WO2017106616A1 - Varicella zoster virus encoding regulatable cas9 nuclease - Google Patents

Abstract

Recombinant Herpesviridae mutants that encode guide RNA sequences and Cas9 nuclease fused with a destabilization domain that effectively controls expression of a Cas9 nuclease fusion protein, and the subsequent inactivation of genes targeted by the guide RNA sequences. The recombinant mutants are useful in the prevention and treatment of infection by T-cell tropic viruses.

Description

VARICELLA ZOSTER VIRUS ENCODING REGULATABLE CAS9 NUCLEASE

RELATED APPLICATIONS

[0001] This applicatiaa claims priority to, and the benefit of, U.S. Provisional Application No. 62/268,867, filed December 17, 2015, me content of which is incorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

[0002] The contents of the text file named "UNCO-007-001WOJST25," which was created on December 14, 2016 and is 2.44KB in size, are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

[0003] This disclosure relates to recombinant Herpesviridac virus mutants that encode guide RNA sequences and cas9 nuclease fused with a destabilization domain mat effectively controls expression of the cas9 nuclease, and therefore the subsequent inactivation of genes targeted by the guide RNA sequences.

BACKGROUND

[0004] Varicella zoster virus (VZV) causes varicella (chickenpox) in children, becomes latent in ganglia along the entire neuraxis (Mahalingam et al., Latent varicella- zoster virus DNA in human trigeminal and thoracic ganglia, 1990 N Engl J Med 323, 627- 630) and reactivates later in life to produce zoster (shingles). Zoster is characterized by pain and vesicular rash. The most common complication of zoster is postherpetic neuralgia (PHN), operationally defined as pain persisting more man 3 months after acute zoster. Zoster patients also develop stroke from unifocal or multifocal VZV vasculopathy, as well as myelitis and zoster paresis, rneningoencephalitis, and ocular complications. Most neurologic complications of zoster are increased in the rapidly expanding aging and iinmuriocompromised populations, especially in AIDS patients. Zostavax immunization reduces zostercases by Sl% andPHNby 66%. It is estimated mat by the year 2030, 65 million people will be over age 65, and by 2050, at least 21 million will be over age 85. least 500,000 zoster patients, almost half of whom will experience PHN. Recently, a VZV subunit vaccine has been shown to significantly reduce the risk of zoster in 50 years and older immunocompetent adults. While the effectiveness of the vaccine in preventing zoster is impressive, its efficiency in preventing neurological diseases particularly in

immunodeficient individuals is yet to be determined. Further, the extent of stroke or disease outcomes of VZV infection of immune-privileged sites such as arteries has not been fully elucidated. Therefore, it is essential to inactivate latent virus genome residing in human ganglia to prevent reactivation. But there is no existing method to inactivate the latent VZV in human ganglia thereby preventing reactivation.

[0005] In 2012, an estimated 1.2 million people in the United States were living with HTV infection, an increase of more than 20% over the previous decade, attributable to continued new infections and a 36.5% decrease in deaths related to the acquired

immunodeficiency syndrome (AIDS). Reported new diagnoses of HIV infection have decreased overall, but there are large disparities among groups; new HIV infections are increasingly concentrated among gay men and younger men. Between 2003 and 2014, the numbers of diagnoses of infections associated with injection-drug use and heterosexual contact decreased by approximately 70% and 40%, respectively. However, between 2003 and 2014, diagnoses increased by 5% among all men who have sex with men, in whom more than two thirds of all diagnoses now occur, and more than doubled among young gay and bisexual men. Blacks continue to account for nearly half of all diagnoses each year, most among gay and bisexual men; the proportion of diagnoses among Hispanic Americans, Asian Americans, and American Indians or Alaska Natives has increased, and the number of diagnoses among people 13 to 24 years of age increased by 43%, also mostly among gay and bisexual men.

[0006] Current therapy for controlling HTV-1 infection and impeding AIDS development (highly active antiretroviral therapy; HAART) includes a mixture of compounds mat suppress various steps of the viral life cycle. HAART profoundly reduces viral replication in cells that support HTV-1 infection and reduces plasma viremia to a minimal level but neither suppresses low-level viral genome expression and replication in tissues nor targets the latently infected cells mat serve as a reservoir for HTV-1, including brain macrophages, microglia, and astrocytes, gut-associated lymphoid cells, and others. HTV-1 persists in approximately 106 cells per patient during HAART, and is linked to Because current therapies are unable to suppress viral gene transcription from integrated proviral DNA or to eliminate the transcriptionally-silent proviral genomes, low-level viral protein production by latently infected cells may contribute to multiple illnesses in the aging HIV-l-infected patient population. Supporting this notion, pathogenic viral proteins including transactivator of transcription (Tat) are present in the cerebrospinal fluid of HIV - 1 -positive patients receiving HAART. To prevent viral protein expression and viral reactivation in latently-infected host cells, new strategies are thus needed to permanently disable the HIV-1 genome by eradicating large segments of integrated proviral DNA.

