WO2018118587A1 - Polynucléotides modifiés pour traitement antiviral - Google Patents

Polynucléotides modifiés pour traitement antiviral Download PDF

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Publication number
WO2018118587A1
WO2018118587A1 PCT/US2017/066115 US2017066115W WO2018118587A1 WO 2018118587 A1 WO2018118587 A1 WO 2018118587A1 US 2017066115 W US2017066115 W US 2017066115W WO 2018118587 A1 WO2018118587 A1 WO 2018118587A1
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composition
guide rna
cas endonuclease
mrna
virus
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PCT/US2017/066115
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English (en)
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Derek D. Sloan
Sarah LEDOUX
Michael Patrick HOUSLEY
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Agenovir Corporation
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    • 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 RNAses, DNAses
    • 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
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • 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
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • 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
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • 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
    • 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
    • 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
    • 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
    • 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
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • 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
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/1133—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 herpetoviridae, e.g. HSV
    • C—CHEMISTRY; METALLURGY
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    • 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 [CRISPRs]
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • 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 disclosure relates to antiviral therapeutics.
  • 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.
  • Medical treatments for viral infections include those that target viral proteins. However, latent or persistent infections may not present those proteins, and thus may evade clearance by such treatments. Viruses may even use latency to evade detection by the host immune system.
  • antiviral therapeutic agents may be recognized as foreign and cleared by the host immune system, or the molecular components of the therapeutic agent may be metabolized inside host cells. Thus, even where some methods are known for treating viral infections, those treatments may not be adequate. Such a treatment may provide short-term relief from some symptoms of the infection but may not be effective against latent or persistent infections, which may later be triggered to become active viral infections with accompanying symptoms.
  • Compositions include a CRISPR-associated (Cas) endonuclease, mRNA encoding the nuclease, and a guide RNA that has a sequence complementary to a sequence from a viral genome, wherein the mRNA or guide RNA includes one or more modified nucleotides, such as 5-methylcytidine or pseudouridine in the mRNa or 2'OMethylated nucleotides and/or locked nucleic acid in the guide RNA, that reduce immunogenicity of the guide RNA or otherwise improve efficacy.
  • the Cas endonuclease cleaves specific targets within the viral genome.
  • the composition may include a Cas polypeptide or a mRNA that encodes a Cas endonuclease.
  • the viral-recognition sequence within the guide RNA directs the Cas endonuclease to target viral sequences in the nucleic acid of the host cell by binding to the viral sequences.
  • the guide RNA , the mRNA, or both include one or more modified nucleotides that improve the therapeutic efficacy of the composition.
  • the modified nucleotide may be 5-methylcytidine or pseudouridine in the mRNA or one or more 2'-0-methyl nucleotides in the guide RNA, which nucleotides may decrease the likelihood that the host's immune system will recognize the RNA as foreign and clear it from the body.
  • the modified nucleotide may increase hybridization specificity of the guide RNA, allowing for lower dosages and minimizing deleterious side effects.
  • the modified nucleotide may be a nucleotide, such as 5-aminoallyluridine, that allows covalent attachment of a non-nucleic acid component to the guide RNA.
  • the non-nucleic acid component may be an antibody, that binds to a viral antigen expressed by the host cell, which facilitates delivery of the guide RNA to a tissue or cell that has been infected by the virus.
  • the covalently-bound non-nucleic acid component may be a molecule, e.g., a cell-penetrating peptide, that promotes entry of the guide RNA into an infected cell and targeting of the guide RNA to a specific subcellular destination to perform its function.
  • a cell-penetrating peptide that promotes entry of the guide RNA into an infected cell and targeting of the guide RNA to a specific subcellular destination to perform its function.
  • Other useful modifications to nucleotides of the guide RNA within the scope of the invention include those that increase the chemical stability, resistance to enzymatic degradation, structural rigidity, or detectability of the guide RNA, alter its melting temperature, or result in increased Cas endonuclease cleavage efficiency.
  • compositions that include a mRNA encoding a Cas endonuclease may also include one or more modified nucleotides.
  • the mRNA may include N6- methyladenosine, which can promote expression of the Cas endonuclease.
  • compositions of the invention are useful to treat patients with viral infections or diseases caused by viral infections and to clear such infections or mitigate their effects. Many medically significant diseases are correlated with transmissible viral infections that include periods of latency or persistence.
  • compositions of the invention target viral nucleic acids, rather than viral proteins, the compositions do not require the virus to be in an active state to combat the infection. On the contrary, the compositions of the invention are effective regardless of stage of the infection cycle or whether the viral genome has integrated into the host genome. In addition, the compositions have specific modifications that improve their usefulness as therapeutics.
