WO2018118586A1 - Antiviral/endogenous combination therapy - Google Patents

Abstract

Compositions include a nuclease and other agents that work together to not only digest viral nucleic acid but also to prevent human genes from being used to maintain the viral episome. Compositions may include a programmable nuclease such as a CRIS PR- associated (Cas) endonuclease that cleaves specific targets within the viral genome. The compositions also include an agent such as a small molecule, siRNA, or a guide RNA with the Cas endonuclease that operates as an inhibitor of episome maintenance, replication, or transcription by preventing expression of a human gene into a protein that aids in maintaining the viral episome.

Description

ANTIVIRAL/ENDOGENOUS COMBINATION THERAPY

Cross -Reference to Related Applications

This application claims the benefit of priority of U.S. Provisional Application No.

62/438,000, filed December 22, 2016, the contents of which are incorporated by reference.

Technical Field

The disclosure relates to antiviral therapeutics.

Background

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.

Summary

The invention comprises 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. For example, 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. 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.

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

endogenous host genes as well as viral genes, 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.

In preferred embodiments, 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). To target the virus, 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. For delivery to tissue such as basal epithelium or mucosal epithelium, 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.

In certain aspects, 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). 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.

In some embodiments, the inhibitor of episome maintenance may be polyamide 1, polyamide 25, aphidicolin, Chirl24, PF477736, roscovitine, or an interferon.

In certain embodiments, 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. Where 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, MAPK3, MAPK8, MAPKAPK2, MCM2, MCM3, MCM4, MCM7, MDM2, MECP2, MGMT, MLH3, MREllA, MTOR, MTOR, MYC, NAP1L1, NBN, NCL, NFBD1, NFKB1, NGF, NOTCH1, NPM1, NRAS, OCT2, ORCl, ORC2, ORC3, ORC4, PARP1, PCNA, PIK3CA, POU2F1, RAD1, RAD17, RAD21, RAD50, RAD51, RAD9A, RAF1, RB, RCOR1, REST, RFC1, RPAl, RPL4, RTELl, RUVBL2, SETMAR, SLC7A11, SMC1, SMC1A, SMC3, STAT3, TDP2, TERF2, TERF2IP, TWIN, TNKS, TOP2B, TOPBPl, TP53BP1, TP53BP1, TP73, TYMS, UNG, WNT1, YWHAB, YWHAE, YWHAG, YWHAH, YWHAQ, YWHAZ, or combinations thereof.

In some embodiments, 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).

In preferred embodiments, 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

papillomavirus (HPV).

In some embodiments, 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 Kaposi's sarcoma-associated herpesvirus (KSHV).

In some embodiments, the human gene is HCFC1, REST, NGF, HDAC1, HDAC2 RCOR1, REST, KDM1A, H3F3A, or POU2F1. The programmable nuclease may be

programmed to cleave nucleic acid of the Herpes Simplex virus (HSV).

Methods and 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).

Brief Description of the Drawings

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.

Detailed Description

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. In the depicted embodiment, 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. For the programmable nuclease 107 or the inhibitor, 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. As shown, 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. In the depicted embodiment, 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. When delivered to a patient, 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. Because the second programmable nuclease digests the human gene used for episomal maintenance, the second nuclease functions as an inhibitor 157 of episomal maintenance. As shown, 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. Most preferably, a nanoparticle such as a liposome or other cationic lipid nanoparticle is used to deliver the active RNP particles. 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.

In some embodiments, 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 inactive

programmable nuclease programmed to bind to a human gene used in episomal maintenance may be valuable in the cases in which cleaving a human gene could bring complications such as cell death or oncogenesis. 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. Once delivered to cells in vivo in a patient, the mRNA 207 is translated by the host (patient) ribosomes to provide the active programmable nuclease. The composition 201 preferably includes a guide sequence 221 that programs the nuclease to cleave viral nucleic acid. Where 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. Here, 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

programmable nuclease. 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.

Any suitable virus may be targeted using compositions and methods of the invention. Suitable 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). Another episomal DNA virus is a papillomavirus, such as human papillomavirus (HPV). Following an active infection, 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). Subsequently, these viruses may be activated to cause a latent infection in the host, resulting in a variety of disorders.

In their episomal states, 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. As an example, 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. For example, in some embodiments, 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.

Additionally or alternatively, 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.

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). To achieve best effect in treating viruses that may be characterized by a latent phase and an active phase, targeting endogenous genes or proteins that contribute to episomal maintenance is done in combination with the use of a programmable nuclease to digest the viral genetic material directly. The composition 201 includes an mRNA 207 that encodes a

programmable nuclease.

The programmable nuclease may be any suitable programmable nuclease. A

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;

structure-guided endonucleases (SGNs); zinc-finger nuclease (ZFNs); transcription activator effector-like nuclease (TALENs); and meganucleases. In preferred embodiments, methods and compositions of the disclosure use a Cas endonuclease. 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. 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. As used herein, "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.

