmable nucleases or nucleic acids may be found to enhance topical delivery of the basal epithelium. It may also be found that certain glycoconjugates (e.g., N-acetylgalactosamine) promote hepatocyte uptake and thus may be useful where a programmable nuclease is programmed to target a hepatitis virus such as HBV. It also may be found that basal epithelium uptake is greatly promoted by a sugar such as fucose or galactose.
  • glycoconjugates e.g., N-acetylgalactosamine
  • FIG. 13 shows a composition 1301 that includes a nanoparticle 415 conjugated to GalNAc 1413.
  • the nanoparticle 415 contains a Cas endonuclease 151 complexed with a gRNA 115.
  • the Cas endonuclease, or an mRNA encoding the Cas endonuclease could be conjugated to GalNAc.
  • FIG. 14 shows a composition 1401 that includes a nanoparticle 415 having an agent comprising fucose 1415 on its outer surface.
  • the nanoparticle may include an mRNA encoding the Cas endonuclease.
  • the Cas endonuclease, or an mRNA encoding the Cas endonuclease could be conjugated to the fucose 1415.
  • FIG. 15 shows a composition 1501 that includes a nanoparticle 415 with galactose 1417 on its outer surface, the nanoparticle 415 containing a Cas endonuclease 151 complexed with a gRNA 115. Additionally or alternatively, the nanoparticle may include an mRNA encoding the Cas endonuclease. Additionally or alternatively, the Cas endonuclease, or an mRNA encoding the Cas endonuclease, could be conjugated to the galactose 1417.
  • programmable nucleases of the invention may be fused with ligands targeting keratins 5 (K5) or 14 (K14), expressed as a K5/14 heterodimer on the basal epithelium.
  • K5/K14 ligand or monoclonal antibodies against K5/14 may be fused to a programmable nuclease of the invention and delivered to target epithelial cells.
  • K5 expression has been reported to increase in HPV-infected dysplastic cells. Accordingly, K5 ligand conjugated programmable nucleases may be well suited to treating HPV infections by preferentially targeting HPV-infected dysplastic cells and delivering HPV genome targeting nucleases as described below.
  • FIG. 16 shows a composition 1601 that includes a nanoparticle 415 with a keratin- binding protein 1419 on its outer surface.
  • the nanoparticle 415 contains a Cas endonuclease 151 complexed with a gRNA 115.
  • FIG. 17 shows a composition 1701 that includes an nanoparticle 415 with a monoclonal antibody 1421 disposed on its outer surface.
  • the nanoparticle 415 contains a Cas endonuclease 151 complexed with a gRNA 115.
  • FIG. 18 shows a composition 1801 that includes nanoparticle 415 with survivin 1423 on its outer surface.
  • the nanoparticle 415 contains a Cas endonuclease 151 complexed with a gRNA 115.
  • FIG. 19 shows a composition 1901 comprising a keratin-binding protein 1419 being transported through epithelial tissue 213 from a site of topical application 133 to a HPV-infected dysplastic cell 1425.
  • FIG. 20 illustrates an exemplary embodiment of a composition of the invention wherein an agent 111 is linked to an RNP 153 comprising a programmable nuclease 107 complexed with a guide RNA 115 as part of a fusion protein 525.
  • FIG. 21 illustrates an exemplary embodiment of a composition 2101 of the invention wherein a programmable nuclease or nucleic acid 605 encoding a programmable nuclease is contained in a non- viral vector 607 such as an LNP coupled to, coated with, comprising, or otherwise associated with one or more agents 111.
  • a non- viral vector 607 such as an LNP coupled to, coated with, comprising, or otherwise associated with one or more agents 111.
  • compositions include, as the programmable nuclease, an RNA-guided nuclease (e.g., Cas9) and at least one gRNA targeting the genome of the virus.
  • an RNA-guided nuclease e.g., Cas9
  • at least one gRNA targeting the genome of the virus e.g., Cas9
  • Suitable targets in viral genomes include, but are not limited to, a portion of a genome or gene of adenovirus, herpes simplex virus, varicella- zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus type 8, human papillomavirus, BK virus, JC virus, smallpox, hepatitis B virus, human bocavirus, parvovirus, B 19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, sever acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, dengue virus, west nile virus, rubella virus, hepatitis E virus, human immunodeficiency virus, influenza virus, guanarito virus, Junin virus, Lassa virus, machupo virus, sabia virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, measles virus,
  • compositions of the invention are provided as antiviral therapeutics that include a modified programmable nuclease programmed to treat an infection by a hepatitis virus, a hepatitis B virus (HBV), an Epstein-Barr virus, a Kaposi's sarcoma-associated herpesvirus (KSHV), a herpes-simplex virus (HSV), a cytomegalovirus (CMV), human papilloma virus (HPV), and Merkel cell polyomavirus.