SUMMARY

[0007] This disclosure provides novel and improved recombinant viral mutants based on the Herpes viridae family of DNA viruses, and methods for their manufacture and use in the treatment of viral infections, particularly latent viral infections, and diseases or disorders caused by or secondary to latent viral infections. The recombinant Herpesviridae viral mutants of this disclosure, when administered in conjunction with trimethoprim, reduce or even eliminate latent viral infections, and reduce or eliminate diseases or disorders caused by or secondary to latent viral infections, including serious neurologic disease caused by varicella zoster virus (VZV), or another herpesvirus, or human immunodeficiency virus (HIV).

[0008] One aspect of this disclosure provides a recombinant Herpesviridae mutant that is composed of a Herpesviridae virus genome recombinantly engineered to include a polynucleotide sequence encoding a Cas9 nuclease fused to a polynucleotide sequence encoding at least one destabilization domain, and at least one polynucleotide sequence encoding at least one guide RNA (gRNA). The Herpesviridae virus genome may be a strain selected from Varicella-zoster virus (VZV), Simian varicella virus, HSV-1, HSV-2, HHV-5 or Cytomegalovirus, HHV5 or Epstein Barr virus, HHV6A and HHV6B or herpes lymphotropic virus, HHV7 or Pityriasis Rosacea, and SHV/HHV8.

[0009] The polynucleotide sequence encoding a Cas9 nuclease may be fused to at least one polynucleotide sequence encoding a nuclear localization signal protein (NLS).

[0010] The destabilization domain may be the Escherichia dihydrofolate reductase

(DHFR) destabilization domain. distinct gRNA sequences.

[0012] Hie polynucleotide sequence encoding at least one gRNA may encode a gRNA targeted to a protein of a T-cell tropic DNA virus.

[0013] The polynucleotide sequence encoding at least one gRNA may encode a gRNA targeted to a polynucleotide sequence encoding viral components of a virus selected from a VZV virus, a Herpes simplex virus type 1, a Herpes simplex virus type 2, an HIV virus, and a Simian varicella virus (SW).

[0014] The polynucleotide sequence encoding at least one gRNA may encode a gRNA targeted to a varicella zoster virus (VZV) or a simian varicella virus (SVV) protein selected from VZV glycoprotein E, VZV viral kinase ORF47, VZV viral kinase ORF66, VZV IE62 protein, VZV IE63 protein, VZV IE70 protein, VZV IE71 protein, VZV DNA polymerase, and a VZV glycoprotein, ORF 63/70, ORF 62/71 , ORF6, ORF28, ORF55, ORF25, ORF26, ORF30, ORF34, ORF 42/45, ORF 43, ORF54, ORF4, ORF5, ORF9A, ORF9, ORF 17, ORF20, ORF21 , ORF22, ORF24, ORF27, ORF29, ORF 31, ORF33, ORF33.5, ORF37, ORF38, ORF39, ORF40, ORF41, ORF44, ORF46, ORF48, ORF50, ORF51, ORF52, ORF53, ORF56, ORF60, ORF61 , ORF62, ORF64, ORF65, ORF66, ORF67, ORF68, and/or ORF69.

[0015] The polynucleotide sequence encoding at least one gRNA may encode a gRNA targeted to a Herpes simplex virus type 1 (HSV-1) protein selected from DNA Polymerase (UL42), DNA Polymerase Catalytic Subunit (UL30), DNA Helicase (UL5), DNA Primase (UL52), 1CP4 (transcriptional regulator), US 1 (host range factor), UL49A (envelope protein), ICPO (transcriptional regulator), ULl, UL8, UL9, UL 14, UL15, UL17, UL18, UL19, UL22, UL25, U126, UL26.5, UL27, UL28, UL29 UL31 , UL34, UL35, UL36, UL37, UL38, UL48, UL49, UL49.5, UL53, UL54, RS I, and/or US6.

[0016] The polynucleotide sequence encoding at least one gRNA may encode a gRNA targeted to a Herpes simplex virus type 2 (HSV-2) protein selected from DNA Polymerase (UL42), DNA Polymerase Catalytic Subunit (UL30), DNA Helicase (UL5), DNA Primase (UL52), ICP4 (transcriptional regulator), US1 (host range factor), UL49A (envelope protein), ICPO (transcriptional regulator), UL 1, UL8, UL9, UL14, UL15, ULl 7, UL18, UL19, UL22, UL25, U126, UL26.5, UL27, UL28, UL29 UL31 , UL34, UL35, UL36, UL37, UL38, UL48, UL49, UL49.5, UL53, UL54, RS I, and/or US6. gRNA targeted to The polynucleotide sequence encoding at least one gRNA may encode a gRNA targeted to a Human immunodeficiency virus (HIV) protein selected from Gag, Pol, gp 160, Tat, Rev, Nef, Vpr, Vlf, and Vpu.