FIG. 3 shows a Cas endonuclease RNP 301. In the depicted embodiment, 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).

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. These are then subdivided even further into type II (e.g., Cas9) and type V (e.g., Cpfl). 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.

C2cl (Class 2, candidate 1) is a type V-B Cas endonuclease that has been found.

Examples of C2cl have been indicated to be functional in E. coli. tracrRNAs (short RNAs that help separate the CRISPR array into individual spacers, or crRNAs) were required. As is the case for Cas9, with C2cl, 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). 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. Thus, 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 (SGN) 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. Similar to ZFNs and TALENS, in the SGN strategy, the two halves of the Fokl endonuclease are brought together by two adjacent targets on opposite strands, in essence creating a 40-bp or longer target sequence. Xu et al., 2016, An alternative novel tool for DNA editing without target sequence limitation: the structure-guided nuclease (SGN), 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 (TALENs) 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. In certain embodiments, 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. The ataxia-telangiectasia mutated (ATM) and ATM and Rad3 -related (ATR) serine/threonine protein kinases are sensors of DNA damage (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. Also, 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

programmable nuclease 107 programmed to cleave viral genetic material, or an mRNA 207 encoding the programmable nuclease. 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. Because treatment of epithelial tissue shows promise in that HPV episomes are lost upon treatment with PA1 or PA25, methods and compositions of the invention that involve topical delivery to basal or mucosal epithelium may be particularly effective to treating high grade, pre-cancerous HPV lesions and avoiding such consequences as cervical or anal cancer. 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. Once delivered to cells in vivo in a patient, 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. Where 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. Here, 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).

The guide sequences (e.g., guide RNAs, or "gRNAs", for a Cas endonuclease) may be designed to cause the programmable nuclease to cleave a specific target. In preferred

embodiments, 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. In relation to EBV, EBNAl is the only nuclear Epstein-Barr virus (EBV) protein expressed in both latent and lytic modes of infection. 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.

To design guide RNA targeting the EBV genome, the EBV reference genome is referred to. 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).

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

programmable nuclease as well as a guide RNA 1321 that programs the programmable nuclease to cleave viral nucleic acid. 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. For example, 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).

Viral episomal maintenance makes use of human genes and their associated protein pathways. For example, with the ATM and ATR pathways, during the very earliest stages of checkpoint activation, DNA damage sensors relay information to members of a family of phosphoinositide 3-kinase related kinases (PIKKs). In mammalian cells, two PIKK family members, ATM (ataxia-telangiectasiamutated) and ATR (ATM and Rad 3-related), play roles in early signal transmission through cell-cycle checkpoints. Ataxia telangiectasia mutated (ATM) is a serine/threonine protein kinase that is recruited and activated by DNA double- strand breaks. It phosphorylates several key proteins that initiate activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis. 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).

In response to a double stranded break (DSB), ataxia telangiectasia mutated serine- protein kinase (ATM) are normally activated. 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). Here, ATM is inhibited by the inhibitor 1361.

In some embodiments, Chk2 is inhibited by an inhibitor 1362. Normally, phosphorylated Chk2 in turn inactivates by phosphorylation cell division cycle 25A phosphatase (Cdc25A). Lack of active Cdc25A results in the accumulation of the phosphorylated (inactive) form of Cdk2, which is incapable to participate in initiation of replication. In the normal pathways, 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. For example, NF-KB activates the transcription of c-Myc (which in turn activates transcription of Cdc25A and tumor suppressor p53.

For UV damage, 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. The activated Chkl then phosphorylates Cdc25A, leading to Gl arrest. Normally, ATR phosphorylates ATR interacting protein (ATRIP), which in turn regulates ATR expression, and is an essential component of the DNA damage checkpoint pathway.

Activation of the Chk-Cdc25A pathways is followed by the p53-mediated maintenance of Gl/S arrest. In the maintenance stage, ATM or ATR phosphorylates Serl5 of p53 directly and Ser20 through activation of Chk2 or Chkl. In addition, 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. Moreover, 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. In this pathway ATM phosphorylation of structural maintenance of chromosomes 1-like 1 protein (SMCl) and Fanconi anemia complementation group D2 protein, isoform 1 (FANCD2), with the help of Nibrin, 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). In addition, 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. Any of the aforementioned participants in the ATR pathway may be inhibited using compositions of the invention. For example, a composition may include an inhibitor 1363 that prevents expression of the NBN gene. Additionally or alternatively, 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). Table 1 gives a listing of genes that are reported to play a role in maintenance of viral episomes. In Table 1, for each row, column 1 names a protein; column 2 gives a known abbreviation for that protein; column 3 gives an accession number for a database where that protein is recorded (OM= Online Mendelian Inheritance in Man; GC = GeneCards)— the accession numbers provide information for retrieving the sequence of the corresponding gene from GenBank; 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.

