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WO2018118586A1 - Polythérapie antivirale/endogène - Google Patents

Polythérapie antivirale/endogène Download PDF

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Publication number
WO2018118586A1
WO2018118586A1 PCT/US2017/066113 US2017066113W WO2018118586A1 WO 2018118586 A1 WO2018118586 A1 WO 2018118586A1 US 2017066113 W US2017066113 W US 2017066113W WO 2018118586 A1 WO2018118586 A1 WO 2018118586A1
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Prior art keywords
composition
virus
nuclease
viral
episome
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • 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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • 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
    • C12N2320/31—Combination therapy
    • 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. 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 a latent or persistent infection may be unaffected by the treatment, and may later re-activate as an active viral infection.
  • compositions that include a nuclease and other agents (as described below) that work together to cleave viral nucleic acid and to prevent human genes from maintaining, propagating, or transcribing the viral episome.
  • Compositions may include a programmable nuclease such as a CRISPR-associated (Cas) endonuclease that cleaves specific targets within the viral genome.
  • the compositions also include an agent, such as a small molecule or a guide RNA associated with the Cas endonuclease, that prevents expression of a human gene into a protein that aids in maintaining the viral episome.
  • a composition may include a Cas endonuclease with guide RNAs that target both viral genetic material and human genes that are used by the virus for episomal maintenance. Additionally or alternatively, a composition may include a programmable nuclease that cleaves the viral genetic material as well as a small molecule, peptide, or other agent that prevents episomal maintenance by interfering with the use of endogenous host proteins to maintain the viral episome.
  • Embodiments of the invention provide compositions that include a programmable nuclease such as a CRISPR-associated (Cas) endonuclease that cleaves specific targets within the viral genome as well as a catalytically inactive (e.g., dCas9) programmable nuclease that prevents expression of a human gene into a protein that aids in maintaining the viral episome.
  • a programmable nuclease such as a CRISPR-associated (Cas) endonuclease that cleaves specific targets within the viral genome as well as a catalytically inactive (e.g., dCas9) programmable nuclease that prevents expression of a human gene into a protein that aids in maintaining the viral episome.
  • the nuclease and the inactive nuclease may each be provided in nanoparticles such as lipid nanoparticles. The nuclease cleaves and thus inactives the
  • compositions and methods of the invention may be particularly effective as antiviral treatments because they both attack the virus directly and stop the virus from using host genes for episomal maintenance. Because compositions and methods are provided to digest
  • the treatments work from two lines of approach to clear viral episomes from host cells. Since the viral episomes are cleared from the cells, the virus is unable to maintain latent or persistent infection. Since the virus is unable to maintain a latent or persistent infection, the viral infection can be fully and finally cleared.
  • methods and compositions are useful for delivery of a Cas endonuclease and a guide RNA targeting a virus as well as a guide RNA targeting a human gene used in maintenance of the viral episome (e.g., SETMAR) or a compound that destabilizes the episome (e.g., polyamide 1).
  • the guide RNA should include a targeting sequence substantially complementary to a site in the viral genome.
  • Preferred viral targets include those with episomal DNA genomes during persistence or latency such as human papillomavirus (HPV), Herpes Simplex virus (HSV), Epstein Barr virus (EBV), Kaposi' s sarcoma-associated herpesvirus (KSHV), hepatitis b virus (HBV), human Herpes virus 6 (HHV6), human Herpes virus 7 (HHV7), Cytomegalovirus (CMV), or Varicella zoster virus (VZV).
  • Compositions of the preferred embodiments are formulated for topical delivery, i.e., so that the human gene is blocked only at the site of delivery rather than systemically.
  • the Cas endonuclease— or an RNA encoding the Cas endonuclease— is preferably delivered via a nanoparticle such as a lipid nanoparticle that includes cationic lipids to encourage tissue and cellular penetration.
  • Delivery of the active, ribonucleoprotein (RNP) form of the Cas endonuclease or the RNA encoding the Cas endonuclease avoids the requirement for nuclear import and transcription.
  • Topical delivery of RNA or RNP to tissue such vaginal or anal tissue is used for preferred targets such as HPV to treat warts, lesions, or even cancers such as cervical cancer.
  • the disclosure includes a composition for treating a viral infection.
  • the composition includes a programmable nuclease programmed to cleave viral nucleic acid or an RNA encoding the programmable nuclease, as well as an inhibitor of episome maintenance.
  • the inhibitor of episome maintenance is preferably an agent that prevents a host-encoded gene from replicating a viral episome.
  • the composition may include a nanoparticle encapsulating at least the programmable nuclease or the RNA encoding the programmable nuclease.
  • the virus may be Herpes Simplex virus (HSV), human Herpes virus 6 (HHV6), human Herpes virus 7 (HHV7), Kaposi's sarcoma-associated herpesvirus (KSHV), Cytomegalovirus (CMV), Epstein Barr virus (EBV), Varicella zoster virus (VZV), human papillomavirus (HPV), or hepatitis b virus (HBV).
  • HSV Herpes Simplex virus
  • HHV6 human Herpes virus 6
  • HHV7 human Herpes virus 7
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • CMV Cytomegalovirus
  • EBV Epstein Barr virus
  • VZV Varicella zoster virus
  • HPV human papillomavirus
  • HBV hepatitis b virus
  • the programmable nuclease may be a zinc-finger nuclease, a transcription activator effector like nuclease, a structure-guided nuclease, a DNA-guided endonuclease, or a CRISPR-associated (Cas) endonuclease.
  • the inhibitor of episome maintenance may be polyamide 1, polyamide 25, aphidicolin, Chirl24, PF477736, roscovitine, or an interferon.
  • the inhibitor of episome maintenance comprises a second programmable nuclease programmed to block expression of a human gene that codes for a protein that maintains the episome.
  • the programmable nuclease is a Cas endonuclease, such as Cas9 or Cpfl
  • the Cas endonuclease may complexed with a first guide RNA with a portion complementary to the genetic material of the virus.
  • the second programmable nuclease may also be a Cas endonuclease, and it may be complexed with a second guide RNA with a portion complementary to the human gene.
  • the human gene may be, for example, AKT1, ATF4, ATM, ATR, ATRIP, BRCA1, BRD2, BRD4, BUB1, CIQBP, CBX1, CDC2, CDC25A, CDC25C, CDC6, CDC7, CDK2, CDK4, CDK6 CDKNIA, CDKNIA, CDT1, CHEKl, CHEK2, CHUK, CK1, CK1D, CLSPN, CREBBP, CTCF, CTCF, CXCR4, EBP2, FANl, FANCC, FANCD2, GADD45A, GADD45B, GADD45G, GSK3A, GSK3B, H3F3A, HCFC1, HDAC1, HDAC2 , HICl, HIF1A, HRAS, HTIM, HUS1, IFIT1, IKBA, JAK1, JAK2, JUN, KAT7, KDM1A, KPNAl, KRAS, MAP2K1, MAPK1, MAPK11, MAPK12, MAPK14, MAPK
  • the human gene NCL, RPL4, BRD4, NAP1L1, ORCl, ORC2, ORC3, ORC4, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, NPM1, NCL, TERF2, TERF2IP, TNKS, CIQBP, KPNAl, EBP2, RPAl, ORCl, ORC2, ORC3, ORC4, CTCF, CDC6, TIPIN, HCFC1, OCT2, MREl lA, RAD50, NBN, ATRIP, RAD9A, HUS 1, RAD1, TOPBPl, CLSPN, CHEKl, TP53BP1, or CDKNIA.
  • the programmable nuclease may be programmed to cleave nucleic acid of the Epstein-Barr virus (EBV).
  • EBV Epstein-Barr virus
  • the human gene is ATM, NBS 1, 53BP1, Chk2, CDC25A, CDK2, ATR, RPAl, HUS 1, RAD1, RAD 17, RAD9A, Chkl, BRCA1, UNG, TDP2, RUVBL2, RTELl, TOPBPl, RAD51, MREl lA, MGMT, p73, MLH3, TYMS, FANl, FANCC, or MTOR.