[0018] Another aspect of this disclosure is a bacterial artificial chromosome (BAC) clone comprising any one of the recombinant Herpesviridae mutants described above.

[0019] This disclosure also provides a mammalian cell that has been infected with a recombinant Herpesviridae mutant described above.

[0020] This disclosure also provides an in vitro cell culture comprising at least one mammalian cell infected with a recombinant Herpesviridae mutant described above.

[0021] This disclosure also provides a polynucleotide encoding a Cas9 nuclease fused to a polynucleotide encoding at least one destabilization domain.

[0022] This disclosure also provides a recombinant fusion protein comprising a

Cas9 nuclease fused to at least one destabilization domain.

[0023] This disclosure also provides a pharmaceutical composition comprising an effective amount of the recombinant Herpesviridae mutants described above and a pharmaceutically acceptable carrier or excipient.

[0024] Another aspect of this disclosure is a method of treating an infection by a T- cell tropic DNA virus by administering a recombinant Herpesviridae mutant described above to a patient in need of such treatment, and administering an amount of trimethoprim to the patient effective to stabilize the cas9 protein expressed by the recombinant

Herpesviridae mutant. In these methods, the infection treated may be a herpes simplex virus type 1 infection, a herpes simplex virus type 2 infection, a varicella virus infection, a human or simian immunodeficiency virus (HIV or SIV) infection, and a latent HIV or varicella zoster virus (VZV) infection. In these methods, the trimethoprim may be administered to the patient for a period between 1 and 20 days. In these methods, the trimethoprim may be administered to the patient at a dose between about 5 mg/kg/day and about 40 mg/kg/day. In these methods, the trimethoprim may be administered to the patient in combination with sulfamethoxazole. In these methods, the patient may be immunocompromised. In these methods, the patient may be 60 year-old or older, and/or is immune deficient secondary to an infection or cancer.

[0025] This Summary is neither intended nor should it be construed as being representative of the full extent and scope of the present disclosure. Moreover, references certain embodiments of the present disclosure and should not necessarily be construed as limiting all embodiments to a particular description. The present disclosure is set forth in various levels of detail in this Summary as well as in the attached drawings and the Description of Embodiments and no limitation as to the scope of the present disclosure is intended by either the inclusion or non-inclusion of elements, components, etc. in this Summary. Additional aspects of the present disclosure will become more readily apparent from the Description of Embodiments, particularly when taken together with the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 shows the production scheme for the preparation of a VZV genome containing gRNAs and Cas9-coding sequences.

[0027] Figure 2 shows a Western blot analysis of protein extracts from Vero cells infected with a recombinant mutant VZV virus in the presence of trimethoprim (TMP) in a concentration range of 0.0 - 0.1 uM.

DESCRIPTION OF EMBODIMENTS

[0028] The present disclosure is drawn to recombinant Herpesviridae mutants engineered to encode guide RNA sequences, and cas9 nuclease fused with a destabilization domain. These mutants provide precise control over expression of the cas9 nuclease and precise inactivation of genes targeted by the guide RNA sequences. These mutants may be used to treat diseases or disorders such as herpes virus infection, including varicella virus infections such as chicken pox, herpes zoster (shingles), and human or simian

immunodeficiency virus (HIV or SIV) infections. These mutants may also be used to treat or prevent latent viral infections, including latent HIV or varicella zoster virus (VZV) infection.

Definitions

[0029] As used herein and in the claims, the singular forms "a," "an," and "the" include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to "an agent" includes a single agent and a plurality of such agents.

[0030] "BAC" refers to a recombinant bacterial artificial chromosome (BAC). microbial nuclease system, and CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage.

[0032] CRISPR is an adaptive immune system that provides protection against mobile genetic elements (e.g., viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type Π CRISPR systems, correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous nuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for nuclease 3-aided processing of pre-crRN A Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, and then trimmed 3 -5' exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNA species. However, guide RNAs can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA molecule. (See, e.g., Jinek M, et. al. 2012 Science 337:816-821 the entire contents of which is hereby incorporated by reference). The tracrRNA and spacer RNA together are often referred to as guide RNA, which is typically between 17 and 20 nucleotides in length. The two RNA species can be joined to form one hybrid RNA molecule referred to herein as "guide RNA" (gRNA). When complexed with CAS9, the CAS9-guide RNA complex will find and specifically cut the correct DNA targets. (Pennisi, E. 2013 Science 341 (6148): 833-836). Thus, reference herein to a gRNA "targeted to" a component, including a specific protein, of a viral genome refers to a CRISPR-Cas system gRNA that hybridizes with the specified target sequence, whereby the gRNA hybridizes to the targeted sequence and the CRISPR-associated Cas9 nuclease cleaves the targeted viral DNA molecule.