A number of human genes have been implicated in maintenance of viral episomes.

Human genes that have been reported to play a role in the maintenance of viral episomes are listed Table 1 and include AKT1, ATF4, ATM, ATR, ATRIP, BRCA1, BRD2, BRD4, BUB1, C1QBP, CBX1, CDC2, CDC25A, CDC25C, CDC6, CDC7, CDK2, CDK4, CDK6 CDKN1A, CDKN1A, CDT1, CHEK1, CHEK2, CHUK, CK1, CK1D, CLSPN, CREBBP, CTCF, CTCF, CXCR4, EBP2, FAN1, 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, MAPK3, MAPK8, MAPKAPK2, MCM2, MCM3, MCM4, MCM7, MDM2, MECP2, MGMT, MLH3, MREllA, MTOR, MTOR, MYC, NAP 1 LI, NBN, NCL, NFBD1, NFKB1, NGF, NOTCH1, NPM1, NRAS, OCT2, ORC1, ORC2, ORC3, ORC4, PARP1, PCNA, PIK3CA, POU2F1, RAD1, RAD17, RAD21, RAD50, RAD51, RAD9A, RAF1, RB, RCOR1, REST, RFC1, RPA1, RPL4, RTEL1, RUVBL2, SETMAR, SLC7A11, SMC1, SMC1A, SMC3, STAT3, TDP2, TERF2, TERF2IP, TWIN, TNKS, TOP2B, TOPBP1, TP53BP1, TP53BP1, TP73, TYMS, UNG, WNT1, YWHAB, YWHAE, YWHAG, YWHAH, YWHAQ, and YWHAZ.

Some categories of genes that contribute to episomal maintenance include DNA damage sensors. DNA damage sensors include Radl7, Radl, Rad9, Rad26, and Husl as well as proliferating cell nuclear antigen (PCNA) and replication factor c (RFC). The p53-dependent increase in p21 expression suppresses cyclin E- and cyclin A-associated cdk2 activities, and thereby prevents Gl-to-S phase progression.

Cells that have incurred DNA double-strand breaks (dsbs) during Gl phase activate p53 primarily via an ATM-dependent pathway. 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.

At the G2 checkpoint, DNA damage leads to the activation of Chkl, which, in turn, phosphorylates the mitosis-promoting phosphatase, Cdc25C. Phosphorylation of Cdc25C by hChkl creates a binding site for 14-3-3 proteins, and, in the 14-3-3-bound form, Cdc25C is either catalytically inhibited or sequestered in the cytoplasm (or both). In any case, the phosphorylated, 14-3-3-bound form of Cdc25C is prohibited from dephosphorylating and activating the mitotic cyclin B Cdc2 kinase, and the damaged cells are effectively blocked from entering mitosis. See Abraham, 2001, Cell cycle checkpoint signaling through the ATM and ATR kinases, Genes & Dev 15:2177-2196; U.S. Pub. 2004/0209239; each incorporated by reference. Any of those proteins may be targeted using an inhibitor of the invention.

Another protein that may be targeted is Metnase. 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).

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).

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. In the depicted embodiment, 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.

Where the programmable nuclease gene 907 codes for Cas endonuclease, 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. 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

complementary to a target in a human gene.

In certain embodiments, 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. 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.

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. In some embodiments, 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. These

embodiments may be preferred where 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. Where 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. In a most preferred embodiment, 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 (e.g., liposomes) 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. Preferably, 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. In the most preferred embodiments, 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.

The composition is then used to cleave 1713 the genetic material of the virus.

Additionally, 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. Preferably 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. Specifically, 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, MAPK3, MAPK8, MAPKAPK2, MCM2, MCM3, MCM4, MCM7, MDM2, MECP2, MGMT, MLH3, MRE11A, MTOR, MTOR, MYC, NAP1L1, NBN, NCL, NFBD1, NFKB1, NGF, NOTCH1, NPM1, NRAS, OCT2, ORC1, ORC2, ORC3, ORC4, PARP1, PCNA, PIK3CA, POU2F1, RAD1, RAD17, RAD21, RAD50, RAD51, RAD9A, RAF1, RB, RCOR1, REST, RFC1, RPA1, RPL4, RTEL1, RUVBL2, SETMAR, SLC7A11, SMCl, SMCIA, SMC3, STAT3, TDP2, TERF2, TERF2IP, TWIN, TNKS, TOP2B, TOPBP1, TP53BP1, TP53BP1, TP73, TYMS, UNG, WNT1, YWHAB, YWHAE, YWHAG, YWHAH, YWHAQ, or YWHAZ. In preferred embodiments, 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).

Example 1: HPV

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-β 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). Thus it may be beneficial to interfere with the expression of uracil DNA glycosylase by cleaving the endogenous human gene UNG. 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.