  • the programmable nuclease is programmed (e.g., by a gRNA) to bind to or cleave a particular target.
  • the target in the viral genome may lie within one or more of a preC promoter in a hepatitis B virus (HBV) genome, an S 1 promoter in the HBV genome, an S2 promoter in the HBV genome, an X promoter in the HBV genome, a viral Cp (C promoter) in an Epstein-Barr virus genome, a minor transcript promoter region in a Kaposi's sarcoma-associated herpesvirus (KSHV) genome, a major transcript promoter in the KSHV genome, an Egr-1 promoter from a herpes-simplex virus (HSV), an ICP 4 promoter from HSV-1, an ICP 10 promoter from HSV-2, a cytomegalovirus (CMV) early enhancer element, a cytomegalovirus immediate-early promoter, an HPV early promoter, and an HPV late promoter.
  • HBV hepatitis B virus
  • S 1 promoter in the HBV genome an S2 promoter in the H
  • the virus is hepatitis B and the gRNA includes one or more of sgHBV-RT, sgHBV-Hbx, sgHBV-Core, and sg-HBV-PerS l.
  • compositions and methods can be used to selectively target the HPV genome or selectively express the targetable nuclease within cells that infected by HPV.
  • Agents may be selected in order to target receptors in types of tissue such as basal epithelium or, infected tissue such as precancerous high grade lesions caused by HPV.
  • methods and compositions of the invention use CRISPR guide RNA sequences targeting the HPV E6 and E7 genes.
  • a composition of the invention, such as a DNA vector encoding cas9, may code for gRNAs that are complementary to specific targets within the HBV genome.
  • compositions for treating a viral infection that includes a programmable nuclease linked to a transport-promoting agent 111 via a linkage through a side chain, N-terminus, or C-terminus of an amino acid in a peptide sequence.
  • the programmable nucleases may be DNA or RNA-guided nuclease.
  • RNA-guided nuclease may be Cas9 or a modified Cas9 having an amino acid sequence at least 90% similar to Cas9.
  • the nuclease may be codon-optimized for the host.
  • the programmable nuclease may be present in ribonucleoprotein form with the nuclease complexed with a guide RNA. A portion of the guide RNA is complementary to a target in a viral genome and not substantially
  • the transport-promoting agent may be attached to the programmable nuclease to form a fusion protein.
  • the agent and the programmable nuclease may be joined through a linker, which may include a disulfide bond; a thioether; an amine bond; a hydrazine linkage; an amide bond; an imidoester; a peptide bond; maleimide; polyethylene glycol (PEG); BM(PEG)n with 1 ⁇ n ⁇ 9; and biotin.
  • Embodiments of the invention provide a programmable nuclease linked to an endocytosis or uptake enhancing agent as a composition for modifying genetic material within a target cell. Such a composition may be preferred for its amenability to known approaches to synthesis.
  • a gene for the agent and a gene for the programmable nuclease may be combined via known genetic engineering techniques. The resulting recombinant gene may be expressed using, for example, E. coli or another suitable medium.
  • a linker segment may be included in the recombinant gene to provide a proteinaceous linker between the programmable nuclease and the agent. The gene is expressed and collected (e.g., purified, isolated, and optionally provided within a pharmaceutical carrier or solution).
  • the programmable nuclease is an RNA- guided nuclease
  • a gRNA is provided to complex with the nuclease.
  • the RNA-guided nuclease portion of the recombinant protein may be present in ribonucleoprotein form with the nuclease complexed with a guide RNA, in which a portion of the guide RNA is complementary to a target in genetic material to be modified.
  • the target may be in a viral genome present in the target host cell and the guide RNA may not be substantially complementary to any part of a human genome.
  • An exemplary flexible linker may include a plurality of glycine residues.
  • the programmable nucleases may be delivered to a cell or tissue using a nucleic acid vector such as a plasmid. Additionally, the invention may provide an antiviral therapy that includes a nucleic acid vector encoding a programmable nuclease for delivery to viral-infected cells.