  • the programmable nuclease may be programmed to cleave nucleic acid of the human
  • HPV papillomavirus
  • the human gene is AKT1, ATF4, BRD2, BRD4, BUB 1, CBX1, CDC25C, CDC6, CDC7, CDK2, CDK6, CDT1, CREBBP, CTCF, GSK3A, GSK3B, HDAC1, HICl, HIF1A, HRAS, HTIM, IKBA, JAK1, JAK2, JUN, KAT7, KRAS, MAP2K1, MAPK1, MAPK11, MAPK12, MAPK14, MAPK3, MAPK8, MAPKAPK2, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, MECP2, MTOR, MYC, NOTCH1, NPM1, ORCl, ORC2, ORC3, ORC4, PARP1, PIK3CA, RB, RFC1, SLC7A11, STAT1, STAT2, STAT3, TIPIN, TOP2B, TP53BP1, or WNTl.
  • the programmable nuclease may be programmed to cleave nucleic acid of
  • the human gene is HCFC1, REST, NGF, HDAC1, HDAC2 RCOR1, REST, KDM1A, H3F3A, or POU2F1.
  • the programmable nuclease may be
  • HSV Herpes Simplex virus
  • compositions of the disclosure may be used in combination with delivery of a chemotherapeutic (Provisional Application Serial No. 62/299,792, filed February 25, 2016) and/or delivery with a compound that prevents NHEJ repair (Provisional Application Serial No. 62/299,839, filed February 25, 2016 are both incorporated by reference).
  • FIG. 1 shows a composition for treating a viral infection.
  • FIG. 2 shows a composition for treating a viral infection, in which the composition includes an mRNA that encodes a programmable nuclease.
  • FIG. 3 shows a Cas endo nuclease ribonucleoprotein (RNP).
  • FIG. 4 shows a composition that includes polyamide 25 and a programmable nuclease programmed to cleave viral genetic material, or an mRNA encoding the nuclease.
  • FIG. 5 shows a composition that includes polyamide 1 and a programmable nuclease programmed to cleave viral genetic material, or an mRNA encoding the programmable nuclease.
  • FIG. 6 shows a composition that includes aphidicolin and a programmable nuclease programmed to cleave viral genetic material, or an mRNA encoding the programmable nuclease.
  • FIG. 7 shows a composition that includes Chirl24 for use as the inhibitor of episome maintenance and a programmable nuclease programmed to cleave viral genetic material, or an mRNA encoding the programmable nuclease.
  • FIG. 8 shows a composition that includes PF477736 and a programmable nuclease programmed to cleave viral genetic material, or an mRNA encoding the programmable nuclease.
  • FIG. 9 shows a composition that includes roscovitine for use as an inhibitor of episome maintenance and a programmable nuclease programmed to cleave viral genetic material, or an mRNA encoding the programmable nuclease.
  • FIG. 10 shows a composition with a programmable nuclease and an agent that functions as an inhibitor of episome maintenance.
  • FIG. 11 shows a programmable nuclease in a nanoparticle.
  • FIG. 12 is a map of the Epstein Barr genome showing key target categories.
  • FIG. 13 shows a composition that includes a messenger RNA and an inhibitor.
  • FIG. 14 shows targeting proteins in the ATM and ATR pathways.
  • FIG. 15 shows an antiviral composition with a DNA vector encoding a nuclease.
  • FIG. 16 shows a method of preparing an antiviral composition.
  • FIG. 17 diagrams a method for treating a viral infection.
  • FIG. 18 is a map of HPV E6 and E7 genes on the HPV genome.
  • FIG. 19 gives the results of an in vitro digestion of HPV with a Cas endonuclease.
  • FIG. 20 shows results of an in vivo digestion of EBV with a Cas endonuclease.
  • FIG. 1 shows a composition 101 for treating a viral infection.
  • the composition 101 includes a programmable nuclease 107 programmed to cleave viral nucleic acid, or an RNA encoding the programmable nuclease.
  • the composition 101 also includes an inhibitor 157 of episome maintenance.
  • the inhibitor 157 of episome maintenance comprises a second programmable nuclease programmed to block expression of a human gene that codes for a protein that maintains the episome.
  • any suitable nuclease may be used.
  • Suitable nucleases include a zinc-finger nuclease, a transcription activator effector like nuclease, structure-guided nuclease, a DNA-guided endonuclease, and a CRISPR-associated (Cas) endonuclease.
  • the programmable nuclease 107 and the inhibitor 157 are each a Cas endonuclease, such as Cas9, Cpfl, or a modified Cas9 or Cpfl.
  • the programmable nuclease 107 includes a guide RNA 121 that programs the nuclease 107 to cleave viral RNA.
  • the guide RNA includes a targeting sequence 127 that is substantially (e.g., at least 60%) complementary to a target in viral nucleic acid.
  • the inhibitor 157 here is a second Cas endonuclease.
  • the second Cas endonuclease includes a second guide RNA 161.
  • the second guide RNA 161 includes a second targeting sequence 167 that is at least substantially complementary to a target in a human gene.
  • the second Cas endo nuclease functions as the inhibitor 157 by binding to the human gene where the second targeting sequence 167 is complementary to the human gene.
  • the composition 101 is effective as an antiviral therapeutic because the second targeting sequence 167 is designed to be complementary to a target within a human gene that codes for a protein that participates in viral episome maintenance and because the targeting sequence 127 causes the programmable nuclease 107 to digest the viral genetic material.
  • the composition 101 may include the programmable nuclease 107 in an active form, e.g., as a Cas endonuclease complexed with a guide RNA 121 as an active ribonucleoprotein (RNP).
  • the inhibitor 157 may also be present as an active RNP, as shown.
  • the RNP particles may be provided in a suitable medium for topical delivery such as a gel, ointment, solution, emulsion etc.
  • compositions and methods of the disclosure may also be used to treat viral infections by delivering a messenger RNA (mRNA) that encodes the programmable nuclease or a DNA vector— such as a plasmid— that includes a gene for nuclease.
  • mRNA messenger RNA
  • DNA vector such as a plasmid
  • the second nuclease is catalytically inactive (e.g., dCas9).
  • the composition may include include a programmable nuclease such as a CRISPR-associated (Cas) endonuclease that cleaves specific targets within the viral genome as well as a catalytically inactive programmable nuclease that prevents expression of a human gene into a protein that aids in maintaining the viral episome.
  • the nuclease and the inactive nuclease may each be provided in nanoparticles such as lipid nanoparticles. The nuclease cleaves and thus inactives the viral genetic material and the catalytically inactive nuclease binds to a human gene involved in episomal maintenance and prevents expression of that gene.
  • a catalytically inactiveally inactive e.g., dCas9
  • the composition is used to deliver, for example, dCas9 as a transcription repressor to inhibit transcription of human gene that used in maintenance of viral episome, without permanently change the human genome.
  • the composition preferably includes two intendent nuclease systems, one for cleaving viral genome and the other for suppressing human gene transcription.
  • FIG. 2 shows a composition 201 for treating a viral infection, in which the composition 201 includes an mRNA 207 that encodes a programmable nuclease.
  • the composition 201 preferably includes a guide sequence 221 that programs the nuclease to cleave viral nucleic acid.
  • the programmable nuclease is, for example, a Cas endonuclease
  • the guide sequence 221 is a guide RNA with a targeting sequence 227 that is substantially complementary to a target within the viral nucleic acid.
  • the depicted embodiment of the composition 201 also includes an inhibitor 261 of episome maintenance.
  • the inhibitor 261 is a second guide sequence 261 that will form a complex with a second instance, or "copy" of the programmable nuclease. It is noted that the mRNA 207 will be translated by host cells into multiple copies, or instances, of the
  • a first copy of the nuclease will form a complex with the guide sequence 221 and cleave viral nucleic acid.
  • a second nuclease will complex with the inhibitor 261 and block expression of a human gene involved in maintenance of the viral episome.
  • compositions of the invention use a programmable nuclease to digest viral nucleic acid and also include an inhibitor of episomal maintenance.
  • Compositions and methods of the disclosure may have particular benefit for the treatment of viruses that are capable of entering a latent or persistent phases or that maintain the viral genetic material in an episome.
  • viruses include, for example, viruses that present episomal DNA including members of the Herpesviridae family such as Herpes Simplex virus (HSV), human Herpes virus 6 (HHV6), human Herpes virus 7 (HHV7), human Herpes virus 8 (HHV8), Cytomegalovirus (CMV), Epstein Barr virus (EBV), and Varicella zoster virus (VZV).