  • the invention provides a composition for topical delivery to treat a viral infection.
  • the composition includes a Cas endonuclease or mRNA encoding a Cas endonuclease and a guide RNA that has a recognition sequence substantially complementary to a target sequence within a genome of a virus and includes a modified nucleotide.
  • the composition includes a Cas endonuclease.
  • the Cas endonuclease is Cas9.
  • the Cas endonuclease may exist in a complex with the guide RNA as an active ribonucleoprotein (RNP).
  • the composition includes a mRNA encoding a Cas endonuclease.
  • the mRNA encodes Cas9.
  • the 5' portion of the mRNA may include N6- methyladenosine.
  • the mRNA may have a 3' end with a triple helical structure. With the triple helical structure, the transcript is efficiently translated in vivo despite the lack of a poly(A) tail.
  • the triple helical structure may be provided by including a sequence substantially similar to that found at a 3' end of the MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) locus.
  • the triple helical structure may include regions with Watson-Crick hydrogen bonds and regions with Hoogsteen hydrogen bonds.
  • the virus may be human papillomavirus, Epstein-Barr virus, hepatitis B virus, herpes simplex virus 1, herpes simplex virus 2, Merkel cell polyomavirus (MCV), varicella zoster virus, cytomegalovirus, or Kaposi's sarcoma-associated herpesvirus.
  • the virus is human papillomavirus.
  • the Cas endonuclease or mRNA encoding a Cas endonuclease is encapsulated in a nanoparticle.
  • the nanoparticle may be a non-viral nanoparticle.
  • the nanoparticle may be a liposome.
  • the nanoparticle may be a lipid nanoparticle.
  • the lipid nanoparticle may include cationic lipids.
  • the guide RNA includes at least one modified nucleotide.
  • the at least one modified nucleotide may be selected from the group consisting of 2'-0- methyladenosine, 2'-0-methylcytidine, 2'-0-methylguanosine, and 2'-0-methyluridine.
  • the modified nucleotide may include 5-methoxyUridine, 2-aminoadenine, 2-thiocytosine, 2- thiothymine, 2-thiouracil, 3-methyladenine, 4-thiouracil, 5,6-dehydrouracil, 5-allylcytosine, 5- allyluracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-ethynylcytosine, 5- ethynyluracil, 5-fluorouracil, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-iodouracil, 5- methylcytosine, 5-methyluracil, 5-propynylcytosine, 5-propynylcytosine, 5-propynyluracil, 5- propynyluracil, 6-O-methylguanine, 6-thioguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7- deazaguan
  • the modified nucleotide includes 5-methylcytosine, pseudouridine, or 5- aminoallyluridine.
  • the guide RNA may include repeating segments that self-assemble into an RNA nano structure.
  • a non-nucleotide group is linked to the guide RNA.
  • the non- nucleotide group may be a cell-penetrating peptide, polyethylene glycol, poly(N-(2- hydroxypropyl)methacrylamide), chitosan, N-acetylgalactosamine, N-acetylglucosamine, lactose, a polypeptide, an antibody, a fluorophore, a pharmaceutical agent, streptavidin, biotin, or albumin.
  • the composition may include an ap tamer.
  • the invention provides a composition for topical delivery to treat a viral infection, and the composition includes a Cas endonuclease or mRNA encoding the Cas endonuclease, a crRNA that includes a recognition sequence substantially complementary to a target sequence within a genome of a virus, and a tracrRNA that binds to the Cas endonuclease, and the crRNA, tracrRNA, or both include a modified nucleotide.
  • the invention provides a method of making an antiviral composition.
  • the method may include providing a Cas endonuclease and a guide RNA, combining them to form a ribonucleoprotein (RNP), and packaging the RNP into a liposome.
  • the method may include providing a mRNA encoding a Cas endonuclease and a guide RNA and packaging the mRNA into a nanoparticle.
  • the guide RNA may also be packaged into the nanoparticle.
  • the nanoparticle may include cationic lipids.
  • the invention provides a method for treating a viral infection.
  • the method entails providing a composition that includes a Cas endonuclease and a guide RNA and delivering the composition to cells infected by a virus.
  • the method entails providing a composition that includes a mRNA encoding a Cas endonuclease and a guide RNA and delivering the composition to cells infected by a virus.
  • FIG. 1 shows an antiviral composition that includes a Cas endonuclease and a guide
  • FIG. 2 shows covalent attachment of a non-nucleotide component to a guide RNA.
  • FIG. 3 shows a mechanism for covalently attaching a non-nucleotide component to a guide RNA that includes 5-aminoallyluridine.