The tyrosyl-DNA -phosphodiesterase 2 protein maintains viral episomes. Thus cleaving the endogenous human gene TDP2 may be effective. TDP2 (aka TTRAP) knockdown has been reported to cause an episomal loss. 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. Where that 20-nt + PAM has no match within the human genome to a certain criteria, then that 20-nt + PAM can be used as the targeting sequence. The match criteria may be the requirement of no perfect match. In a preferred embodiment, 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%. In a much preferred embodiment, 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. In certain embodiments, 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.

Example 2: EBV

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

corresponding endogenous human genes. 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. ATR is activated in response to the presence of persistent ssDNA that results from replication stress. RPA (replication protein A) binds this ssDNA and recruits ATRIP (ATR interacting partner) and ATR. 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. For example, a DDR mediated increase in stability of p53 can lead to increased expression of CDK inhibitor p21, which arrests cells in Gl. Likewise, Chkl and Chk2 phosphorylation inhibits Cdc25 family members, which are important for progression at several stages of the cell cycle.

To inhibit EBV transcription, it may be valuable to target or inhibit cell epigenetic modifying enzymes: DNA methyltransferases (DNA MTase, DNMT) and histone methyl transferases (HMT), Ezh2 and Suv420h.

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 (HCF1), a component of the mixed-lineage leukemia (MLL) histone methyltransferase complex; transcription factor OCT2 (octamer-binding transcription factor 2); and BRD4.

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.

Example 3: Kaposi's sarcoma-associated herpesvirus (KSHV)

During latent phase, the viral episome of Kaposi's sarcoma-associated herpesvirus (KSHV) is tethered to the host chromosome and replicates once during every cell division. Latency-associated nuclear antigen (LANA) 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.

Endogenous human genes that participate in maintenance of the KSHV episome have been reported to include AKT1, ATF4, BRD2, BRD4, BUB1, CBX1, CDC25C, CDC6, CDC7, CDK2, CDK6, CDTl, CREBBP, CTCF, GSK3A, GSK3B, HDACl, HICl, HIFIA, 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, ORC1, ORC2, ORC3, ORC4, PARP1, PIK3CA, RB, RFC1, SLC7A11, STAT1, STAT2, STAT3, TWIN, TOP2B, TP53BP1, and WNT1. 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 KSHV genome, as well as an agent that inhibits expression or function of an endogenous human gene product such as one or more of AKT1, ATF4, BRD2, BRD4, BUBl, CBXl, CDC25C, CDC6, CDC7, CDK2, CDK6, CDTl, 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, ORC1, ORC2, ORC3, ORC4, PARP1, PIK3CA, RB, RFC1, SLC7A11, STAT1, STAT2, STAT3, TIPIN, TOP2B, TP53BP1, and WNT1.

Example 4: HSV

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

POU2F1.

Example 5: HBV

To inhibiti HBV cccDNA transcription, 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. Thus, embodiments provide a composition for treating an HBV infection. The composition includes a programmable nuclease that cleaves viral nucleic acid or an RNA encoding the programmable nuclease; and

an inhibitor of episome maintenance, replication, or transcription. 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

CAP20, CDKN1 A,

CIP1 , MDA-6,

CDK-interacting SDI1 , WAF1 , cell cycle protein 1 P21 GC: 1026 P21 CIP1 CDKN1 A regulation

CDKN1 A, CAP20,

CIP1 , MDA-6, cell cycle kinase inhibitor 1 A P21 OM: 1 16899 SDI1 , WAF1 CDKN1 A regulation

DUP, RIS2,

chromatin licensing

DNA replication and DNA

factor Cdt1 CDT1 GC: 81620 replication factor 1 CDT1 licensing factor checkpoint kinase DNA damage 1 Chk1 GC: 1 1 1 1 CHEK1 , hChkl CHEK1 response

CHEK2, CDS1 ,

HuCdsl , LFS2,

checkpoint kinase PP1425, RAD53, DNA damage 2 Chk2 GC: 1 1200 hChk2 CHEK2 response inhibitor of nuclear

factor kappa-b CHUK, IKBKA, epidermal subunit alpha IKKA GC: 1 147 NFKBIKA, TCF16 CHUK differentiation casein kinase I,

alpha 1 CKI OM: 600505 CSNK1 A1 , CKI CK1 DNA damage casein kinase 1 , CSNK1 D, HCKID, DNA damage delta CK1 5 OM: 600864 CSNK1 D CK1 D response

checkpoint claspin CLSPN GC: 63967 claspin CLSPN regulator

CREBBP,

CREB-binding AW558298, transcriptional protein CBP GC: 1387 KAT3A, RSTS CREBBP activator

CCCTC-binding

factor CTCF GC: 10664 MRD21 CTCF transcription factor

Transcriptional MRD21 , CCCTC- repressor CTCF CTCF GC: 10664 binding factor CTCF transcription factor