  • HSV Herpes Simplex virus
  • HHV6 human Herpes virus 6
  • HHV7 human Herpes virus 7
  • HHV8 human Herpes virus 8
  • CMV Cytomegalovirus
  • EBV Epstein Barr virus
  • VZV Varicella zoster virus
  • 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, HHV6 and HHV7).
  • 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, HHV6 and HHV7).
  • these viruses may be activated to cause a latent infection in the host, resulting in a variety of disorders.
  • these viruses each contain an episomal maintenance (EM) element, which is typically a segment of between about 200 to 1000 nucleotides of viral DNA that is involved in maintaining and replicating the virus in the host cell.
  • EM episomal maintenance
  • one such episomal maintenance element is a viral origin of replication, e.g., the oriP of EBV, the OriS or Ori L of HSV.
  • Other episomal maintenance elements are referred to as terminal repeat (TR) sequences, such as those in HSV, HHV6, HHV7 and HHV8.
  • HPV's episomal maintenance sequence is referred to as a long-control region (LCR).
  • Still other terms for the EM elements include autonomous replicating sequence and matrix attachment region.
  • the DNA sequences of EM sequences for these and other viruses may all be found published in Genbank.
  • Methods and compositions of the disclosure use a programmable nuclease to digest nucleic acid of the virus, thereby rendering the virus incapable of replication or infection of the host patient.
  • Methods and compositions of the disclosure also use an inhibitor of episomal maintenance. Any suitable approach to disrupting or blocking the mechanism by which the viral episome is maintained in the infected cell may be used.
  • infected cells are treated by delivering a Cas9 endonuclease topically, to tissue, along with guide RNA that causes the nuclease to cleave the viral genome and guide RNA that causes the nuclease to cleave down endogenous (e.g., host or human) genes required for viral episome maintenance.
  • the programmable nuclease may be delivered with an agent, such as a small molecule or peptide, that destabilizes the episome directly or that interferes with endogenous proteins that contribute to episomal maintenance.
  • an agent such as a small molecule or peptide
  • Any suitable endogenous gene or protein may be targeted.
  • Potential suitable targets include the Ataxia- telangiectasia mutated (ATM) and ataxia- telangiectasia Rad3 -related (ATR) pathways (HPV).
  • ATM Ataxia- telangiectasia mutated
  • ATR ataxia- telangiectasia Rad3 -related pathway
  • the programmable nuclease may be any suitable programmable nuclease.
  • programmable nuclease is a molecule that can be designed to, or "programmed” to, cleave a nuclease in a sequence- specific manner.
  • Programmable nucleases includes CRISPR-associated (Cas) nucleases, such as Cas9, Cpfl, C2cl, C2c3, and C2c2; argonautes such as NgAgo;
  • Cas endonucleases 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.
  • tracrRNA 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.
  • Guide RNA and a Cas endonuclease form an active ribonucleoprotein (RNP) complex that cleaves the target nucleic acid.
  • RNP active ribonucleoprotein
  • FIG. 3 shows a Cas endonuclease RNP 301.
  • the Cas endonuclease RNP 301 includes a Cas9 protein 307 and an sgRNA 321.
  • the sgRNA 321 forms the RNP 301 with Cas9 protein 307, and the RNP 301 finds the target by hybridization of a targeting sequence 327 to the intended target.
  • the RNP will cleave when the target is found next to a sequence known as protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • Cas endonucleases are programmed to target a specific viral nucleic acid by providing a gRNA that includes a ⁇ 20-bp targeting sequence that is substantially complementary to a target adjacent to a PAM in viral nucleic acid.
  • the targetable sequences include, among others, 5 -X 20NGG-3 ' or 5 ' -X 20NAG-3 ' ; where X 20 is substantially complementary to the targeting sequence in the gRNA 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.
  • CRISPR systems with single- subunit effectors are known as Class 2.
  • Cas endonucleases include Cas9, Cpfl, C2cl, C2c3, and C2c2, and modified versions of Cas9, Cpfl, C2cl, C2c3, and C2c2, such as nuclease with a amino acid sequence that is different, but at least about 85% similar to, an amino acid sequence of wild-type Cas9, Cpfl, C2cl, C2c3, or C2c2, or a Cas9, Cpfl, C2cl, C2c3, or C2c2 protein with a 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 other molecule or agent.
  • Cas endonucleases include Cas9, Cpfl, C2cl, C2c3, and C2c2, and modified versions of Cas9, Cpfl, C2cl, C2c3, and C2c2, such as nuclease with a amino
  • C2cl (Class 2, candidate 1) is a type V-B Cas endonuclease that has been found.
  • C2cl examples have been indicated 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 Cpf 1 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.
  • 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 DNA-guided programmable nucleases that may be included in compositions of the invention. Gao et al., 2016, DNA-guided genome editing using the Natronobacterium gregoryi Argonaute, Nat Biotech 34:768-73 is incorporated by reference.
  • Structure-guided endonuclease include a type of programmable, DNA guided nuclease.
  • Structure-guided nuclease-mediated DNA editing uses an engineered SGN comprising FEN-1, which recognizes a 3' "flap" structure (consisting of a double- stranded helix where one strand is shorter, creating a flap at the end), and the cleavage domain of the Fokl endonuclease.
  • FEN-1 uses a guide DNA comprising a (minimum) 20 base-pair (bp) complementary sequence to the target site where the 3 ' end has a single -base mismatch creating an unpaired base, forming the "flap" structure.
  • SGN structure-guided nuclease
  • Genome Biol 17: 186 is incorporated by reference.
  • Key features of SGNs include an FEN-1 fusion that uses DNA oligomers to target a specific locus. Also, targeting using an SGN has a tendency to create larger deletions than with certain other nucleases on the order of several hundreds to thousands of bases. SGN targeting has been shown work in an animal model.
  • 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.
  • the 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.
  • Methods and compositions of the disclosure use a programmable nuclease to digest nucleic acid of the virus— thereby rendering the virus incapable of replication or infection of the host patient— and also use an inhibitor of episomal maintenance.
  • the inhibitor of episomal maintenance may be an agent, such as a small molecule or peptide, that destabilizes the episome directly or that interferes with endogenous proteins that contribute to episomal maintenance. Any suitable agent that interferes with maintenance of the episome may be included in compositions and methods of the disclosure. Suitable agents that prevent viral episome maintenance may include, for example, polyamide 25, polyamide 1, aphidicolin, Chirl24, PF477736, and roscovitine.
  • DNA viruses have a relationship with DNA damage response (DDR) pathways, which are one component of cellular antiviral defense. Infected cells use mechanisms to eliminate foreign (viral) DNA. The virus seeks to avoid elimination e.g., through the use of the host DDR machinery.
  • DDR DNA damage response
  • ATM ataxia-telangiectasia mutated
  • ATR ATM and Rad3 -related serine/threonine protein kinases
  • ATM is chiefly involved with responding to double-stranded DNA breaks; ATR responds to a variety of DNA insults).
  • the Chk2 and Chkl kinases in the ATM and ATR pathways coordinate the DDR. ATM activation has been implicated in productive HPV DNA replication and stable episome maintenance.
  • Stable episomal maintenance refers to the ability of cells to maintain a constant copy number of viral episomes over time.
  • Two DNA binding compounds, polyamide 1 (PA1) and polyamide 25 (PA25) have been reported to cause substantial loss of episomes.
  • PA1 polyamide 1
  • PA25 polyamide 25
  • aphidicolin as well as inhibitors of ATR and Chkl may act as inhibitors of episomal maintenance.
  • FIG. 4 shows a composition 401 that includes polyamide 25 (PA25) 461 and a programmable nuclease 107 programmed to cleave viral genetic material, or an mRNA 207 encoding the nuclease.
  • Polyamides are minor groove DNA binding agents derived from the natural product distamycin A.
  • PA25 is a 16 ring polyamide.
  • FIG. 5 shows a composition 501 that includes polyamide 1 (PA1) 551 and a
  • PA1 is a large 12 ring polyamide. It is reported that polyamide 1 (PA1) and polyamide 25 (PA25)— two N-methylpyrrole-imidazole polyamides of the hairpin type— exhibit anti-HPV activity. Both polyamides are reported to have antiviral activity against a variety of viral genotypes when tested on cells maintaining HPV episomes. Treatment of epithelia engineered in organotypic cultures with these compounds causes a dose- dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus.