  • FIG. 4 shows a ribonucleoprotein (RNP) that includes a Cas endonuclease and a guide
  • FIG. 5 shows an antiviral composition that includes a nanoparticle in which a
  • RNP ribonucleoprotein
  • FIG. 6 shows an antiviral composition that includes a guide RNA and a Cas
  • FIG. 7 shows an antiviral composition that includes a guide RNA and a Cas
  • FIG. 8 shows an antiviral composition that includes a nanoparticle in which a Cas endonuclease-encoding mRNA and a guide RNA have been packaged.
  • FIG. 9 shows an antiviral composition that includes a guide RNA and a nanoparticle in which a Cas endonuclease-encoding mRNA has been packaged.
  • FIG. 10 shows an antiviral composition that includes a Cas endonuclease-encoding DNA and a guide RNA.
  • FIG. 11 shows an antiviral composition that includes a Cas endonuclease, a crRNA, and a tracrRNA.
  • FIG. 12 is a diagram of a method of preparing an antiviral composition.
  • FIG. 13 is a diagram of a method of treating a viral infection.
  • Embodiments of the invention relate to the use of guide RNAs with modified nucleotides in compositions with Cas endonucleases to improve the antiviral therapeutic properties of those compositions.
  • the administration of unmodified therapeutic RNAs can trigger an immune response from the host, resulting in elimination of the foreign RNAs and abrogation of their therapeutic effect.
  • the incorporation of certain modified nucleotides, such as 5- methylcytidine and pseudouridine, into RNA decreases its immunogenicity. Consequently, guide RNAs that contain, for example, 2'OMethylated nucleotides may be more effective and have reduced side effects because they can be administered at lower doses.
  • an mRNA encoding a programmable nuclease as an antiviral therapeutic may include, for example, one or more of 5-methoxyUridine, 5-methylcytidine, or pseudouridine and be more effective and have reduced side effects because it can be administered at lower doses than an unmodified mRNA.
  • 5- methylcytidine and pseudouridine are examples of modified nucleotides that decrease the immunogenicity of mRNAs
  • 2'OMehthylated nucleotides are examples of modified nucleotides that decrease the immunogenicity of guide RNAs, but other modified nucleotides that have a similar effect are understood to be within the scope of the invention.
  • the invention also encompasses guide RNA and mRNA that include modified nucleotides, such as 5-aminoallyluridine, that provide RNA with specific reactive groups.
  • modified nucleotides such as 5-aminoallyluridine
  • the incorporation of 5-aminoallyluridine into an RNA facilitates covalent attachment of other molecules to a specific site in the RNA without constraining the structure of the RNA or impairing its ability to anneal to complementary nucleic acids.
  • 5-aminoallyluridine is discussed here as an example of a modified nucleotide that confers a reactive group to guide RNAs, other modified nucleotides that have a similar effect are understood to be within the scope of the invention.
  • FIG. 1 shows a composition 101 for treating a viral infection.
  • the composition 101 shown includes a Cas endonuclease 103 and a guide RNA 105 that binds to the Cas
  • the guide RNA 105 includes a recognition sequence 107 substantially complementary to a target sequence within a genome of a virus.
  • the guide RNA 105 includes at least one modified nucleotide 111.
  • the guide RNA 105 includes the modified nucleotides 111 at its 5' and 3' ends, but the modified nucleotides may be at any position within the guide RNA 105.
  • Any Cas endonuclease 103 may be used in the composition 101.
  • the guide RNA 105 may be covalently linked to the Cas endonuclease 103.
  • Cas endonucleases make up a family of RNA-guided endonucleases that function 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 clustered regularly interspaced short palindromic repeat (CRISPR).
  • CRISPR clustered regularly interspaced short palindromic repeat
  • 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 is also transcribed from the locus and contributes to the processing of pre-crRNA.
  • the crRNA and tracrRNA have been shown to be combinable into a single guide RNA.
  • guide RNA and "gRNA” refer to either format.
  • the gRNA forms a ribonucleoprotein (RNP) complex with a CRISPR-associated (Cas) endonuclease, and the RNP cleaves a target that includes a portion complementary to the protospacer sequence in the gRNA and a sequence known as protospacer adjacent motif (PAM).
  • RNP ribonucleoprotein
  • Cas CRISPR-associated endonuclease
  • Cas endonucleases 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.
  • Guide RNAs of the invention include recognition sequences that are substantially complementary (e.g., at least 50%, 60%, 70%, 80%, or 90%) to sequences in a viral genome or viral nucleic acid.
  • Guide RNAs of the invention may have recognition sequences that are not substantially complementary to any sequence in the host genome, e.g., the human genome, to ensure that the Cas endonuclease does not target and cleave non- viral sequences.