CD184, D2S201 E,

FB22, HM89,

LAP3, LCR1 ,

C-X-C chemokine LESTR, NPY3R,

receptor type 4 CXCR4 OM: 162643 WHIM, fusin CXCR4 ubiquitin ligand

EBNA1 -binding enables EBV protein homolog EBP2 OM: 61443 EBNA1 BP2 EBP2 latency

FANCD2/FANCI- associated KIAA1018, KMIN,

nuclease 1 FAN1 GC: 22909 MTMR15 FAN1 endonuclease

Fanconi anemia Face, FA3, mir- group C protein FANCC GC: 2176 3074-1 FANCC DNA repair protein Fanconi anemia

complementation

group D2 protein, FA-D2, FA4,

isoform 1 FANCD2 GC: 2177 FACD, FAD FANCD2 DNA repair protein

Growth arrest and

DNA damage- inducible protein GADD45 GADD45

alpha A GC: 1647 DDIT1 A stress response

Growth arrest and

DNA damage- GADD45 GADD45

inducible, beta B GC: 461 6 MYD1 18 B stress response

Growth arrest and

DNA damage- inducible protein GADD45 GADD45

gamma G GC: 10912 DDIT2, GRP17 G stress response

Glycogen cell proliferation synthase kinase 3 GSK-3 OM: 606784 GSK3A GSK3A and signalling glycogen synthase

kinase 3 beta GSK3B GC: 2932 GSK-3 GSK3B signal transducer

H3 histone, family H3F3A OM: 601 128 H3F3, Histone H3 H3F3A histone

HCFC1 , CFF,

MRX3, PPP1 R89, transcription

Host cell factor C1 HCF1 GC: 3054 VCAF HCFC1 co regulator

Histone GON-10, HD1 , histone deacetylase 1 HDAC1 OM: 601241 RPD3 HDAC1 modification

Histone histone deacetylase 2 HDAC2 GC: 3066 HD2, RPD3, YAF1 HDAC2 modification hypermethylated methylated in in cancer 1 HIC1 GC: 3090 ZBTB29, ZNF901 HIC1 cancer

HIF-1 -alpha,

Hypoxia-inducible MOP1 , PASD8,

factor 1 -alpha HIF1 A GC: 3091 bHLHe78 HIF1 A transcription factor

C-BASE/HAS, cell division

GTPase Hras HRAS OM: 190020 CTLO, p21 ras HRAS regulation

cell cycle timeless homolog hTIM OM: 603887 Tim, timeless HTIM regulation

Checkpoint protein

HUS1 HUS1 GC: 3364 hHUS1 HUS1 checkpoint clamp

IFN-induced

protein with ISG56, P56, C56,

tetratricopeptide G10P1 , IFI-56K, interferion induced repeats 1 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,

Janus kinase 1 JAK1 GC: 371 6 JTK3 JAK1 signal transducer

Janus kinase 2 JAK2 GC: 371 7 JTK10, THCYT3 JAK2 signal transducer c-Jun JUN OM: 1651690 AP-1 , AP1 JUN transcription factor histone

acetyltransferase KAT7, HBOA, licensing and DNA KAT7 HB01 GC: 1 1 143 MYST2, ZZC2HC7 KAT7 replication

Lysine-specific AOF2, BHC1 10,

histone KDM1 , LSD1 ,

demethylase 1 A KDM1 A GC: 23028 CPRF KDM1 A demethylase IPOA5, NPI-1 ,

importin KPNA1 GC: 3836 RCH2 KPNA1 transport protein

V-Ki-ras2 Kirsten

rat sarcoma viral

oncogene CFC2, NS, NS3, cell division homolog KRAS GC: 3845 RALD, GTPase KRAS regulation mitogen-activated

protein kinase CFC3, MEK1 , 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

protein kinase 1 1 MAPK1 1 OM: 602898 PRKM1 1 , p38-beta MAPK1 1 stress response mitogen-activated ERK6, p38-gamma,

protein kinase 12 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

JNK1 , PRKM8,

AI849689, JNK,

mitogen-activated Prkm8, SAPK1 , transcription protein kinase 8 MAPK8 OM: 601 158 JNK-46 MAPK8 regulation

MAP kinase- activated protein MAPKAP MK-2, MK2, MAPKAP MAP kinase kinase 2 K2 GC: 9261 MAPKAP-K2 K2 cascade

DNA replication BM28, CCNL1 ,

licensing factor CDCL1 , D3S3194, replication MCM2 MCM2 OM: 1 16945 MITOTIN MCM2 licensing factor

DNA replication

licensing factor HCC5, P1 -P1 .h, mini-chromosome MCM3 MCM3 GC: 4172 RLFB MCM3 maintenance