[0033] The term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease comprising a Cas9 protein, or a functional fragment thereof. A Cas9 nuclease is also referred to sometimes as a CRISPR-associated nuclease. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., Ferretti J. J., et al., Complete genome sequence of an Ml strain of Streptococcus pyogenes. Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); Deltcheva E, 2011 Nature 471:602-607; and Jinek M, et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. 2012 Science 337:816-821, the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilics. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, et al., The tracrKNA and Cas9 families oftype llCRISPR-Cas immunity systems. 2013 RNA Biology 10:5, 726-737; the entire contents of which is incorporated herein by reference. In some embodiments, proteins comprising Cas9, or functional fragments thereof, are referred to as "Cas9 variants." A Cas9 variant shares homology to Cas9, or a fragment thereof while retaining gRNA-guided endonuclease activity. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to wild type Cas9, while retaining gRNA-guided endonuclease activity. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to the corresponding fragment of wild type Cas9, while retaining gRNA-guided endonuclease activity. Wild type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC017053.1). The Cas9 nuclease useful in the recombination viral mutant constructs of this disclosure may also be codon-optimized for the desired cell type in which the Cas9 is to be expressed.

[0034] "Destabilization domain" refers to a polypeptide that, when fused to the coding sequence of a protein such as a gene product required for virus replication, causes the fused protein to be degraded unless a particular condition is met (e.g., presence of an antibiotic) which prevents or inhibits the degradation otherwise elicited by the

destabilization domains. Preferably the domain is linked at or proximate to the 5'- or 3'-end of a Herpesviridae gene involved in replication, e.g., VZV or SW gene 62, gene 63, gene 70 or gene 71. recombinant Herpes viridae virus mutant of this disclosure that expresses at least one gRNA and Cas9 nuclease to cut host DNA at a location(s) specified by the gRNA, which amount of viral mutant is obtained when exposed to conditions that provide for the stable expression of Cas9 nuclease. The inducible conditions that permit stable expression of Cas9 nuclease may comprise the administration of the antibiotic trimethoprim, which, when administered proximate to the viral mutant, or even years after administration of the viral mutant, results in the stable expression of the Cas9 nuclease and thereby the cutting of DNA at site(s) dictated by the gRNA(s) encoded by the viral mutant. The effective amount of the recombinant Herpes viridae viral mutants of this disclosure required for effective expression of Cas9 and site-specific DNA cutting may vary given the particular viral strain, or the weight and/or health of the individual treated.

[0036] As used herein, the term "encode" refers broadly to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first. The second molecule may have a chemical structure that is different from the chemical nature of the first molecule. For example, in some aspects, the term "encode" describes the process of semi-conservative DNA replication, where one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase. In other aspects, a DNA molecule can encode an RNA molecule (e.g., by the process of transcription that uses a DNA-dependent RNA polymerase enzyme). Also, an RNA molecule can encode a polypeptide, as in the process of translation. When used to describe the process of translation, the term "encode" also extends to the triplet codon that encodes an amino acid. In some aspects, an RNA molecule can encode a DNA molecule, e.g., by the process of reverse transcription incorporating an RNA-dependent DNA polymerase. In another aspect, a DNA molecule can encode a polypeptide, where it is understood that "encode" as used in that case incorporates both the processes of transcription and translation.

[0037] "Escherichia coli DHFR destabilization domain" or "Escherichia coli DHFR destabilization gene" refers to a sequence derived from the E coli dihydrofolate reductase (DHFR) polypeptide which when fused (to the C- or N-terminus of the polypeptide) or incorporated into proteins such as viral genes encoding polypeptides involved or required for viral replication ma}' cause degradation of the protein unless an effective amount of the chemical method to regulate protein stability in the mammalian central nervous system. 2010 Chem Biol. 17(9):981-8). The sequence of exemplary DHFR destabilization domain nucleotide and protein sequences are contained in SEQ ID NO: 1 and 2 preceding the claims. Preferably the DHFR destabilization domain will comprise a polypeptide at least 80% identical to SEQ ID NO: 2, more preferably at least 85% identical, still more preferably at least 90% identical and even more preferably at least 95, 96, 97, 98 or 99% identical thereto.