  • the nucleic acid vector is a plasmid and the encoded nuclease is an RNA-guided nuclease such as Cas9, a modified Cas9 (at least 90% similar to wt Cas9), Cpfl, or a modified Cpfl .
  • the programmable nuclease may be encoded as part of a recombinant gene on the plasmid.
  • the plasmid or other nucleic acid encoding the programmable nuclease may be associated with a uptake or endocytosis enhancing agent such as contained in an LNP coated with an endocytosis enhancing agent of the invention or contained in a capsid comprising, for example, LI and/or L2 capsid proteins of HPV.
  • a cellular or tissue uptake enhancing agent may be linked to a programmable nuclease through a linker.
  • a linker may be chosen for its properties. For example, for a polypeptide linker (e.g., within a recombinant fusion protein) to be flexible it may be provided with a plurality of glycine resides (e.g., > 30% or > 50%). For a more rigid polypeptide linker, it may be desirable to include a plurality of proline residues.
  • the linker may be biodegradable.
  • the linker is cleavable.
  • the linker may include an enzyme cleavage region.
  • an enzyme cleave region can be the target of a protease.
  • the agent is non-covalently bound to the programmable nuclease.
  • the programmable nuclease or the agent may be biotinylated and the agent may thus be non-covalently bound to the programmable nuclease through a
  • a composition for treating a viral infection may include a programmable nuclease covalently linked to a uptake enhancing agent through a protein linker.
  • Some recombinant fusion proteins are composed of two or more functional domains joined by linker peptides.
  • the linker serves to connect the proteins, and also provide many other functions, such as maintaining cooperative inter-domain interactions or preserving biological activity.
  • the natural length of linkers in multi-domain proteins is about 6 to 10 residues on average.
  • Preferred residues for linkers include threonine (Thr), serine (Ser), proline (Pro), glycine (Gly), aspartic acid (Asp), lysine (Lys), glutamine (Gin), asparagine (Asn), and alanine (Ala), arginine (Arg), phenylalanine (Phe), and glutamic acid.
  • residues are polar (charged or uncharged).
  • Proline may be included to give the linker rigidity. It is though that the lack of an amide hydrogen, as well as the cyclic side chain, limit proline's ability to participate in promiscuous hydrogen bonding and restrict its flexibility.
  • the small, polar amino acids, such as Thr, Ser, and Gly are thought to be favorable for providing good flexibility due to their small sizes, and also help maintain stability of the linker structure in the aqueous solvent through formation of hydrogen bonds with water.
  • the linker may include a plurality of glycine residues. In some embodiments, the linker comprises a plurality of threonine and serine residues.
  • a composition for treating a viral infection may include a programmable nuclease linked to an uptake enhancing agent (e.g., a target cell surface protein binding agent) through a nonprotein chemical linker.
  • an uptake enhancing agent e.g., a target cell surface protein binding agent
  • a linker may provide functionality such as flexibility, rigidity (e.g., even a mixture of both flexibility and rigidity at different points along it), solubility, cleavage targets, binding targets, others, or combinations thereof.
  • the linker is included to provide a spacer arm.
  • the spacer arm is the chemical chain between two groups.
  • the length of a spacer arm (e.g., in angstroms) determines how flexible a conjugate will be. Longer spacer arms have greater flexibility, reduced steric hindrance, and offer more sites for potential nonspecific binding. Spacer arms can range from zero length to > 100 angstroms.
  • the molecular composition of a crosslinkers spacer arm can affect solubility and nonspecific binding.
  • linkers have spacer arms that contain hydrocarbon chains or polyethylene glycol (PEG) chains. Hydrocarbon chains are not water soluble and typically require an organic solvent such as DMSO or DMF for suspension. Those crosslinkers are suited for penetrating the cell membrane and performing intercellular crosslinking because they are hydrophobic and uncharged. If a charged sulfonate group is added to the termini of such crosslinkers, a water soluble analogue is formed.
  • PEG polyethylene glycol
  • Certain exemplary categories of cross-linkers use bismaleimide-activated PEG
  • BM(PEG)n bis(succinimidyl) PEG
  • BS(PEG)n bis(succinimidyl) PEG
  • BM(PEG)n cross-links sulfhydryls and BS(PEG)n cross-links amines, although variations will be understood by one of skill in the art.