  • Cas endonucleases include Cas9, Cpfl, C2cl, C2c3, and C2c2, and modified versions of Cas9, Cpfl, C2cl, C2c3, and C2c2, such as an endonuclease with an amino acid sequence that is different, but at least about 85%, at least about 90%, or at least about 95% identical to, an amino acid sequence of wild-type Cas9, Cpfl, C2cl, C2c3, or C2c2, or a Cas9, Cpfl, C2cl, C2c3, or C2c2 protein.
  • Cas endonucleases also include the such proteins linked to an accessory element such as another polypeptide or protein domain (e.g., within a recombinant fusion protein or linked via an amino acid side-chain) or
  • C2cl (Class 2, candidate 1) is a type V-B Cas endonuclease that has been found to be functional in E. coli.
  • tracrRNAs short RNAs that help separate the CRISPR array into individual spacers, or crRNAs
  • the tracrRNA may be fused to the crRNA to make a single short guide, or sgRNA.
  • C2cl targets DNA with a 5' PAM sequence TTN.
  • C2c3 (Class 2, candidate 3) is a type V-C Cas endonuclease that clusters with C2cl and Cpfl within type V. C2c2 was found in metagenomic sequences, and the species is not known.
  • C2c2 (Class 2, candidate 2) is a type VI Cas endonuclease. C2c2 has been indicated to make mature crRNAs in E. coli. See Shmakov, 2015, Discovery and functional characterization of diverse class 2 CRISPR-Cas systems, Mol Cell 60(3):385-397, incorporated by reference.
  • viruses include, for example, viruses that present episomal DNA including members of the Herpesviridae family such as herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), human herpes viruses 6A and 6B (HHV-6A and HHV-6B), human herpes virus 7 (HHV-7), human herpes virus 8 or Kaposi's sarcoma virus (HHV-8 or KHSV), cytomegalovirus (CMV), Epstein Barr virus (EBV), and varicella-zoster virus (VZV).
  • Another episomal DNA virus is a papillomavirus, such as human papillomavirus (HPV).
  • these viruses reside episomally in the host in certain cell types, including epithelial cells (e.g., EBV and HBV), ganglia (e.g., VZV and HSV). monocytes (e.g., CMV), endothelial cells (e.g., HHV8) and lymphocytes, particularly B lymphocytes (e.g., EBV, HHV-6 and HHV-7).
  • epithelial cells e.g., EBV and HBV
  • ganglia e.g., VZV and HSV
  • monocytes e.g., CMV
  • endothelial cells e.g., HHV8
  • lymphocytes particularly B lymphocytes (e.g., EBV, HHV-6 and HHV-7).
  • these viruses may be activated to cause an active infection in the host, resulting in a variety of disorders.
  • the compositions of the invention target sequences from a viral genome, e.g., RNA or
  • a nucleotide is composed of a nucleoside component, which consists of a base and pentose ring, and a phosphate group.
  • Naturally-occurring polynucleotides such as RNA and DNA, comprise chains of nucleotides linked by phosphodiester bonds.
  • a polynucleotide chain may exist as a monomer or in a complex with another polynucleotide as a result of hydrogen bonding between bases on different chains.
  • Polynucleotides can be synthesized enzymatically in a template-directed process by DNA and RNA polymerases. Alternatively, polynucleotides can be synthesized chemically without a template by sequentially joining specific nucleotides in a desired sequence.
  • RNA polymerases use as the building blocks for RNA synthesis are riboadenosine (also called “adenosine), ribocytidine (also called “cytidine”), riboguanosine (also called “guanosine”), and ribouridine (also called “uridine”).
  • riboadenosine also called “adenosine
  • ribocytidine also called “cytidine”
  • riboguanosine also called “guanosine”
  • ribouridine also called “uridine”
  • Each of these nucleosides consists of the pentose ring ribose with a base attached to the first-position carbon of the ribose.
  • Adenosine has the base adenine
  • cytidine has the base cytosine
  • guanosine has the base guanine
  • uridine has the base uracil.
  • deoxyriboadenosine also called “deoxyadenosine”
  • deoxyribocytidine also called “deoxycytidine”
  • deoxyriboguanosine also called “deoxyguanosine”
  • deoxyribothymidine also called “thymidine”
  • thymidine deoxyribothymidine
  • Deoxyadenosine has the base adenine; deoxycytidine has the base cytosine; deoxyguanosine has the base guanine; and thymidine has the base thymine.
  • modified nucleotide refers to a nucleotide that differs in structure from one of the nucleotide subunits in a newly-synthesized nucleic acid (e.g., newly transcribed crRNA or tracrRNA), i.e., a nucleotide that differs in structure from one of nucleotides described above.
  • a modified nucleotide may differ chemically from one of the nucleotides described above in its base, pentose ring, nucleoside, or phosphate group.