  • FIG. 6 shows a composition 601 that includes aphidicolin 601 and a programmable nuclease 107 programmed to cleave viral genetic material, or an mRNA 207 encoding the programmable nuclease.
  • Aphidicolin may be used as an inhibitor of episome maintenance.
  • Aphidicolin is a tetracyclic diterpene antibiotic isolated from the fungus Cephalosporum aphidicola. Aphidicolin has antiviral and antimitotic properties. Aphidicolin is a reversible inhibitor of eukaryotic nuclear DNA replication. It blocks the cell cycle at early S phase.
  • Aphidicolin is a specific inhibitor of DNA polymerase A,D in eukaryotic cells
  • FIG. 7 shows a composition 701 that includes Chirl24 751 for use as the inhibitor of episome maintenance and a programmable nuclease 107 programmed to cleave viral genetic material, or an mRNA 207 encoding the programmable nuclease.
  • Chirl24 may be used as an inhibitor of episome maintenance.
  • Chkl kinase is a critical regulator of both S and G(2)-M phase cell cycle checkpoints in response to DNA damage.
  • Chirl24 is an inhibitor of Chkl.
  • FIG. 8 shows a composition 801 that includes PF477736 851 and a programmable nuclease 107 programmed to cleave viral genetic material, or an mRNA 207 encoding the programmable nuclease.
  • PF477736 may act as an inhibitor of episome maintenance because PF477736 is an inhibitor of Chkl.
  • FIG. 9 shows a composition 901 that includes roscovitine 951 for use as an inhibitor of episome maintenance and a programmable nuclease 107 programmed to cleave viral genetic material, or an mRNA 207 encoding the programmable nuclease.
  • Roscovitine may be used as an inhibitor of episome maintenance.
  • Roscovitine is a cyclin-dependent kinase (CDK) inhibitor that preferentially inhibits multiple enzyme targets including CDK2, CDK7 and CDK9, which alter the growth phase or state within the cell cycle of treated cells.
  • Roscovitine is a 2,6,9-substituted purine analog. It inhibits CDK2/E, CDK2/A, CDK7 and CDK9.
  • FIG. 10 shows a composition 1001 for treating a viral infection.
  • the composition 1001 includes a programmable nuclease 1007 programmed to cleave viral nucleic acid or an RNA encoding the programmable nuclease and an inhibitor 1061 of episome maintenance.
  • the composition preferably includes a nanoparticle 1071 encapsulating at least the programmable nuclease 1007 or the RNA encoding the programmable nuclease.
  • the inhibitor 1061 of episome maintenance may include, for example, polyamide 1, polyamide 25, aphidicolin, Chirl24, PF477736, roscovitine, or an interferon such as interferon a.
  • Interferon a is a pharmaceutical drug containing several naturally occurring IFN-a subtypes.
  • Any suitable nanoparticle 1071 may be used in the composition 1001. In certain embodiment, the nanoparticle is a lipid nanoparticle.
  • FIG. 11 shows a composition 1101 for treating a viral infection, in which the composition 1101 includes a programmable nuclease 1107.
  • the programmable nuclease cleaves viral genetic material under the guidance of a guide sequence (e.g., a gRNA).
  • the composition 1101 preferably includes a guide sequence 1121 that programs the nuclease to cleave viral nucleic acid.
  • the programmable nuclease is, for example, a Cas endonuclease
  • the guide sequence 1121 is a guide RNA with a targeting sequence that is substantially complementary to a target within the viral nucleic acid.
  • the depicted embodiment of the composition 1101 also includes an inhibitor 1161 of episome maintenance.
  • the inhibitor 1161 may be a second guide sequence specific to human gene or an agent or drug that interferes with viral episome maintenance.
  • a first copy of the nuclease 1107 will form a complex with the guide sequence 1121 and cleave viral nucleic acid.
  • Preferred viral targets include those with episomal DNA genomes during persistence or latency such as human papillomavirus (HPV), Herpes Simplex virus (HSV), Epstein Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), hepatitis b virus (HBV), human Herpes virus 6 (HHV6), human Herpes virus 7 (HHV7), Cytomegalovirus (CMV), or Varicella zoster virus (VZV).
  • HPV human papillomavirus
  • HSV Herpes Simplex virus
  • EBV Epstein Barr virus
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • HBV herpes virus 6
  • HHV7 human Herpes virus 7
  • CMV Cytomegalovirus
  • VZV Varicella zoster virus
  • the guide sequences may be designed to cause the programmable nuclease to cleave a specific target.
  • gRNAs guide RNAs, or "gRNAs"
  • At least one instance of a programmable nuclease is programmed to cleave viral genetic material
  • FIG. 12 is a map of the Epstein Barr genome and is used to illustrate how the guide sequence may be designed.
  • the map shown in FIG. 12 shows certain features in the EBV genome that may be targeted with a programmable nuclease.
  • the marks "#", and "+” are used to indicate features that are related to viral structure, transformation, and latency, respectively.
  • Guide RNAs that target the EBV genome are used in compositions according to certain embodiments. Within a genome of interest, such as EBV, selected regions, or genes are targeted. For example, six regions can be targeted with seven guide RNA designs for different genome editing purposes.
  • EBNAl is the only nuclear Epstein-Barr virus (EBV) protein expressed in both latent and lytic modes of infection.
  • EBNA1 While EBNAl is known to play several important roles in latent infection, EBNA1 is crucial for many EBV functions including gene regulation and latent genome replication. Therefore, guide RNAs sgEBV4 and sgEBV5 were selected to target both ends of the EBNA1 coding region in order to excise this whole region of the genome. These "structural" targets enable systematic digestion of the EBV genome into smaller pieces.
  • EBNA3C and LMPl are essential for host cell transformation, and guide RNAs sgEBV3 and sgEBV7 were designed to target the 5' exons of these two proteins respectively.
  • EBNA1 is crucial for many EBV functions including gene regulation and latent genome replication.
  • Guide RNA sgEBV4 and sgEBV5 are targeted to both ends of the EBNA1 coding region in order to excise that region of the genome.
  • Guide RNAs sgEBVl, 2 and 6 fall in repeat regions, so that the success rate of at least one CRISPR cut is multiplied.
  • Those "structural" targets enable systematic digestion of the EBV genome into smaller pieces.
  • EBNA3C and LMPl are essential for host cell transformation, and guide RNAs sgEBV3 and sgEBV7 are designed to target the 5' exons of these two proteins respectively.
  • the double-strand DNA breaks generated by a programmable nuclease may be repaired with small deletions. Those deletions will disrupt the protein coding and hence create knockout effects.
  • Suitable targets include viruses such as Herpes Simplex virus (HSV), human Herpes virus 6 (HHV6), human Herpes virus 7 (HHV7), Kaposi's sarcoma-associated herpesvirus (KSHV), Cytomegalovirus (CMV), Epstein Barr virus (EBV), Varicella zoster virus (VZV), human papillomavirus (HPV), or hepatitis b virus (HBV).
  • HSV Herpes Simplex virus
  • HHV6 human Herpes virus 6
  • HHV7 human Herpes virus 7
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • CMV Cytomegalovirus
  • EBV Epstein Barr virus
  • VZV Varicella zoster virus
  • HPV human papillo
  • FIG. 13 illustrates a preferred embodiment that includes a composition 1301 for treating a viral infection.
  • the composition 1301 includes a messenger RNA 1037 encoding a
  • the composition also includes an inhibitor 1361 of episome maintenance.
  • the inhibitor 1361 of episome maintenance may include an agent that prevents a host-encoded gene from replicating a viral episome. Additionally or alternatively, the inhibitor 1361 may include a second guide RNA that programs additional copies of the nuclease to cleave the host encoded gene.
  • the composition 1301 also includes a nanoparticle 1371 encapsulating at least the RNA 1307 encoding the programmable nuclease.
  • the guide RNA 1321, the inhibitor 1361, both, or neither may also be encapsulated within the nanoparticle 1371 or within addition instances of the nanoparticle.
  • the inhibitor 1361 may be an agent that interferes with an ability of the host proteins to participate in maintenance of the viral episome.
  • the inhibitor 1361 may be a checkpoint kinase inhibitor.
  • the inhibitor may include poly amide 1, polyamide 25, aphidicolin, Chirl24, PF477736, roscovitine, or an interferon.