[0038] As used herein, an expression control element refers to a region which controls the expression of a target gene product in an expression cassette or viral mutant construct. Specifically, it refers to a region comprising a promoter. The expression regulatory region may further comprise various regulatory sequences involved in a transcriptional regulation. The phrase "operably linked" describes a relationship wherein the components operably linked function in their intended manner. Particularly, a nucleic acid sequence encoding a protein may be operably linked to expression control sequences (also referred to as regulatory sequences; e.g., promoter, enhancer, silencer sequence, etc.) so as to retain proper transcriptional regulation. In one instance, a polynucleotide sequence encoding a cas9 nuclease may be operably linked to an expression control sequence to effectively promote or 'drive' expression of the cas9 nuclease.

[0039] "Gene essential to viral replication" or "Gene essential to herpesvirus replication" herein refers to a gene present in a virus, e.g., a herpesvirus, the expression of which alone or in association with another viral gene is required for the virus to replicate and maintain its normal life cy cle. Herpesviruses have been well studied, in particular those that infect humans, and there are a number of genes in each of Herpes simplex viruses 1 and 2, varicella-zoster virus, EBV (Epstein-Barr virus), human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, and Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) which have been identified to be essential to virus replication.

[0040] "Herpesviridae" or herpesviruses refers to a large family of DNA viruses that cause diseases in animals, including humans. The members of this family are also known as herpesviruses. The family name is derived from the Greek word herpein ("to creep"), referring to the latent, recurring infections typical of this group of viruses. Herpesviridae can cause latent or lytic infections. There are more than 130 herpesviruses, and some are from mammals, birds, fish, reptiles, amphibians, and mollusks. Of these there are eight (Epstein-Barr virus), human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, and Kaposi's sarcoma-associated herpesvirus. Of these eight, there are at least five species of Herpesviridae which are extremely widespread among humans, HSV-1, which causes facial/oral cold-sores, HSV-2 (genital herpes), Varicella zoster virus, which causes chicken- pox and shingles, Epstein-Barr virus, which causes mononucleosis (glandular fever) and Cytomegalovirus - which are extremely widespread among humans. More than 90% of adults have been infected with at least one of these, and a latent form of the virus remains in most people.

[0041] "PHN" refers to postherpetic neuralgia (PHN).

[0042] The terms "polynucleotide," "polynucleotide sequence," and "nucleic acid sequence" are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double- stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Polynucleotides can comprise deoxyribonucleotides and ribonucleotides including both naturally occurring molecules and synthetic analogues, and any combination these. The polynucleotides provided herein also encompass all forms of sequences including, but not limited to, single- stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

[0043] "Recombinant" in reference to a nucleic acid or polypeptide or virus indicates that the material (e.g., a recombinant nucleic acid, gene, polynucleotide, polypeptide, etc.) has been altered by human intervention. This includes the recombinant polynucleotides comprising the various components of the viral mutant constructs of this disclosure. The terms "recombinant polynucleotide" and "recombinant viral mutant construct" are used interchangeably herein. A recombinant construct comprises an artificial or heterologous combination of nucleic acid sequences, e.g., regulatory and coding sequences mat are not found together in nature. Generally, the arrangement of parts of a recombinant molecule is not a native configuration, or the primary sequence of the recombinant polynucleotide or polypeptide has in some way been manipulated. A naturally occurring nucleotide sequence becomes a recombinant polynucleotide if it is removed from the native location from which it originated (e.g., a chromosome), or if it is transcribed from a recombinant DNA construct. A gene open reading frame is a recombinant molecule if that nucleic acid mutant (even if mat ORF has the same nucleotide sequence as the naturally occurring gene). Protocols and reagents to produce recombinant molecules, especially rocoinbinant nucleic acids, are well known to one of ordinary skill in the ait In some embodiments, the term "reconibinant cell line" refers to any cell line containing a recombinant nucleic acid, mat is to say, a nucleic acid mat is not native to mat host cell.

[0044] "Varicella zoster virus (VZV)" is an alphaherpesvirus that is in the same subfamily as herpes simplex virus (HSV) 1 and 2. VZV is a member of varicellovirus genus, along with equine herpesvirus 1 and 4, pseudorabies virus, and bovine herpesvirus 1 and 5. Ceropithccine herpesvirus 9 (simian varicella virus) is the virus most homologous to VZV. The complete sequence of the VZV genome was determined by (Davison AJ, and Scott I, The complete DNA sequence of varicella-zoster virus. 1986 J Gen Virol. 67: 1759- 1816). The prototype strain, VZV Dumas, is 124,884 base pairs in length. The genome consists of a unique long region (UL) bounded by terminal long CTRL) and internal long (URL) repeats, and a unique short region (US) bounded by internal short (IRS), and terminal short (TRS) repeats (Figure 1). The US region can orientate either of two directions, while the UL region rarely changes its orientation; thus, there are usually two isomers of the genome in infected cells. The genome is linear in virions with an unpaired nucleotide at each end. (Cohen, J. I. The Varicella-Zoster Virus Genome.2010 Current Topics in Microbiology and Immunology, 342, 1-14). "Simian varicella virus or SW" is the simian counterpart of VZV and like VZV is a neurotropic alphaherpesvirus which causes varicella in simians and like VZV virus becomes latent in ganglia along the entire ncuraxis, and may reactivate during immunosuppression resulting in zoster like symptoms.