  • a modified nucleotide may differ chemically from one of the nucleotides described above in its base, pentose ring, nucleoside, or phosphate group.
  • a modified nucleotide may be linked to an adjacent subunit by a chemical bond other than a phosphodiester bond.
  • Some modified nucleotides e.g., 5-methylcytidine, pseudouridine, and N6-methyladenosine, occur in nature as a result of enzymatic modification of the nucleotide subunit subsequent to nucleic acid synthesis.
  • Other modified nucleotides described herein have not been found in naturally-occurring nucleic acids.
  • a modified nucleotide may have an altered nucleoside, i.e., any nucleoside other than deoxyriboadenosine, deoxyribocytidine, deoxyriboguanosine, deoxyribothymidine,
  • riboadenosine ribocytidine, riboguanosine, or ribouridine.
  • a modified nucleotide may have an altered base.
  • a modified nucleotide may have 2-aminoadenine, 2-thiocytosine, 2-thiothymine, 2-thiouracil, 3-methyladenine, 4- thiouracil, 5,6-dehydrouracil, 5-allylcytosine, 5-allyluracil, 5-aminoallylcytosine, 5- aminoallyluracil, 5-bromouracil, 5-ethynylcytosine, 5-ethynyluracil, 5-fluorouracil, 5- hydroxymethylcytosine, 5-hydroxymethyluracil, 5-iodouracil, 5-methylcytosine, 5-methyluracil, 5-propynylcytosine, 5-propynylcytosine, 5-propynyluracil, 5-propynyluracil, 6-0- methylguanine, 6-thioguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7
  • a modified nucleotide may have an altered pentose ring.
  • a modified nucleotide may have 2'-0-methyl ribose, 2'-0-4'-C methylene ribose, or 2'-0-4'-C ethylene ribose.
  • a modified nucleotide may have a pentose ring that differs from other pentose rings in the molecule.
  • a modified nucleotide in predominantly RNA molecule may have a 2-deoxyribose ring
  • a modified nucleotide in a predominantly DNA molecule may have a ribose ring.
  • a modified nucleotide may have an unmodified nucleoside that is linked to an adjacent nucleoside via a covalent attachment other than a phosphodiester bond, such as a
  • modified nucleotides include 5-methylcytidine, pseudouridine, 2'-0- methyladenosine, 2'-0-methylcytidine, 2'-0-methylguanosine, 2'-0-methyluridine, 2' -O-methyl 3 '-phosphorothioate adenosine, 2' -O-methyl 3 '-phosphorothioate cytidine, 2' -O-methyl 3'- phosphorothioate guanosine, 2' -O-methyl 3 '-phosphorothioate uridine, and N6-methyladenosine.
  • the guide RNAs of the invention may include one or more modified nucleotides.
  • a guide RNA may include modified nucleotides at any position in the guide RNA sequence.
  • the modified nucleotide(s) may be near the 5' end of the guide RNA.
  • the first one or more nucleotides at the 5' end of the guide RNA may be modified, or modified nucleotides may be clustered, but not necessarily contiguous with one another, in a region proximal to the 5' end.
  • the last one or more nucleotides at the 3' end may be modified, or modified nucleotides may be clustered, but not necessarily contiguous with one another, in a region proximal to the 3' end.
  • Modified nucleotides may also be at internal positions of the guide RNA, either in consecutive positions, non-contiguous clusters, or interspersed throughout the length of the guide RNA.
  • FIG. 2 shows the covalent attachment of a non-nucleotide component 217 to a modified nucleotide 211 in a guide RNA 205.
  • the modified nucleotide 211 includes a reactive group that forms a covalent bond to attach the non-nucleotide component 217 to the guide RNA 205.
  • the reaction occurs specifically with the reactive group of the modified nucleotide, so the site of attachment is determined by the position of the modified nucleotide 211 within the guide RNA 205.
  • the modified nucleotide 211 shown here is near the 5' end of the guide RNA 205, but the modified nucleotide 211 may be at any position in the guide RNA 205.
  • FIG. 3 shows a mechanism for covalently attaching a non-nucleotide component 305 to a guide RNA.
  • the modified nucleotide 5-aminoallyluridine 313 is shown as a subunit of RNA.
  • 5-aminoallyluridine 313 has an allylamine group that can react with a N- hydroxysuccinimide ester group to form a peptide bond.
  • the non-nucleotide component 305 coupled to N-hydroxysuccinimide reacts with 5-aminoallyluridine 313 to attach the non- nucleotide 305 component to the 5-aminoallyluridine 313 subunit of the RNA.
  • the non-nucleotide component may be a cell-penetrating peptide.