  • an uptake enhancing or endocytosis promoting agent may be attached to a programmable nuclease, optionally through a linker, at an amino acid with a side chain comprising an amine, a carboxyl, a sulfhydryl, or a carbonyl.
  • the agent or the linker may be attached to the programmable nuclease at an amino acid in the nuclease such as lysine, cysteine, aspartic acid, or glutamic acid.
  • a programmable nuclease is linked to an uptake enhancing or endocytosis promoting agent through a click reaction product such as one more five-membered rings or acyclic derivatives thereof.
  • Click chemistry includes a class of biocompatible reactions intended primarily to join substrates of choice with specific biomolecules. Click chemistry provides methods joining small modular units. In general, click reactions usually join a biomolecule and an uptake promoting agent. Typical click reactions occur in one pot, are not disturbed by water, make unremarkable byproducts, and are driving quickly and irreversibly to high yield of a single click reaction product, with high reaction specificity (in some cases, with both regio- and stereo-specificity).
  • the Azide-Alkyne Huisgen Cycloaddition is a 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to give a 1,2,3-triazole.
  • the 1,3-dipolar cycloaddition is a chemical reaction between a 1,3-dipole and a dipolarophile to form a five-membered ring.
  • Linking a programmable nuclease to an agent via click chemistry can create a linker that includes, as the click reaction product, one or more five-membered rings or acyclic derivatives thereof.
  • This 1,3-dipolar cycloaddition is an important route to the regio- and stereoselective synthesis of five-membered heterocycles and their ring-opened acyclic derivatives.
  • the 1,3-dipolar cycloaddition between organic azides and terminal alkynes may proceed by a copper(I) -catalyzed version of the Huisgen reaction, CuAAC (for Copper-catalyzed Azide-Alkyne Cycloaddition), which proceeds readily in mild conditions that can approximate physiological conditions.
  • Click chemistry may be bioorthogonal: azides and alkynes are typically not found in biomolecules discussed herein and can be selectively reacted.
  • FIG. 22 shows steps of a method 1202 of preparing antiviral compositions.
  • the method 1202 includes the steps of providing 1205 a programmable nuclease and providing 1209 an uptake enhancing agent.
  • the method 1202 includes the step of providing 1213 a linker.
  • the method 1202 includes the steps of providing the programmable nuclease 107 linked to the uptake promoting agent 111, optionally through the linker.
  • the composition includes a recombinant fusion protein that includes the programmable nuclease 107 and the uptake promoting agent 111.
  • the method 1202 includes providing the linked programmable nuclease 107 and the uptake promoting agent 111 by synthesizing the recombinant fusion protein from a recombinant gene.
  • the composition includes the programmable nuclease 107 linked to the uptake promoting agent 111 via a chemical linker.
  • the method 1202 includes providing the linked programmable nuclease 107 and the uptake promoting agent 111 by performing the appropriate chemical reaction including any of those described elsewhere herein.
  • the method 1202 includes providing the linked programmable nuclease 107 and uptake promoting agent 111 with a suitable carrier such as a liposome, a solution, cream, ointment, or other pharmaceutically or therapeutically acceptable carrier.
  • a suitable carrier such as a liposome, a solution, cream, ointment, or other pharmaceutically or therapeutically acceptable carrier.
  • the invention provides an active Cas9 RNP and a delivery agent that promotes tissue or cellular uptake, e.g., distribution through tissue, endocytosis, cell penetration, or nuclear localization
  • exemplary suitable delivery agents may include one or a combination of transferrin; insulin; IgA; a gp340-binding agent; HPV LI capsid protein; HPV L2 capsid protein; a sugar; survivin; an antibody against K5 or K14; or a protease targeting Fl lR/JAM-A, E-cadherin, Claudin-1, TJPl (ZO-1), Occludin, keratin, desmosomes, or integrins.
  • compositions of the invention may be delivered by any suitable method include subcutaneously, transdermal, by hydrodynamic gene delivery, topically, or any other suitable method.
  • the composition is provided a carrier and is suitable for topical application to the human skin.
  • the composition may be introduced into the cell in situ by delivery to tissue in a host. Introducing the composition into the host cell may include delivering the composition non-systemically to a local reservoir of the viral infection in the host, for example, topically.
  • FIG. 23 shows steps of a method 1302 of treating a viral infection.