  • the inhibitor 1361 is preferably selected to target certain potential targets, such as proteins in the Ataxia-telangiectasia mutated (ATM) and ataxia- telangiectasia Rad3 -related (ATR) pathways (e.g., where the virus is HPV).
  • ATM Ataxia-telangiectasia mutated
  • ATR ataxia- telangiectasia Rad3 -related pathway
  • Viral episomal maintenance makes use of human genes and their associated protein pathways.
  • DNA damage sensors relay information to members of a family of phosphoinositide 3-kinase related kinases (PIKKs).
  • PIKKs phosphoinositide 3-kinase related kinases
  • ATM ataxia-telangiectasiamutated
  • ATM ATR
  • ATM and Rad 3-related phosphoinositide 3-kinase related kinases
  • ATM ataxia-telangiectasiamutated
  • ATM Ataxia telangiectasia mutated
  • ATM targets include the tumor suppressors p53, CHK2, BRCA1, NBS 1 and H2AX.
  • FIG. 14 shows targeting certain exemplary proteins in the ATM and ATR pathways.
  • Infected tissue may be treated with e.g., a Cas endonuclease complexed with a first RNA with a portion complementary to the genetic material of the virus and an inhibitor 1461 of a point in the ATM or ATR pathway (for example, second programmable nuclease such as a Cas endonuclease complexed with a second RNA with a portion complementary to the human gene).
  • ATM serine- protein kinase
  • the reaction that initiates the Gl/S arrest is phosphorylation of cell cycle checkpoint kinase 2 (Chk2) or cell cycle checkpoint kinase 1 (Chkl) by ATM.
  • Nuclear factor with BRCT domains protein 1 (NFBD1) may participates in transfer signal from ATM to Chk2 and other regulators (e.g. p53 , and breast and ovarian cancer susceptibility protein 1 (Brcal).
  • NFBD1 nuclear factor with BRCT domains protein 1
  • Chk2 is inhibited by an inhibitor 1362.
  • phosphorylated Chk2 in turn inactivates by phosphorylation cell division cycle 25A phosphatase (Cdc25A).
  • Cdc25A phosphorylation cell division cycle 25A phosphatase
  • Lack of active Cdc25A results in the accumulation of the phosphorylated (inactive) form of Cdk2, which is incapable to participate in initiation of replication.
  • 14-3-3 proteins participate in regulation activity of some elements Gl/S checkpoint pathway (e.g. Chkl, Cdc25A and p53).
  • ATM may normally regulate signaling cascades involving nuclear factor- kappaB (NF-KB), a transcription factor that is upstream of a wide variety of stress-responsive genes.
  • NF-KB nuclear factor- kappaB
  • NF-KB nuclear factor- kappaB
  • NF-KB nuclear factor- kappaB
  • NF-KB nuclear factor- kappaB
  • the signal leads to phosphorylation of serine/threonine-protein kinase Chkl by ataxia telangiectasia and Rad3 related protein kinase (ATR) with a participation cell cycle checkpoint control cell cycle regulator RAD9 and claspin.
  • ATR ataxia telangiectasia and Rad3 related protein kinase
  • the activated Chkl then phosphorylates Cdc25A, leading to Gl arrest.
  • ATR phosphorylates ATR interacting protein (ATRIP), which in turn regulates ATR expression, and is an essential component of the DNA damage checkpoint pathway.
  • ATR ATR interacting protein
  • Activation of the Chk-Cdc25A pathways is followed by the p53-mediated maintenance of Gl/S arrest.
  • ATM or ATR phosphorylates Serl5 of p53 directly and Ser20 through activation of Chk2 or Chkl.
  • the essential elements of p53 regulation are ubiquitination and sumoylation.
  • Phosphorylated p53 activates its target genes, including cyclin-dependent kinase inhibitor 1A (p21), which binds to cyclin-dependent kinase 2 (Cdk2) and cyclin-dependent kinase 4 (Cdk4). It inhibits binding between Cdk and cyclins.
  • the DNA damage activates p53 via inhibition its repressor - the ubiquitin-protein ligase E3 MDM2.
  • the intra-S-phase checkpoint is activated by damage encountered during the S phase or by unrepaired damage that escapes the S/G2 checkpoint and leads to a block in replication.
  • ATM phosphorylation of structural maintenance of chromosomes 1-like 1 protein (SMCl) and Fanconi anemia complementation group D2 protein, isoform 1 (FANCD2) leads to inhibition of replication. It supposed, that phosphorylation of SMCl results to the repression sister chromatid cohesion.
  • FANCD2 may participate in inhibition of replication via activation Brcal.
  • Brcal is phosphorylated by ATR (perhaps, with the aid of BML) or ATM, and activates transcription of growth arrest and DNA-damage-inducible transcripts alpha and beta (GADD45 alpha/beta).
  • GADD45 alpha/beta the transcription of GADD45 alpha/beta may be regulated by p53.
  • GADD45 alpha/beta was found to bind to proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication and repair. p21 blocks the ability of PCNA to bind with Gadd45.
  • PCNA proliferating cell nuclear antigen
  • Any of the aforementioned participants in the ATR pathway may be inhibited using compositions of the invention.
  • a composition may include an inhibitor 1363 that prevents expression of the NBN gene.
  • the composition may include an inhibitor 1364 that prevents expression of the ATR gene. It has been reported that the ATM and ATR pathways participate in maintaining viral episomes. Any suitable gene or protein of the ATM or ATR pathways may be targeted using compositions and methods of the disclosure as an inhibitor of episomal maintenance.
  • the inhibitor may be programmable nuclease, or a nucleic acid encoding the same, that is programmed to cleave an endogenous human gene that participates in maintenance of the viral episome (e.g., the NBN gene, the ATR gene, or any other gene for a protein of the ATM or ATR pathways).
  • an endogenous human gene that participates in maintenance of the viral episome e.g., the NBN gene, the ATR gene, or any other gene for a protein of the ATM or ATR pathways.
  • Table 1 gives a listing of genes that are reported to play a role in maintenance of viral episomes.
  • column 1 names a protein
  • column 2 gives a known abbreviation for that protein
  • column 4 gives known aliases for the protein;
  • column 5 gives the accepted gene name of the gene that encodes the protein; and
  • column 6 gives a brief statement of a category of function of the protein.
  • DNA damage sensors include Radl7, Radl, Rad9, Rad26, and Husl as well as proliferating cell nuclear antigen (PCNA) and replication factor c (RFC).
  • PCNA proliferating cell nuclear antigen
  • RRC replication factor c
  • dsbs DNA double-strand breaks
  • ATM regulates p53 accumulation by indirect pathways involving the Chk2-mediated phosphorylation of Ser 20 on p53, by promoting casein kinase-I-dependent phosphorylation of Ser 18, and by directly phosphorylating MDM2 on Ser 395.
  • ATR may influence Ser 20 phosphorylation through activation of Chkl.
  • S-phase checkpoint repair proteins include NBS 1, Mrel l, Rad50, and 53BP1.
  • Metnase is encoded by the gene SETMAR. Metnase is a component of the human non-homologous end-joining repair pathway is reported to maintain EBV episomes.
  • Certain embodiments of the invention use a programmable nuclease such as a Cas endonuclease and an agent to inhibit episomal maintenance such as an episome destabilizer compound like polyamide 1 or 25 (PA1, PA25), aphidicolin (reported to reduce HPV episome stability).
  • a programmable nuclease such as a Cas endonuclease and an agent to inhibit episomal maintenance
  • an episome destabilizer compound like polyamide 1 or 25 (PA1, PA25), aphidicolin (reported to reduce HPV episome stability).
  • PA1 and PA25 are polyamides characterized by a hairpin structure. PA1 and PA 25 may be delivered to eliminate HPV 16 episomes. Additionally, PA25 is a treatment against cancer- causing forms of HPV. PA1 and PA 25 interact with sequences in the long control region (LCR) of HPV16 (7348-122).
  • LCR long control region
  • FIG. 15 diagrams a composition 900 for treating a viral infection that includes a nucleic acid vector 901 (e.g., a plasmid) encoding a programmable nuclease for delivery to viral-infected cells.
  • the nucleic acid vector 901 is a plasmid that includes a gene 927 (e.g., a Cas endonuclease) preferably under control of a promoter 939.
  • the plasmid may also include a viral origin of replication 935 to support maintenance of the plasmid preferentially in viral-infected cells.