DNA replication

licensing factor CDC21 , NKCD, genome

MCM4 MCM4 GC: 4173 CDC54 MCM4 replication

DNA replication CDC47, P85MCM,

licensing factor PNAS146, genome

MCM7 MCM7 GC: 4176 PPP1 R104 MCM7 replication mouse double ACTFS, HDMX,

minute 2 homolog MDM2 GC: 4193 hdm2 MDM2 p53 regulator

AUTSX3, MRX16,

methyl CpG PPMX, RS, RTS, expression binding protein 2 MECP2 GC: 4204 RTT MECP2 regulation

06-alkylguanine

DNA AGT, AI267024,

alkyltransferase MGMT GC: 4255 AGAT MGMT DNA repair

DNA mismatch HNPCC7, mutL

repair protein Mlh3 MLH3 GC: 27030 homolog 3 MLH3 DNA repair

Double-strand

break repair ATLD, HNGS1 ,

protein MRE1 1 A MRE1 1 A GC: 4361 MRE1 1 MRE1 1 A DS break repair mechanistic target FRAP, RAFT1 , cell growth of rapamycin MTOR GC: 2475 RAPT1 , SKS MTOR regulation mechanistic target FRAP, RAFT1 , cell cycle of rapamycin mTOR OM: 601231 RAPT1 , SKS MTOR regulation MRTL, MYCC, transcription c-Myc MYC GC: 4609 bHLHe39, v-myc MYC activator

Nucleosome

Assembly Protein

1 NAPL1 GC: 4673 NAP1 , NRP NAP1 L1 DNA replication

NBN, AT-V1 , ATV,

NBS, P95, nibrin,

nibrin NBS1 OM: 602667 NBS1 NBN DS break repair nucleolin NCL GC: 4691 C23 NCL ribsome synthesis nuclear factor with

BRCT domians transcriptional protein 1 NFBD1 OM: 607593 MDC1 NFBD1 transactivator nuclear factor

kappa-light-chain- enhancer of transcription activated B cells NF-kB OM: 16401 1 p105, p50 NFKB1 controller

Nerve growth Beta-HSAN5,

factor NGF OM: 162030 NGFB NGF growth factor

Notch homolog 1 , Mis6, N1 , Tan1 , lin- translocation- 12, AOSS, AOVD1 ,

associated NOTCH1 GC: 4851 hN1 NOTCH1 cell fate regulator ribosome nucleophosmin NPM GC: 4869 B23, NPM1 NPM1 synthesis

Neuroblastoma

RAS viral

oncogene ALPS4, CMNS, N- cell division homolog NRAS GC: 4893 ras, NCMS, NS6 NRAS regulation octamer-binding

transcription factor POU2F2, OTF2,

2 OCT2 OM: 164176 Oct-2 OCT2 transcription factor

Origin recognition

complex subunit 1 ORC1 GC: 4998 HSORC1 L, PARC1 ORC1 DNA replication

Origin recognition

complex subunit 2 ORC2 GC: 4999 ORC2L ORC2 DNA replication

Origin recognition

complex subunit 3 ORC3 GC: 23595 LAT, LATHEO ORC3 DNA replication

Origin recognition

complex subunit 4 ORC4 GC: 5000 ORC4L ORC4 DNA replication poly (ADP-ribose) DNA damage polymerase 1 PARP1 GC: 142 ADPRT, PPOL PARP1 response proliferating cell

nuclear antigen PCNA OM: 176740 ATLD2 PCNA DNA replication phosphatidylinosit

ol-4,5- CLOVE, CWS5,

bisphosphate 3- MCAP, MCM,

kinase PIK3CA OM: 171834 p1 10-alpha PIK3CA oncogene

POU domain,

class 2,

transcription factor

1 POU2F1 GC: 5451 OCT1 , OTF1 POU2F1 transcription factor

Cell cycle

checkpoint protein HREC1 , RAD1 cell cycle

RAD1 RAD1 GC: 581 0 homolog RAD1 checkpoint

Cell cycle CCYC, HR24L, cell cycle checkpoint protein RAD1 7 GC: 5884 RAD17SP RAD1 7 checkpoint RAD17

ds break repair CDLS4, HR21 , regulates protein rad21 HMCD1 , NXP1 , chromatid homolog RAD21 GC: 5885 SCC1 RAD21 separation

DNA repair protein

RAD50 RAD50 GC: 101 1 1 NBSLD, hRad50 RAD50 DS break repair

DNA repair protein BRCC5, FANCR,

RAD51 RAD51 OM: 17961 7 MRMV2, RECA RAD51 DNA break repair

Cell cycle Rad9 checkpoint

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

RB, OSRC,

retinoblastoma PPP1 R130, p1 05- protein pRb GC: 5925 Rb, pp1 10 RB tumor supressor