  • the method 1301 includes the steps of providing 1305 a programmable nuclease and providing 1309 a protease targeting a paracellular adhesion protein in the target tissue.
  • the method includes then administering 1313 the protease and the programmable nuclease to the target tissue.
  • compositions of the invention may be delivered to an affected area of the skin in an acceptable topical carrier such as any acceptable formulation that can be applied to the skin surface for topical, dermal, intradermal, or transdermal delivery of a medicament.
  • an acceptable topical carrier such as any acceptable formulation that can be applied to the skin surface for topical, dermal, intradermal, or transdermal delivery of a medicament.
  • Topical formulations of the invention are prepared by mixing the composition with a topical carrier according to methods known in the art.
  • topical carriers useful for topical delivery of the agent described herein can be any carrier known in the art for topically administering pharmaceuticals, for example, but not limited to, acceptable solvents, such as a polyalcohol or water; emulsions (either oil-in-water or water- in-oil emulsions), such as creams or lotions; micro emulsions; gels; ointments; liposomes;
  • the topical carrier used to deliver the compositions described herein is an emulsion, gel, or ointment.
  • Emulsions such as creams and lotions are suitable topical formulations for use in accordance with the invention.
  • An emulsion has at least two immiscible phases, one phase dispersed in the other as droplets ranging in diameter from 0.1 ⁇ to 100 ⁇ .
  • An emulsifying agent is typically included to improve stability.
  • the topical carrier is a gel, for example, a two-phase gel or a single-phase gel.
  • Gels are semisolid systems consisting of suspensions of small inorganic particles or large organic molecules interpenetrated by a liquid. When the gel mass comprises a network of small discrete inorganic particles, it is classified as a two-phase gel.
  • Single-phase gels consist of organic macromolecules distributed uniformly throughout a liquid such that no apparent boundaries exist between the dispersed macromolecules and the liquid.
  • Polymer thickeners (gelling agents) that may be used include those known to one skilled in the art, such as hydrophilic and hydro-alcoholic gelling agents frequently used in the cosmetic and pharmaceutical industries.
  • the gelling agent comprises between about 0.2% to about 4% by weight of the composition.
  • the agent may be cross -linked acrylic acid polymers that are given the name carbomer. These polymers dissolve in water and form a clear or slightly hazy gel upon neutralization with a caustic material such as sodium hydroxide, potassium hydroxide, or other amine bases.
  • a caustic material such as sodium hydroxide, potassium hydroxide, or other amine bases.
  • the topical carrier is an ointment.
  • Ointments are oleaginous semisolids that contain little if any water.
  • the ointment is hydrocarbon based, such as a wax, petrolatum, or gelled mineral oil.
  • the topical carrier used in the topical formulations of the invention is an aqueous solution or suspension, preferably, an aqueous solution.
  • aqueous solution preferably, an aqueous solution.
  • Well-known ophthalmic solutions and suspensions are suitable topical carriers for use in the invention.
  • the pH of the aqueous topical formulations of the invention are preferably within the range of from about 6 to about 8.
  • an effective amount of a buffer is included.
  • the buffering agent is present in the aqueous topical formulation in an amount of from about 0.05 to about 1 weight percent of the formulation.
  • Tonicity- adjusting agents can be included in the aqueous topical formulations of the invention.
  • the topical formulations of the invention can include acceptable excipients such as protectives, adsorbents, demulcents, emollients, preservatives, antioxidants, moisturizers, buffering agents, solubilizing agents, skin-penetration agents, and surfactants.
  • acceptable excipients such as protectives, adsorbents, demulcents, emollients, preservatives, antioxidants, moisturizers, buffering agents, solubilizing agents, skin-penetration agents, and surfactants.
  • Suitable protectives and adsorbents include, but are not limited to, dusting powders, zinc sterate, collodion, dimethicone, silicones, zinc carbonate, aloe vera gel and other aloe products, vitamin E oil, allatoin, glycerin, petrolatum, and zinc oxide.
  • Suitable demulcents include, but are not limited to, benzoin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, and polyvinyl alcohol.
  • Suitable emollients include, but are not limited to, animal and vegetable fats and oils, myristyl alcohol, alum, and aluminum acetate.