  • the cell- penetrating peptide may be BMV Gag-(7-25), D-Tat, FBP, FHV Coat-(35-49), HTLV-II Rex-(4- 16), MAP, meganin, MPG, MPG(ANLS), Penetratin, Pep-1, Pep-2, poly(arginine-alanine), poly(arginine-glycine), poly(arginine-methionine), poly (arginine- serine), poly(arginine- threonine), polyarginine, polylysine, PTD-4, PTD-5, R9-Tat, SBP, SynB l, SynB3, TD-1, or TAT.
  • the non-nucleotide component may be an organic polymer, saccharide, polypeptide, antibody, fluorophore, or pharmaceutical agent.
  • the organic polymer may be polyethylene glycol, poly(N-(2-hydroxypropyl)methacrylamide), or chitosan.
  • the saccharide may be N- acetylgalactosamine, N-acetylglucosamine, or lactose.
  • the non-nucleotide component may be bio tin or albumin.
  • a modified nucleotide may do one or more of the following: reduce immunogenicity of a polynucleotide; alter (e.g., increase or decrease) hybridization specificity of a polynucleotide; facilitate delivery of a polynucleotide to a tissue or cell that has been infected by a virus; facilitate entry of a polynucleotide into a cell; facilitate targeting of a polynucleotide to a specific subcellular destination (e.g.., the nucleus); facilitate detection of a polynucleotide; alter (e.g., increase or decrease) the chemical stability of a polynucleotide as a free molecule or in a complex with another nucleic acid; alter (e.g., increase or decrease) resistance of
  • polynucleotide to enzymatic degradation when the polynucleotide is a free molecule or in a complex with another nucleic acid alter (e.g., increase or decrease) the melting temperature of a polynucleotide in complex with another nucleic acid; alter (e.g., increase or decrease) the melting temperature of different regions within the polynucleotide; alter (e.g., increase or decrease) the structural rigidity of a polynucleotide as a free molecule or in complex with another nucleic acid; alter (e.g., increase or decrease) activity of the Cas endonuclease; or alter (e.g., increase or decrease) the half-life of a polynucleotide in serum.
  • FIG. 4 shows a ribonucleoprotein (RNP) 413 that includes a Cas endonuclease 403 and guide RNA 405. Because both the guide RNA 405 and the Cas endonuclease 403 are functional in the RNP 413, the complex may be called an active RNP.
  • RNP ribonucleoprotein
  • FIG. 5 illustrates an embodiment of a composition for treating a viral infection.
  • the composition includes a Cas endonuclease 503 and a guide RNA 505 encapsulated in a nanoparticle 519.
  • the Cas endonuclease 503 and guide RNA 505 may be complexed in a RNP 513 as shown in the illustration.
  • the Cas endonuclease 503 may be encapsulated within the nanoparticle 519, and the guide RNA 503 may be bound to an outer surface of the nanoparticle 519 or separate from the nanoparticle 519.
  • the guide RNA 505 is encapsulated within the nanoparticle 519, and the Cas endonuclease 503 is bound to the outer surface of the nanoparticle 519 or is separate from the nanoparticle 519.
  • the nanoparticle may be any nanoparticle suitable for delivery of the Cas endonuclease and guide RNA.
  • the nanoparticle may be a non- viral nanoparticle.
  • the nanoparticle may be a cationic lipid nanoparticle.
  • the cationic lipids may form a complex with a nucleic acid, e.g., RNA or DNA, or polynucleotide of the composition.
  • RNAs with modified nucleotides in combination with mRNA that encodes a Cas endonuclease to create antiviral compositions with improved therapeutic properties.
  • naturally- occurring mRNAs of nuclear genes have 7-methylguanosine attached via a 5'-5' triphosphate linkage at their 5' ends (the "5' cap"), and their 3' ends include a string of 100 or more
  • the poly(A) tail mRNAs that lack 5' caps entirely are poorly translated, while those that have only a 5' triphosphate group (with no 7-methylguanosine) are immunogenic.
  • Poly(A) tails are critical for translation and stability of mRNAs. In the in vitro methods typically used to synthesize therapeutic mRNAs, however, addition of 5' caps and poly(A) tails is technically challenging. Consequently, the invention provides mRNAs that include modifications that circumvent the requirement for 5' caps and poly(A) tails.
  • the mRNAs may include at their 5' ends a modified nucleotide, such as N6-methyladenosine, that boosts cap-independent translation of mRNA.
  • the mRNA may also include sequences at their 3' ends that promote formation of RNA triplexes, which protect RNA from degradation.
  • FIG. 6 shows a composition 601 for treating a viral infection.
  • the composition 601 shown includes a mRNA 603 encoding a Cas endonuclease and a guide RNA 605 that binds to the Cas endonuclease.