REST corepressor neural

1 CoREST GC: 231 86 RCOR1 , RCOR RCOR1 differentiation

RE1 -Silencing

Transcription

factor 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

Replication protein

A 70 kDa DNA- HSSB, MST075,

binding subunit RPA1 GC: 61 1 7 REPA1 , RF-A RPA1 DNA repair

60S ribosomal

protein L4 RPL4 GC: 6124 L4 RPL4 ribosome protein regulator of

telomere

elongation

helicase 1 RTEL1 OM: 608833 NHL, C20orf41 RTEL1 telomere regulator

ECP51 , INO80J,

RuvB-like 2 (E. REPTIN, TIH2,

coli) RUVBL2 GC: 10856 TIP48 RUVBL2 helicase

Histone-lysine N- METNASE,

methyltransferase SETMAR GC: 641 9 HsMaM , Marl SETMAR NHEJ pathways solute carrier

family 7 member

1 1 SLC7A1 1 OM: 607933 XCT, xCT, CCBR1 SLC7A1 1 signalling structural

maintenance of regulates chromosomes chromatid protein 1 SMC1 PDB: 1 W1 W CHL10 SMC1 separation structural

maintenance of

chromosomes 1 - CDLS2, DXS423E, chromosome like 1 protein SMC1 A GC: 8243 SMC1 SMC1 A segregation structural

maintenance of regulates chromosomes chromatid protein SMC3 Entrez: 853371 J 1049 SMC3 separation

Signal transducer

and activator of ADMIO, ADRF,

transcription 3 STAT3 OM: 102582 HIES STAT3 transcription factor Tyrosyl-DNA

Phosphodiesteras TRAF, TTRAP,

e 2 TDP2 OM: 605764 HTDP2, EAP2 TDP2 DNA repair telomeric repeat

binding factor 2 TERF2 GC: 7014 TRBF2, TRF2 TERF2 telomere protein

TERF2 interacting

protein TERF2IP OM: 605061 Rap1 homolog TERF2IP telomere binding

TIMELESS- cell cycle interacting protein TIPIN GC: 54962 TIPIN TIPIN regulation

ARTD5, PARP-5a,

TIN1 , TNKS1 ,

Tankyrase-1 TNKS GC: 8658 pART5 TNKS DNA replication

DNA

topoisomerase 2- TOPI IB, top2beta,

beta TOP2B GC: 7155 ΤοροΙΙβ TOP2B topisomerase

DNA

topoisomerase 2- binding protein 1 TOPBP1 GC: 1 1073 TOP2BP1 TOPBP1 DNA repair p53-binding p202, TP53,

protein 1 53BP1 GC: 7158 TDRD30 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

Proto-oncogene BMND16, INT1 , canonical Wnt protein Wnt-1 WNT1 GC: 7471 OI15 WNT1 pathway

YWHAB, GW128, member of 14-3-3

14-3-3 alpha/beta YWHAB GC: 7529 HS1 YWHAB proteins

YWHAE, HEL2,

KCIP-1 , MDCR, member of 14-3-3

14-3-3 epsilon YWHAE GC: 7531 MDS YWHAE proteins

member of 14-3-3

14-3-3 gamma YWHAG GC: 7532 YWHAG, PP1 R1 70 YWHAG proteins

member of 14-3-3

14-3-3 eta YWHAH GC: 7533 YWHAH, YWHA1 YWHAH proteins

member of 14-3-3

14-3-3 theta YWHAQ GC: 10971 YWHAQ, 1 C5, HS1 YWHAQ proteins

member of 14-3-3

14-3-3 zeta YWHAZ GC: 7534 YWHAZ, HEL4 YWHAZ proteins

Claims

What is claimed is:

1. A composition for treating a viral infection, the composition comprising:

a programmable nuclease that cleaves viral nucleic acid or an RNA encoding the programmable nuclease; and

an inhibitor of episome maintenance, replication, or transcription.

2. The composition of claim 1, wherein the inhibitor of episome maintenance comprises an agent that prevents a virus or host-encoded gene from maintaining, replicating or transcribing a viral episome.

3. The composition of claim 2, further comprising a nanoparticle encapsulating at least the programmable nuclease or the RNA encoding the programmable nuclease.

4. The composition of claim 3, wherein the inhibitor of episome maintenance, replication, or transcription comprises one selected from the group consisting of: polyamide 1, polyamide 25, aphidicolin, Chirl24, PF477736, roscovitine, 6-azacytidine, decitabine, gemcitabine, interferons alpha and gamma (IFNoc, IFNy), tumor necrosis factor alpha (TNFoc), IL-6, and a lymphotoxin beta receptor (LT R) agonist.

5. The composition of claim 4, wherein said virus is selected from the group consisting of 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), Merkel cell polyomavirus (MCV), and hepatitis b virus (HBV).