Viral Mutant Constructs

[0045] This disclosure provides novel recombinant^ engineered herpesvirus mutants mat include a polynucleotide sequence encoding a cas9 nuclease and at least one destabilization domain, as well as a polynucleotide sequence encoding at least one guide RNA. These recombinant sequences are engineered to express a cas9-destabilization domain fusion protein and CRISPR RNA guide strand segments). Because the cas9 nuclease is fused to a destabilization domain, the cas9 nuclease is destabilized and quickly degraded, without acting to cleave specific nucleic acid sequences as directed by the guide RNA (gRNA). Under conditions that stabilize the destabilization domain, such as the addition of a drug mat stabilizes the destabilization domain, the cas9 nuclease is not specificity of the gRNA.

[0046] Hie herpes virus genome that is recombinant-}' engineered to produce mutant

Herpesviridae in this disclosure may be any live Herpesviridae strain, including HSV-1, HSV-2, cytomegalovirus, human Varicella zoster virus, Epstein Barr virus, HHv-6a or HHV6b, HHV-7 or HHV-8. The virus may be a live attenuated varicella vaccine, the Oka strain that was developed by Takahashi and his colleagues in Japan the early 1970s (Takahashi et al., 1974). Such viruses may include Herpesviridae which infect humans and non-human animals. The Herpesviridae may comprise Herpesviridae types that infect humans and reactivate after initial infection. There are 8 herpesvirus types which are known to infect humans: Herpes simplex viruses 1 and 2, varicella-zoster virus, EBV (Epstein-Barr virus), human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, and Kaposi's sarcoma- associated herpesvirus (also known as human herpesvirus 8 or HHV/8). In particular, these Herpesviridae viruses may include HSV-1, HSV-2, Varicella zoster virus, Epstein-Barr virus, and Cytomegalovirus.

[0047] The Herpesviridae may also be a strain which infects animals, as these strains will be useful in the agricultural industry and veterinary applications. In this regard, there are more than 130 herpesviruses which infect different animals including mammals, birds, fish, reptiles, amphibians, and mollusks.

[0048] A common attribute of different Herpesviridae virus strains is their propensity to become latent in specific cell types after initial infection and to reactivate after the initial infection, sometimes many years after the initial infection. For HSV-1, HSV-2 and VZV, the primarily target cell is mucoepithelial cells, but these strains are all known to sequester or become latent in neurons. By contrast, for HHV-4 or Epstein Barr virus, the primary target cell is B cells and epithelial cells, and these strains are known to become latent in B cells. HHV-5 or cytomegalovirus initially infects monocytes, lymphocytes and epithelial cells and may become latent in monocy tes and lymphocytes after initial infection. The primary target cells for HHV-6 or Roseolovirus or herpes h/mphotropic virus are T cells and this virus may become latent in such T cells after the initial infection. Similarly, the primary target cells for HHV-7 or Pityriasis Rosacea virus is T cells and this virus may become latent in the T cells in an infected individual many years after the initial viral exposure. Finally, the primary target cells for HHV-8 or Kaposi's sarcoma-associated become latent in B cells after the initial viral infection.

[0049] Based on this common property of latency of these herpes viruses, the recombinant Herpesviridae viral mutants of this disclosure may be used to establish an integrated expression system in the mammalian host cells listed above. In the absence of conditions that stabilize the protein destabilization domain, no Cas9 nuclease activity is detected. But in the presence of conditions that stabilize the destabilization domain, the Cas9 nuclease acts to cleave DNA of the host cell in which the viral expression system is residing, at a site or sites dictated by one or more single gRNA strand(s), which are also encoded and expressed by the integrated expression system of this disclosure.

[0050] Due to the stable, long-term integration of the Herpesviridae virus strains composing the recombinant viral mutant constructs of this disclosure, the integrated viral expression systems may be stabilized to 'trigger' the Cas9 nuclease activity at the target sites dictated by the gRNA strand(s) many years or even decades after the initial infection of the individual human, animal, or plant with the recombinant viral mutant constructs of this disclosure.

[0051] The polynucleotide sequence encoding a Cas9 nuclease and at least one destabilization domain, as well as a polynucleotide sequence encoding the gRNA(s), may be inserted into the Herpesviridae virus genome into a region or specific location in the viral genome that is not essential for virus replication. For example, these polynucleotide sequences may be inserted into the Unique Long (UL) region and/or the Unique Short (US) region of the VZV genome. The polynucleotides encoding the Cas9 nuclease-destabilization domain(s) fusion protein, and the polynucleotides encoding the gRNA(s) may be inserted together into the same location in the viral genome, or these polynucleotide sequences may be inserted separately, at different locations in the viral genome.