  • the plasmid may also include at least two of a guide RNA segment 955, which includes portions that correspond to targets in genetic material of a virus and portions that correspond to targets in at least one human gene.
  • a guide RNA segment 955 When the guide RNA segment 955 is transcribed, the product is one gRNA with a portion substantially complementary to a target in viral genetic material, preferably with no match in a human genome, and a second gRNA with a portion substantially
  • vector 901 is a plasmid, programmable nuclease gene 907 codes for a Cas endonuclease (e.g., Cas9 or a modified version of Cas9 that is at least a 95% match to Cas9).
  • the guide RNA segment 955 includes a first 20 nucleotide segment that is at least a 70% match to a segment in a genome of a virus adjacent to a protospacer adjacent motif (PAM) (e.g., NGG); and the viral origin of replication 935 is an origin of replication from the genome of a virus.
  • PAM protospacer adjacent motif
  • the virus may be selected from Human papillomavirus (HPV), Hepatitis B virsus, Cytomegalovirus, herpes simplex virus, Epstein Barr virus, for example. These certain embodiments may be preferred where the nucleic acid vector 901 is part of an antiviral therapeutic composition to be delivered to infected cells.
  • HPV Human papillomavirus
  • Hepatitis B virsus Hepatitis B virsus
  • Cytomegalovirus cytotomegalovirus
  • herpes simplex virus herpes simplex virus
  • Epstein Barr virus Epstein Barr virus
  • the programmable nuclease segment 907 may preferably code for an RNA-guided nuclease such as Cas9, a modified Cas9 (at least 90% similar to wt Cas9), Cpfl, or a modified Cpfl.
  • the guide RNA segment 955 and the viral origin of replication 935 are omitted.
  • Any suitable promoter 939 e.g., U6 promoter may be included.
  • the gene 927 is to be expressed e.g., in culture (for example, in E. coli, yeast, or a Lactobacillus) to produce a nuclease for use in an antiviral therapeutic composition.
  • the programmable nuclease segment 907 codes for an RNA- guided nuclease
  • the expressed protein is preferably complexed with a gRNA to form into an active ribonucleoprotein (RNP).
  • FIG. 16 shows preparation of an antiviral composition.
  • the programmable nuclease and guide RNA 121 are obtained. Those elements are formed in an RNP 1607.
  • the guide RNA 121 includes a targeting sequence substantially complementary to a target site within an HPV genome.
  • An inhibitor of episomal maintenance 1661 is obtained.
  • the RNP 1607 and the inhibitor 1661 are packaged with nanoparticles 2037 to form a composition 101.
  • the composition 101 may further include any suitable carrier fluid, cream, or gel e.g., for topical delivery.
  • the composition 101 is delivered topically to a site in tissue in a patient.
  • the nanoparticles penetrate tissue, preferably to the basal epithelium or mucosal epithelium where, for example, the virus is HPV.
  • the nanoparticles 2037 deliver the RNP 1607 molecules to infected cells 2679, where the RNP 1607 then cleaves viral DNA 2051.
  • FIG. 17 diagrams a method 1701 for treating a viral infection.
  • the method 1701 includes providing 1705 a composition that includes a programmable nuclease programmed to cleave viral nucleic acid or an RNA encoding the programmable nuclease and an inhibitor of episome maintenance, e.g., an agent that prevents a host-encoded gene from replicating a viral episome.
  • the programmable nuclease or the RNA encoding the programmable nuclease are encapsulated in a nanoparticle such as a lipid nanoparticle that includes cationic lipids.
  • the composition is delivered to cells infected by a virus.
  • the composition is delivered topically to avoid systemic distribution.
  • the virus may be, for example, Herpes Simplex virus (HSV), human Herpes virus 6 (HHV6), human Herpes virus 7 (HHV7), Kaposi's sarcoma-associated herpesvirus (KSHV), Cytomegalovirus (CMV), Epstein Barr virus (EBV), Varicella zoster virus (VZV), human papillomavirus (HPV), or hepatitis b virus (HBV).
  • the programmable nuclease is preferably a Cas endonuclease programmed to cleave genetic material of the virus.
  • composition is then used to cleave 1713 the genetic material of the virus.
  • the composition is used to prevent 1719 episome maintenance by interfering with a the ability of an endogenous gene to contribute to maintenance of the viral episome.
  • the inhibitor of episome maintenance is a second programmable nuclease programmed to block expression of a human gene that codes for a protein that maintains the episome.
  • the Cas endonuclease is complexed with a first RNA having a portion complementary to the genetic material of the virus and the second programmable nuclease is a Cas endonuclease complexed with a second RNA that itself has a portion complementary to the human gene.
  • the human gene may be AKT1, ATF4, ATM, ATR, ATRIP, BRCA1, BRD2, BRD4, BUB1, C1QBP, CBX1, CDC2, CDC25A, CDC25C, CDC6, CDC7, CDK2, CDK4, CDK6 CDKN1A, CDKN1A, CDT1, CHEKl, CHEK2, CHUK, CKl, CKID, CLSPN, CREBBP, CTCF, CTCF, CXCR4, EBP2, FANl, FANCC, FANCD2, GADD45A, GADD45B, GADD45G, GSK3A, GSK3B, H3F3A, HCFC1, HDAC1, HDAC2 , HICl, HIF1A, HRAS, HTIM, HUS1, IFIT1, IKBA, JAK1, JAK2, JUN, KAT7, KDM1A, KPNA1, KRAS, MAP2K1, MAPK1, MAPK11, MAPK12, MAPK14, MAPK
  • the human gene is selected from the group consisting of: ATM, NBS 1, 53BP1, Chk2, CDC25A, CDK2, ATR, RPA1, HUS 1, RAD1, RAD 17, RAD9A, Chkl, BRCA1, UNG, TDP2, RUVBL2, RTEL1, TOPBP1, RAD51, MRE11A, MGMT, p73, MLH3, TYMS, FANl, FANCC, and MTOR and the virus is human papillomavirus (HPV).
  • HPV human papillomavirus
  • the APOBEC3 family of 7 proteins mediates HPV episome loss.
  • An inhibitor of episome maintenance may include an APOBEC3 expression vector.
  • Treatment of W12 cells with interferon- ⁇ results in up-regulation of ABOBEC3 gene expression and hypermutation of the HPV 16 E2 gene in a manner dependent upon inhibition of uracil DNA glycosylase (which repairs the APOBEC3-mediated mutation).
  • uracil DNA glycosylase which repairs the APOBEC3-mediated mutation.
  • Synthetic homologs of distamycin A (N-methylpyrrole-imidazole polyamides) designed to bind to AT -rich regions within the origin of replication of HPV, possess the ability to induce loss of HPV episomes.
  • Polyamides may be used as inhibitors of episome maintenance. Knockdown or inhibition of ATR or CHK1 may be found to cause significant loss of HPV episomes. So may delivery of aphidicolin.
  • TDP2 (aka TTRAP) knockdown has been reported to cause an episomal loss.
  • RUVBL2 and RTEL1 Two helicases involved in homologous recombination, RUVBL2 and RTEL1, have been reported to stabilize HPV episomes: knockdown of each may result in a greater than 3-fold loss of HPV genomes.
  • HPV replication foci also recruit factors from both the ATM and ATR pathways, such as TopBPl, Rad51, pNBS l, MRN, RPA, BRCA1, and 53BP1.
  • Genes that participate in maintenance of the HPV episome have been reported to include ATM, NBS 1, 53BP1, Chk2, CDC25A, CDK2, ATR, RPA1, HUS 1, RAD1, RAD 17, RAD9A, Chkl, BRCA1, UNG, TDP2, RUVBL2, RTEL1, TOPBP1, RAD51, MRE11A, MGMT, p73, MLH3, TYMS, FAN1, FANCC, and MTOR.
  • Compositions include inhibitors of endogenous human genes that contribute to maintenance of viral episomes as well as programmable nucleases programmed to cleave genetic material of the virus.
  • the inhibitor may be a programmable nuclease programmed to cleave an endogenous human gene that contributes to episomal maintenance.
  • FIG. 18 is a map of HPV E6 and E7 genes on the HPV gene.
  • the HPV E6 and E7 genes have been used as targets using programmable nucleases in antiviral treatments. Since E6 and E7 proteins may be oncogenic it may be valuable to target their respective genes for destructions by the nuclease.