6. The composition of claim 3, wherein the programmable nuclease is one selected from the group consisting of a zinc-finger nuclease, a transcription activator effector like nuclease, structure-guided nuclease, a DNA-guided endonuclease, and a CRISPR-associated (Cas) endonuclease.

7. The composition of claim 6, wherein 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.

8. The composition of claim 7, wherein the Cas endonuclease is complexed with a first RNA with a portion complementary to the genetic material of the virus and further wherein the second programmable nuclease is a Cas endonuclease complexed with a second RNA with a portion complementary to the human gene.

9. The composition of claim 8, wherein the human gene is one selected from the group consisting of: AKTl, ATF4, ATM, ATR, ATRIP, BRCAl, BRD2, BRD4, BUBl, CIQBP, CBP, CBXl, CDC2, CDC25A, CDC25C, CDC6, CDC7, CDK2, CDK4, CDK6, CDKN1A, CDKN1A, CDT1, CHEK1, CHEK2, CHUK, CK1, CK1D, CLSPN, CREB, CREBBP, CTCF, CTCF, CXCR4, DDB1, EBP2, EZH2, FAN1, FANCC, FANCD2, GADD45A, GADD45B, GADD45G, GCN5, GSK3A, GSK3B, H3F3A, HCFC1, HDAC1, HDAC2 , MCI, HIF1A, HRAS,hSirtl, HTIM, HUS1, IFIT1, IKBA, JAK1, JAK2, JUN, KAT7, KDM1A, KPNA1, KRAS, MAP2K1, MAPK1, MAPK11, MAPK12, MAPK14, MAPK3, MAPK8, MAPKAPK2, MCM2, MCM3, MCM4, MCM7, MDM2, MECP2, MEF 2D, MGMT MLH3, MRE11A, MTOR, MTOR, MYC, NAP 1 LI, NBN, NCL, NFBD1, NFKB1, NGF, NOTCH1, NPM1, NRAS, OCT2, ORC1, ORC2, ORC3, ORC4, p300, PARP1, PCAF, PCNA, PIK3CA, POU2F1, PRMT 1, RAD1, RAD17, RAD21, RAD50, RAD51, RAD9A, RAF1, RB, RCOR1, REST, RFC1, RPA1, RPL4, RTEL1, RUVBL2, SETMAR, SLC7A11, SirTl, SMC1, SMC1A, SMC3, STAT1/2, STAT3, Suv4-20h, TDP2, TERF2, TERF2IP, TWIN, TNKS, TOP2B, TOPBP1, TP53BP1, TP53BP1, TP73, TYMS, UNG, WNT1, YWHAB, YWHAE,

YWHAG, YWHAH, YWHAQ, and YWHAZ.

10. The composition of claim 8, wherein the human gene is selected from the group consisting of: NCL, RPL4, BRD4, NAP1L1, ORC1, ORC2, ORC3, ORC4, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, NPM1, NCL, TERF2, TERF2IP, TNKS, CIQBP, KPNA1, EBP2, RPA1, ORC1, ORC2, ORC3, ORC4, CTCF, CDC6, TIPIN, HCFC1, OCT2, MRE11A, RAD50, NBN, ATRIP, RAD9A, HUS 1, RAD1, TOPBP1, CLSPN, CHEK1, TP53BP1, DNA MTase, DNMT, HMT, Ezh2, Suv420h, MEF 2D, RBP, and CDKN1A.

11. The composition of claim 10, wherein the virus is Epstein-Barr virus (EBV).

12. The composition of claim 8, wherein 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, FAN1, FANCC, and MTOR.

13. The composition of claim 12, wherein the virus is human papillomavirus (HPV).

14. The composition of claim 8, wherein the human gene is selected from the group consisting of AKT1, ATF4, BRD2, BRD4, BUB 1, CBX1, CDC25C, CDC6, CDC7, CDK2, CDK6, CDT1, CREBBP, CTCF, GSK3A, GSK3B, HDAC1, HICl, HIF1A, HRAS, HTIM, 1KB A, JAK1, JAK2, JUN, KAT7, KRAS, MAP2K1, MAPK1, MAPK11, MAPK12, MAPK14, MAPK3, MAPK8, MAPKAPK2, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, MECP2, MTOR, MYC, NOTCH1, NPM1, ORC1, ORC2, ORC3, ORC4, PARP1, PIK3CA, RB, RFC1,

SLC7A11, STAT1, STAT2, STAT3, TIPIN, TOP2B, TP53BP1, and WNT1.

15. The composition of claim 14, wherein the virus is KSHV.

16. The composition of claim 8, wherein the human gene is selected from the group consisting of HCFC1, REST, NGF, HDAC1, HDAC2 RCOR1, REST, KDM1A, H3F3A, and POU2F1.

17. The composition of claim 16, wherein the virus is HSV.

18. The composition of claim 6, wherein the inhibitor of episome maintenance comprises an siRNA complementary to the human gene.