[0052] Each of the polynucleotides encoding the Cas9 nuclease-destabilization domain(s) fusion protein and the polynucleotides encoding the gRN A(s) may be located into the viral genome under the control of an expression control sequence or promoter. As noted above, these polynucleotide sequences may be located at the same site, or in separate sites, in the viral genome, and similarly, the expression of these polynucleotide sequences may be driven by (i.e., "operably linked to") the same promoter or by separate promoters. This may include, for example, a recombinantly engineered Herpesviridae viral mutant in distinct promoters.

[0053] The expression control element may also include various regulatory sequences involved in transcriptional regulation of the polynucleotides inserted into the viral genome constructs of this disclosure. The expression control element(s) may be any promoters that function in a mammalian cell. The expression control element(s) may be a polymerase II promoter or a polymerase in promoter. Examples of useful promoter elements include a constitutive expression promoter such as CMV promoter, SV40 promoter, EF-la promoter, CAG promoter, and PGK promoter; U3 promoter; U6 promoter; HI promoter; and the like. In addition to these promoters, a known inducible promoter, a tissue- or organ-specific promoter, a time-specific promoter, or variant sequences having functional equivalence thereto, may be used in the recombinant viral mutant constructs of this disclosure. Examples of promoters that may be inserted into the viral genomes to drive expression of the gRNA polynucleotide sequences include a U6 or an HI RNA polymerase III promoter, a simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), human Ubiquitin C promoter (UBC), human elongation factor la promoter (EF1 A), mouse phosphogly cerate kinase 1 promoter (PGK), and chicken a β- Actin promoter coupled with CMV early enhancer (C AGG), or a varicella zoster virus (VZV) IE63 promoter. Examples of promoters that may be inserted into the viral genomes to drive expression of the polynucleotide sequence encoding a Cas9 nuclease include a U6 or an HI RNA polymerase III promoter, a simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), human Ubiquitin C promoter (UBC), human elongation factor la promoter (EF1 A), mouse phosphogly cerate kinase 1 promoter (PGK), and chicken β-Actin promoter coupled with CMV early enhancer (CAGG), or a varicella zoster virus (VZV) IE63 promoter.

[0054] As described above, the Cas9 nuclease is a well-known and commercially available RNA-guided endonuclease that catalyzes site-specific cleavage of double stranded DNA. The cleavage site is located adjacent to the target 3-base sequence (NGG) PAM (Protospacer Adjacent Motif) (Jinek M. et al., 2012 Science. 816-821). The PAM sequence, NGG, must follow the targeted region on the opposite strand of the DNA with respect to the region containing the complementary gRNA sequence. A useful nuclear localization signal (NLS), linked to Cas9 nuclease is the S. pyogenes Simian virus 40 (SV40) T antigen nuclear of the cas9 protein.

[0055] Thus, the polynucleotide sequence encoding a Cas9 nuclease inserted into the recombinant Herpesviridae mutants of this disclosure may be fused or 'linked' to include at least one polynucleotide sequence encoding a nuclear localization signal protein (NLS). The polynucleotide sequence encoding a Cas9 nuclease may be fused to at least one polynucleotide sequence encoding a nuclear localization signal protein (NLS) at each end of the polynucleotide sequence encoding the Cas9 nuclease.

[0056] Additionally, the polynucleotide sequence encoding a Cas9 nuclease inserted into the recombinant Herpesviridae mutants of this disclosure is fused or 'linked' to include at least one destabilization domain. As described above, the destabilization domain is a polypeptide which, when fused to the cas9 nuclease causes the cas9 nuclease to be degraded unless a particular condition is met in the cell. When this intracellular condition is met (e.g., presence of an antibiotic) the degradation of the cas9 nuclease is prevented. The destabilization domain may be linked to the cas9 nuclease at, or proximate to, the amino terminus or the carboxy terminus (or both) of the cas9 protein to form a cas9-destabilization domain fusion protein that is expressed from the recombinant Herpesviridae virus mutant constructs of this disclosure.