  • the guide RNA 605 includes a recognition sequence 607 substantially complementary to a target sequence within a genome of a virus.
  • the guide RNA 605 also includes at least one modified nucleotide 611.
  • the guide RNA 605 includes the modified nucleotides 611 at its 5' and 3' ends, but the modified nucleotides 611 may be at any position within the guide RNA 605.
  • the mRNA 603 may encode any Cas
  • the guide RNA 605 may be covalently linked to the mRNA 603 encoding a Cas endonuclease.
  • FIG. 7 shows a composition 701 for treating a viral infection.
  • the composition 701 includes a guide RNA 705 and a mRNA 703 that encodes a Cas endonuclease.
  • the guide RNA 705 includes a recognition sequence 707 substantially complementary to a target sequence within a genome of a virus.
  • the guide RNA 705 and mRNA 703 both include one or modified nucleotides 711. In the example shown, the modified nucleotides 711 of the guide RNA 705 and mRNA 703 may be different.
  • the mRNA may contain N6-methyladenosine, methylcytosine, 5-methoxyUridine, or pseudouridine, and the guide RNA may contain one or more 2 OMethylated nucleotides.
  • the modified nucleotides 711 in the guide RNA 705 and mRNA 703 may be the same.
  • RNA molecules of the invention may include repeating segments that self-assemble into an RNA nanostructure.
  • the RNA nanostructure may be a hexamer, dimer, trimer, hexamer, triangle, tecto square, linear array, triangle array, nanoring, polyhedron, microsponge, hand-in-hand nanoparticle, foot-to-foot nanoparticle, origami tile, or origami tube.
  • the RNA molecules may include sequences at their 3' ends that promote triplex formation.
  • the mRNA may exist as a single molecule with a triple helical structure at the 3' end. With the triple helical structure, the transcript is efficiently translated in vivo despite the lack of a poly(A) tail.
  • the triple helical structure may be provided by including a sequence substantially similar to that found at a 3' end of the MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) locus.
  • the triple helical structure may include regions with Watson-Crick hydrogen bonds and regions with Hoogsteen hydrogen bonds. See Wilusz, 2012, A triple helix stabilizes the 3' ends of long noncoding RNAs that lack poly(A) tails, Genes & Dev 26:2392-2407, incorporated by a reference.
  • FIG. 8 illustrates an embodiment of a composition 801 for treating a viral infection.
  • the composition 801 includes a mRNA 803 encoding a Cas endonuclease and a guide RNA 805 encapsulated in a nanoparticle 819.
  • FIG. 9 illustrates an embodiment of a composition 901 for treating a viral infection.
  • the composition 901 includes a mRNA 903 encoding a Cas endonuclease encapsulated in a nanoparticle 901 and a guide RNA 905.
  • the nanoparticle may contain one or more components of the composition.
  • the nanoparticle may contain one or more of a Cas endonuclease, a mRNA encoding a Cas endonuclease, a DNA encoding a Cas endonuclease, and a guide RNA.
  • FIG. 10 shows a composition 1001 for treating a viral infection.
  • the composition 1001 shown includes a DNA 1003 encoding a Cas endonuclease and a guide RNA 1005 that binds to the Cas endonuclease.
  • the guide RNA 1005 includes a recognition sequence 1007 substantially complementary to a target sequence within a genome of a virus.
  • the guide RNA 1005 includes at least one modified nucleotide 1011.
  • the guide RNA 1005 includes modified nucleotides 1011 at its 5' and 3' ends, but the modified nucleotides 1011 may be at any position within the guide RNA 1005.
  • the DNA 1003 may encode any Cas endonuclease.
  • the composition may include an aptamer.
  • An aptamer is an oligonucleotide or peptide molecule that binds to a specific target molecule.
  • the aptamer may be an oligonucleotide or peptide.
  • the aptamer may be covalently linked to the guide RNA, Cas endonuclease, or mRNA encoding a Cas endonuclease. If an oligonucleotide aptamer is used, the aptamer may include a segment that is complementary to a sequence in the guide RNA or mRNA encoding a Cas endonuclease.
  • compositions of the invention may be delivered by any suitable method include subcutaneously, transdermal, by hydrodynamic gene delivery, topically, or by any other suitable method. Preferably, the compositions are delivered topically.
  • 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.
  • 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 well-known methods in the art, for example, methods provided by standard reference texts such as, REMINGTON: THE SCIENCE AND PRACTCE OF PHARMACY 1577-1591, 1672-1673, 866-885(Alfonso R. Gennaro ed.); Ghosh, T. K.; et al. TRANSDERMAL AND TOPICAL DRUG DELIVERY SYSTEMS (1997).