  • To design a guide RNA each gene is scanned for the protospacer adjacent motif (PAM) of the nuclease (e.g., 5'-NGG-3' for Cas9). For each candidate PAM found within a gene, the 20 nt that are adjacent to the PAM are read and compared to a human genome.
  • PAM protospacer adjacent motif
  • 20-nt + PAM has no match within the human genome to a certain criteria
  • 20-nt + PAM can be used as the targeting sequence.
  • the match criteria may be the requirement of no perfect match.
  • the targeting sequence is 20-nt + PAM (e.g., 23-nt for Cas9) for which there is no 23 nt string within a human genome that matches > 70%.
  • the targeting sequence is 20-nt + PAM for which there is no 20 nt string within the human genome that is followed by the PAM and wherein the 20 nt of human genome matches the 20 nt of targeting sequence by > 70% (e.g., if Cas9 is the nuclease, a 20 nt string of human genome with 14 or more matching bases followed by the PAM would rule out use of a given targeting sequence).
  • the use of a targetable nuclease to cleave an HPV genome has been shown in an in vitro CRISPR endonuclease assay.
  • a genetically encoded gRNA scaffold was provided for transcription by a T7 phage polymerase.
  • T7 in vitro transcription produced the complete guide RNA with scaffold. Flanking regions of the genome targets were PCR amplified from HPV 18 genomic DNA (sold under the trademark 45152D by ATCC of Manassas, VA). Cas9 protein (from PNA Bio of Thousand Oaks, CA), guide RNA and target DNA were mixed and incubated for in vitro endonuclease assay. High endonuclease activities were revealed by DNA gel electrophoresis of the digested DNA.
  • FIG. 19 gives the results of the in vitro CRISPR endonuclease assay.
  • Four lanes show the results of PCR amplicon of the E6-E7 region, and the products of in vitro CRISPR treated amplicons.
  • Lanes 2-4 each show difference relative to control.
  • Lane 3 shows cleavage of the HPV genomic DNA into three fragments of distinct masses. Since the gRNA is designed to match within the E6 or E7 gene, expression of the corresponding proteins may be stopped by nuclease cleavage.
  • compositions and methods can be used to selectively express the targetable nuclease within cells that infected by HPV. It is understood that HPV infects keratinocytes. See e.g., Bossens, 1992, J Gen Virol 73:3269, incorporated by reference.
  • compositions are formulated for topical delivery to basal epithelium or mucosal epithelium, and include lipid nanoparticles, which include cationic lipids.
  • the nanoparticles are used to deliver the programmable nuclease, or mRNA encoding the same, that is programmed to cleave the viral genome as well as the agent that inhibits maintenance of the viral episome by interfering with expression or function of an endogenous human gene product.
  • Genes that participate in maintenance of the EBV episome have been reported to include NCL, RPL4, BRD4, NAP1L1, ORC1, ORC2, ORC3, ORC4, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, NPM1, NCL, TERF2, TERF2IP, TNKS, C1QBP, KPNA1, EBP2, RPA1, ORC1, ORC2, ORC3, ORC4, CTCF, CDC6, TIPIN, HCFC1, OCT2, MRE11A, RAD50, NBN, ATRIP, RAD9A, HUS 1, RAD1, TOPBP1, CLSPN, CHEK1, TP53BP1, and CDKN1A. Also included are proteins of the double- stranded DNA repair (DDR) pathway and their
  • Proteins of the DDR include: the MRE11-Rad50-Nbsl complex (MRN) or replication protein A (RPA); the Ataxia telangiectasia mutated (ATM) and Ataxia telangiectasia and Rad3 related (ATR) transducer kinases.
  • MRN MRE11-Rad50-Nbsl complex
  • RPA replication protein A
  • ATM Ataxia telangiectasia mutated
  • ATR Ataxia telangiectasia and Rad3 related transducer kinases.
  • ATR is activated in response to the presence of persistent ssDNA that results from replication stress.
  • RPA replication protein A
  • ATRIP ATR interacting partner
  • the ring-shaped 9-1- 1 complex (consisting of Rad9, Husl, Radl) is loaded onto collapsed replication forks and recruits TopBPl (Topoisomerase II-binding protein 1), a multifaceted factor essential for maintaining genomic stability and facilitating DNA replication by recruiting replication factors to replication forks.
  • 9-1-1 further enlists claspin, which recruits the kinase, Chkl.
  • ATR- dependent phosphorylation of Chkl activates a cell cycle checkpoint and facilitates stabilization of replication forks.
  • the DDR pathway not only functions to accurately repair DNA, but must also regulate the cell cycle, pausing it to allow repair to be completed.
  • the DDR activates specific downstream cell cycle checkpoints during each phase of the cell cycle to maintain DNA integrity.
  • a DDR mediated increase in stability of p53 can lead to increased expression of CDK inhibitor p21, which arrests cells in Gl.
  • CDK inhibitor p21 which arrests cells in Gl.
  • Chkl and Chk2 phosphorylation inhibits Cdc25 family members, which are important for progression at several stages of the cell cycle.
  • DNA MTase DNA methyltransferases
  • HMT histone methyl transferases
  • EBV BZLF 1 To inhibit EBV transcription, it may be valuable to target or inhibit cell DNA binding proteins, MEF 2D and RBP, that associate with histone deacetylases and maintain repressive chromatin at the EBV BZLF 1.
  • Proteins that maintain the EBV episome may further include nucleolin (NCL); ribosome protein L4 (RPL4); CTCF; Bromodomain Protein 4 (BRD4); Nucleosome Assembly Protein 1 (NAP1); the cell Origin Recognition Complex; the Mini Chromosome Maintenance complex; Nucleophosmin (NPM1); Nucleolin (NCL); telomeric repeat binding factor 2 (TRF2); TRF2- interacting protein hRapl ; the telomere-associated poly(ADP-ribose) polymerase (Tankyrase); p32/Tat-associated protein; Rchl/importin [alpha]; EBP2; replication protein A (RPA); cellular licensing proteins encoded by the MCM complex; the cellular origin recognition complex (ORC); CCCTC-binding factor (CTCF); cohesion; Cdc6; the MCM complex, and MCM2, MCM3, and MCM7; TRF2; Timeless (Tim); Host cell factor 1 (HCF
  • FIG. 20 shows results of an in vivo digestion of EBV with a Cas endonuclease.
  • the gel shows a large deletion induced by a guide RNA sgEBV2.
  • Lane 1-3 are before, 5 days after, and 7 days after sgEBV2 treatment, respectively.
  • a composition for treatment of a viral infection may include a programmable nuclease such as a Cas endonuclease and a guide RNA with a targeting sequence substantially complementary to a target within the EBV genome, as well as an agent that inhibits expression or function of an endogenous human gene product such as one of those listed above.
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • Latency-associated nuclear antigen is a predominant multifunctional nuclear protein expressed during latency, which plays a central role in episome tethering, replication and perpetual segregation of the episomes during cell division.
  • LANA has been shown to modulate multiple cellular signaling pathways and recruits various cellular proteins such as chromatin modifying enzymes, replication factors, transcription factors, and cellular mitotic framework to maintain a successful latent infection, tumor suppressors, p53 and pRb, transcription factors such as ATF4/CREB2 and STAT3, cellular signal transducer, GSK-3B, chromatin-binding proteins such as HP1, histone H2A/B, MeCP2, and Brd4.
  • the majority of the pathways activated after KSHV infections include, JNK, MEK- ERK, and p38 mitogen activated protein kinase (MAPK). The establishment of persistent infection depends on the activation of those cellular signaling pathways.
  • a composition for treatment of a viral infection may include
  • an endogenous human gene product such as one or more of AKT1, ATF4, BRD2, BRD4, BUBl, CBXl,
  • HSV episome Genes that participate in maintenance of the HSV episome have been reported to include HCFC1, REST, NGF, HDAC1, HDAC2 RCOR1, REST, KDM1A, H3F3A, and POU2F1.