  • Suitable preservatives include, but are not limited to, quaternary ammonium agents, such as benzalkonium chloride, benzethonium chloride, cetrimide, dequalinium chloride, and cetylpyridinium chloride; mercurial agents, such as phenylmercuric nitrate, phenylmercuric acetate, and thimerosal; alcoholic agents, for example, chlorobutanol, phenylethyl alcohol, and benzyl alcohol; antibacterial esters, for example, esters of parahydroxybenzoic acid; and other anti-microbial agents such as chlorhexidine, chlorocresol, benzoic acid and polymyxin.
  • quaternary ammonium agents such as benzalkonium chloride, benzethonium chloride, cetrimide, dequalinium chloride, and cetylpyridinium chloride
  • mercurial agents such as phenylmercuric nitrate, phenyl
  • Chlorine dioxide preferably, stabilized chlorine dioxide
  • Suitable antioxidants include, but are not limited to, ascorbic acid and its esters, sodium bisulfite, butylated
  • moisturizers include, but are not limited to, glycerin, sorbitol, polyethylene glycols, urea, and propylene glycol.
  • Suitable buffering agents for use in the invention include, but are not limited to, acetate buffers, citrate buffers, phosphate buffers, lactic acid buffers, and borate buffers.
  • Suitable solubilizing agents include, but are not limited to, quaternary ammonium chlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbates.
  • Suitable skin-penetration agents include, but are not limited to, ethyl alcohol, isopropyl alcohol, octylphenylpolyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N-decylmethylsulfoxide, fatty acid esters (e.g., isopropyl myristate, methyl laurate, glycerol monooleate, and propylene glycol monooleate); and N-methyl pyrrolidone.
  • a Cas endonuclease can cleave at a sequence complementary to the guide RNA.
  • Native Cas9 uses a guide RNA composed of two disparate RNAs that associate to make the guide, which includes the CRISPR RNA (crRNA) and the trans-activating RNA (tracrRNA).
  • crRNA CRISPR RNA
  • tracrRNA trans-activating RNA
  • Cas9 targeting may be simplified through the engineering of a chimeric single guide RNA (sgRNA).
  • sgRNA single guide RNA
  • Cas9 contain RNase H and HNH endonuclease homologous domains which are responsible for cleavages of two target DNA strands, respectively.
  • the sequence similar to RNase H has a RuvC fold (one member of RNase H family) and the HNH region folds as T4 Endo VII (one member of HNH endonuclease family).
  • RuvC fold one member of RNase H family
  • T4 Endo VII one member of HNH endonuclease family
  • HNH domain is responsible for complementary sequence cleavage of target DNA
  • RuvC is responsible for the non-complementary sequence.
  • Methods and materials of the invention use a plasmid that includes a cas9 gene and at least one gene for a short guide RNA (sgRNA). The ssRNA is complementary to a portion of the viral genome.
  • sgRNA short guide RNA
  • the plasmid may contain genes for one or more sgRNAs targeting locations in the hepatitis B genome such as PreS 1, DR1, DR2, a reverse transcriptase (RT) domain of polymerase, an Hbx, and the core ORF.
  • the one or more sgRNAs comprise one selected from the group consisting of sgHBV-Core and sgHBV-PreS l. Incorporation by Reference

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Abstract

Les compositions et les procédés de traitement d'infections virales utilisent une nucléase programmable programmée pour digérer un génome viral et un agent d'administration qui favorise la pénétration de la composition à partir d'un site d'administration topique, à travers un tissu ou des cellules, et dans des cellules infectées. Les compositions peuvent comprendre une nucléase programmable telle qu'une endonucléase Cas qui peut être administrée sous la forme d'une ribonucléoprotéine active (RNP) ou d'un acide ribonucléique messager (ARNm) codant pour la nucléase conditionnée de préférence dans une nanoparticule telle qu'une nanoparticule lipidique.
PCT/US2017/066049 2016-12-22 2017-12-13 Administration de thérapies antivirales Ceased WO2018118567A1 (fr)

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US20110038941A1 (en) * 2007-12-27 2011-02-17 The Ohio State University Research Foundation Lipid Nanoparticle Compositions and Methods of Making and Using the Same
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230045095A1 (en) * 2021-06-23 2023-02-09 Massachusetts Institute Of Technology Compositions, Methods and Systems for the Delivery of Gene Editing Material to Cells
WO2022271548A3 (fr) * 2021-06-23 2023-03-09 Massachusetts Institute Of Technology Compositions, procédés et systèmes pour l'administration d'un matériel d'édition de gènes à des cellules

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