  • the topical carriers useful for topical delivery of the compound 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; powders; and aqueous solutions or suspensions, such as standard ophthalmic preparations.
  • acceptable solvents such as a polyalcohol or water
  • emulsions either oil-in-water or water-in-oil emulsions
  • creams or lotions such as creams or lotions
  • micro emulsions such as creams or lotions
  • gels such as ointments
  • liposomes such as liposomes
  • powders such as standard ophthalmic preparations.
  • aqueous solutions or suspensions such as standard ophthal
  • 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
  • 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.
  • 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 compounds, 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 compounds 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.
  • FIG. 11 illustrates a composition 1101 for treating a viral infection.
  • the composition 1101 includes a Cas endonuclease 1103 and a two-part guide RNA that includes a crRNA 1153 and tracrRNA 1155.
  • the crRNA 1153 includes a recognition sequence 1107 substantially complementary to a target sequence within a genome of a virus.
  • the both the crRNA 1153 and tracrRNA 1155 include a modified nucleotide 1111.
  • only the crRNA 1153 has a modified nucleotide 1111.
  • only the tracrRNA 1155 has a modified nucleotide 1111.
  • FIG. 12 shows a method 1201 of preparing an antiviral composition.
  • a Cas endonuclease 1203 and guide RNA 1205 that has a recognition sequence (not shown) and at least one modified nucleotide (not shown) are combined 1221 to form a RNP 1213.
  • the RNP 1213 is then packaged 1223 into a nanoparticle 1219, e.g., a liposome.
  • the composition may further include any suitable carrier fluid, cream, or gel e.g., for topical delivery.
  • RNA and DNA molecules can be made by template-directed synthesis using an RNA or DNA polymerase, and one or more nucleotides can subsequently be modified, e.g., by use of an appropriate enzyme.
  • RNA and DNA molecules can be made chemically by solid-phase synthesis.
  • modified nucleotides can be incorporated during synthesis.
  • unmodified nucleotides can be used for solid-phase synthesis in a first step, and nucleotides of the RNA or DNA molecules can be modified in a second step.
  • FIG. 13 shows a diagram of a method 1301 for treating a viral infection.
  • the method 1301 includes providing 1331 a composition that includes a Cas endonuclease and a guide RNA that has a recognition sequence substantially complementary to a target sequence within a genome of a virus and includes at least one modified nucleotide.
  • the composition is delivered 1333 to cells infected by a virus.
  • the Cas endonuclease and guide RNA enter 1335 the cells.
  • the recognition sequence of the guide RNA directs the Cas endonuclease to DNA sequences derived from the viral genome.
  • the Cas endonuclease specifically cleaves 1337 viral genetic material, preventing further propagation of the virus inside cells and allowing the body to eliminate the infection.
  • compositions may be delivered by any means to target cells infected by a virus.
  • the composition is delivered topically to avoid systemic distribution.
  • Topical delivery is useful to target cells infected by HPV, HSV-1, HSV-2, HHV-6A, HHV-6B, HHV-7, HHV- 8/KHSV, CMV, EBV, or VZV.
  • These viruses can infect cells in tissues of the mouth, throat, lips, tongue, genitalia (e.g., penis, vagina), anus, or colon. Consequently, infections of these tissue are amenable to topical delivery of anti-viral therapeutics.
  • compositions may be used to treat diseases or conditions associated with a viral infection.
  • the compositions are used to treat diseases or conditions associated with one of the viruses listed above.
  • the compositions may be used to treat cancer (e.g., cervical, vaginal, penile, anal, oral, tongue, or throat cancer), herpes, hepatitis, mononucleosis, chickenpox, or shingles
  • compositions do not rely on detection of viral proteins, they are useful for treating viral infections in both active and latent states.
  • Viruses such as HPV, HSV-1, HSV-2, HHV-6A, HHV-6B, HHV-7, HHV-8/KHSV, CMV, EBV, and VZV result in persistent, latent infections, so the compositions are useful for treating infections with these viruses during the latent phase of their infection cycles.

Abstract

L'invention concerne des compositions antivirales qui utilisent une endonucléase associée à CRISPR (Cas) et un ARN guide qui agissent ensemble pour éliminer l'acide nucléique viral présent dans des cellules hôtes, par exemple des cellules humaines. L'ARN guide comprend une séquence cible complémentaire d'une séquence cible située au sein du génome d'un virus et au moins un nucléotide modifié. L'endonucléase Cas peut être fournie sous forme d'ARNm, et l'ARNm ou l'ARN guide, ou les deux, comprennent un nucléotide modifié ou n'existant pas dans la nature. L'invention concerne également des procédés de préparation et des méthodes d'utilisation de ces compositions.
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