  • a composition for treatment of a viral infection may include a programmable nuclease such as a Cas endonuclease— or an mRNA encoding the same— and a guide RNA with a targeting sequence substantially complementary to a target within the KSHV genome, as well as an agent that inhibits expression or function of an endogenous human gene product such as one or more oi HCFCl, REST, NGF, HDAC1, HDAC2 RCOR1, REST, KDM1A, H3F3A, and
  • compositions are provided to target or inhibit: HBV HBx (inhibit with CRISPR/Cas9); cellular damaged DNA binding protein 1 (DDB l) in the E3 ubiquitin ligase complex, which HBx binds for enhanced transcription; cell transcription factors: CREB, ATF, and STAT1/2; cell histone modifying enzymes: CBP, p300, PCAF/GCN5, HDAC1, hSirtl, and PRMT1 ; or combinations thereof.
  • HBV HBx inhibitor with CRISPR/Cas9
  • DDB l cellular damaged DNA binding protein 1
  • HBx binds for enhanced transcription
  • cell transcription factors CREB, ATF, and STAT1/2
  • cell histone modifying enzymes CBP, p300, PCAF/GCN5, HDAC1, hSirtl, and PRMT1 ; or combinations thereof.
  • the programmable nuclease may preferably be programmed to cleave HBx.
  • the inhibitor may be provided as a second programmable nuclease programmed to cleave a gene for DDB l, CREB, ATF, STAT 1/2, a cell histonemodifying enzyme, CBP, p300, PCAF/GCN5, HDAC1, hSirtl, PRMT1.
  • cell division cycle regulates cell 6 Cdc6 Entrez: 853244 YJL194W CDC6 division cell division cycle regulates cell 7 Cdc7 Entrez: 831 7 HsHskl CDC7 division cyclin-dependent cell cycle kinase 2 CDK2 GC: 101 7 CDKN2, p33 CDK2 regulation cyclin-dependent cell cycle kinase 4 CDK4 OM: 123829 CMM3, PSK-J3 CDK4 regulation cyclin-dependent regulates cell kinase 6 CDK6 GC: 1021 MCPH12, PLSTIRE CDK6 division
  • CDKN1 A CDKN1 A, CAP20,
  • Cdt1 CDT1 GC 81620 replication factor 1 CDT1 licensing factor checkpoint kinase DNA damage 1 Chk1 GC: 1 1 1 1 1 CHEK1 , hChkl CHEK1 response
  • IKBKA epidermal subunit alpha IKKA GC: 1 147 NFKBIKA, TCF16 CHUK differentiation casein kinase I,
  • CREB-binding AW558298 transcriptional protein CBP GC: 1387 KAT3A, RSTS CREBBP activator
  • CTCF GC 10664 MRD21 CTCF transcription factor
  • EBNA1 -binding enables EBV protein homolog EBP2 OM: 61443 EBNA1 BP2 EBP2 latency
  • Fanconi anemia Face FA3, mir- group C protein FANCC GC: 2176 3074-1 FANCC DNA repair protein Fanconi anemia
  • beta B GC 461 6 MYD1 18 B stress response
  • H3 histone family H3F3A OM: 601 128 H3F3, Histone H3 H3F3A histone
  • Host cell factor C1 HCF1 GC 3054 VCAF HCFC1 co regulator
  • GTPase Hras HRAS OM 190020 CTLO, p21 ras HRAS regulation
  • HUS1 HUS1 GC 3364 hHUS1 HUS1 checkpoint clamp
  • IFIT1 GC 3434 IFNAI1
  • RNM561 IFIT1 protein inhibitor of kappa NFKBIA MAD-3
  • transcription factor b IkBa GC 4792 NFKBI IKBA inhibitor
  • JAK1 A JAK1 B,
  • MAP kinase kinase 1 MAP2K1 GC 5604 MKK1 MAP2K1 cascade mitogen-activated regulates cell protein kinase 1 MAPK1 OM: 176948 ERK2, PRKM2 MAPK1 division mitogen-activated
  • MAPK12 OM 602399 SAPK3 MAPK12 stress response mitogen-activated CSPB1 , p38, p38- protein kinase 14
  • MAPK14 OM 600289 alpha MAPK14 stress response mitogen-activated regulates cell protein kinase 3
  • MAPK3 OM 601795 PRKM3, ERK1 MAPK3 division
  • MCM7 MCM7 GC 4176 PPP1 R104 MCM7 replication mouse double ACTFS, HDMX,
  • NAPL1 GC 4673 NAP1 , NRP NAP1 L1 DNA replication
  • BRCT domians transcriptional protein 1 NFBD1 OM 607593 MDC1 NFBD1 transactivator nuclear factor
  • NF-kB OM 16401 1 p105, p50 NFKB1 controller
  • NGF OM 162030 NGFB NGF growth factor
  • Notch homolog 1 Mis6, N1 , Tan1 , lin- translocation- 12, AOSS, AOVD1 ,
  • NOTCH1 GC 4851 hN1 NOTCH1 cell fate regulator ribosome nucleophosmin NPM GC: 4869 B23, NPM1 NPM1 synthesis
  • ORC4 GC 5000 ORC4L ORC4 DNA replication poly (ADP-ribose) DNA damage polymerase 1
  • PARP1 GC 142 ADPRT, PPOL PARP1 response proliferating cell
  • PCNA OM 176740 ATLD2 PCNA DNA replication phosphatidylinosit
  • checkpoint protein HREC1 checkpoint protein HREC1 , RAD1 cell cycle
  • RAD50 RAD50 GC 101 1 1 NBSLD, hRad50 RAD50 DS break repair
  • RAD51 RAD51 OM 17961 7 MRMV2, RECA RAD51 DNA break repair
  • checkpoint control clamp component cell cycle protein RAD9A RAD9A GC 5883 A RAD9A checkpoint proto-oncogene c- c-Raf, Raf-1 ,
  • RAF RAF1 OM 164760 CMD1 NN, NS5 RAF1 ERK pathway
  • REST GC 5978 XBR, REST4, WT6 REST transcription factor replication factor c A1 , MHCBFB, PO- subunit 1
  • RFC1 GC 5981 GA, RECC1 , RFC RFC1 DNA repair
  • binding subunit RPA1 GC 61 1 7 REPA1 , RF-A RPA1 DNA repair
  • helicase 1 RTEL1 OM 608833 NHL, C20orf41 RTEL1 telomere regulator
  • RuvB-like 2 (E. REPTIN, TIH2,
  • binding factor 2 TERF2 GC 7014 TRBF2, TRF2 TERF2 telomere protein
  • TIMELESS- cell cycle interacting protein TIPIN GC 54962 TIPIN TIPIN regulation
  • Tankyrase-1 TNKS GC 8658 pART5 TNKS DNA replication
  • topoisomerase 2- binding protein 1 TOPBP1 GC: 1 1073 TOP2BP1 TOPBP1 DNA repair p53-binding p202, TP53,
  • TP53BP1 binds p53 tumor protein 53 p53 OM: 191 170 TP53, BCC7, LFS1 TP53BP1 tumor supressor cell cycle tumor protein 73 p73 OM: 601990 TP73 TP73 regulation
  • Thymidylate nucleotide synthetase TYMS OM 188350 HST422, TMS, TS TYMS synthesis uracil DNA base excision glycosylase UNG GC: 7374 UDG, DGU, HIGM4 UNG repair

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Abstract

L'invention concerne des compositions comprenant une nucléase et d'autres agents qui agissent ensemble non seulement en vue de digérer l'acide nucléique viral, mais également en vue d'empêcher que des gènes humains ne soient utilisés en vue de maintenir l'épisome viral. Les compositions peuvent comprendre une nucléase programmable telle qu'une endonucléase associée à CRISPR (Cas), qui clive des cibles spécifiques dans le génome viral. Les compositions comprennent également un agent tel qu'une petite molécule, un siARN, ou un ARN guide avec l'endonucléase Cas, qui fonctionne comme un inhibiteur du maintien, de la réplication ou de la transcription de l'épisome, en empêchant l'expression d'un gène humain en une protéine qui aide à maintenir l'épisome viral.
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US20190269682A1 (en) * 2016-09-12 2019-09-05 University Of Florida Research Foundation, Incorporated Use of atr and chk1 inhibitor compounds
US11730734B2 (en) * 2016-09-12 2023-08-22 University Of Florida Research Foundation, Incorporated Use of ATR and Chk1 inhibitor compounds
CN113373148A (zh) * 2021-06-16 2021-09-10 中国人民解放军军事科学院军事医学研究院 一种调控app表达的靶标位点序列及其在防治ad上的应用
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