[0057] Thus, the recombinant Herpesviridae virus mutant constructs of this disclosure include at least one polynucleotide sequence encoding a destabilization domain. The polynucleotide sequence encoding a destabilization domain may be located the 5' end, or the 3' end, or both ends, of the polynucleotide sequence encoding the cas9 nuclease, such that the expression of the cas9 nuclease from the recombinant Herpesviridae viral mutant constructs of this disclosure produces a cas9-destabilization domain fusion protein comprising a destabilization domain linked to the cas9 nuclease at, or proximate to, the amino terminus or the carboxy terminus (or both) of the cas9 protein. The polynucleotide sequence encoding a destabilization domain may be located 5' or 'upstream' to the polynucleotide sequence encoding the Cas9 nuclease, such that expression of the polynucleotides produces a protein destabilization domain fused to the amino-terminus of the Cas9 nuclease. Alternatively or additionally, the polynucleotide sequence encoding a destabilization domain may be located 3' or 'downstream^" to the polynucleotide sequence encoding the Cas9 nuclease, such that expression of the polynucleotides produces a protein destabilization domain fused to the carboxy-terminus of the Cas9 nuclease. responsive elements and/or the FRAP and rapamycin systems that have been developed for use in gene transfer paradigms (Guo ZS, et al., Gene transfer: the challenge of regulated gene expression. 2008 Trends Mol Med 14: 410-418; Stieger et al., In vivo gene regulation using tetracycline-regulatable systems. 2009 Adv Drug Deliv Rev 61: 527-541; Pollock R, Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral mutant. 2000 Proc Natl Acad Sci USA 97: 13221-13226). The destabilization domain may include the Escherichia coli DHFR destabilization domain encoded by the Escherichia coli DHFR destabilization gene, which is a sequence derived from the E coli dihydrofolate reductase (DHFR) polypeptide. When fused to the C- or N-terminus of the cas9 nuclease, or incorporated into a cas9 nuclease protein, this DHFR polypeptide may cause degradation of the cas9 protein unless an effective amount of the antibiotic trimethoprim, or an equivalent is present (see Tai, et al., Destabilizing Domains Mediate Reversible Transgene Expression in the Brain. 2012 PLOS One 7(9):e46269: DOI: 10.1371/journal.pone.0046269). The sequence of exemplary DHFR destabilization domain polynucleotide and protein sequences are set forth in Example 1 of this disclosure. The DHFR destabilization domain may comprise a polypeptide at least 80% identical to this protein sequence, preferably at least 85% identical, at least 90% identical and even more preferably at least 95, 96, 97, 98, or 99% identical thereto.

[0059] As described above, guide RNAs direct Cas9 nucleases to cut DNA at a specific genomic location. Therefore, in addition to expression of the Cas9 nuclease, the CRISPR-Cas9 system requires a specific RNA moiety to recruit and direct the nuclease activity. These guide RNAs may be in the form of the combination of a chemically synthesized trans-activating CRISPR RNA (tracrRNA) plus a chemically synthesized CRISPR RNA (crRN A) designed to cleave the viral gene target site, or in the form of an expressed single guide RNA that consists of both the crRNA and tracrRNA as a single polynucleotide construct. The design of an effective, functional guide RNA achieves the desired and very specific gene excision or 'knockout.' Numerous commercial entities now offer services and algorithms to design and/or test functional, specific guide RNAs designed to target a gene of interest.

[0060] The recombinant Herpes viridae mutant constructs of this disclosure may include two or more distinct gRNA sequences. Such use of multiple gRNA sequences may include gRNA sequences targeted to the same component of a viral genome, or to separate use of two or more gRNA polynucleotide sequences (i.e., multiplexed gRNA sequences) has been shown to increase the specificity of the Cas9 nuclease/gRNA DNA

cutting/excision system and reduce off-target cutting (Hu, W. et al., RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection, 2014 PNAS 111 (31): 11461 -66). Varicella virus strains not only infect and replicate in primary T- lymphocytes. Such viruses are said to be T-cell tropic. Thus, the at least one gRNA polynucleotide sequence may be targeted to polynucleotide sequences encoding viral components (such as viral protein(s)) of a T-cell tropic DNA virus.

[0061] The polynucleotide sequence(s) encoding at least one gRNA may encode at least one gRNA targeted to polynucleotide sequences encoding viral components of a virus selected from a VZV, a Herpes simplex virus type 1, a Herpes simplex virus type 2, an HTV virus, and a Simian varicella virus (S W). Such viral components targeted by the gRNA polynucleotide sequence may encode a viral protein selected from a VZV viral protein, a Herpes simplex virus type 1 protein, a Herpes simplex virus type 2 protein, an HIV viral protein, and a Simian varicella virus (SW) viral protein.

[0062] The at least one polynucleotide sequence encoding at least one gRNA may encode at least one gRNA targeted to a varicella zoster virus (VZV) or a simian varicella virus (SW) protein selected from VZV/SVV glycoprotein E, VZV/SVV viral kinase ORF47, VZV/SW viral kinase ORF66, VZV/SW IE62 protein, VZV/SW IE63 protein, VZV/SW IE70 protein, VZV/SW IE71 protein, VZV/SW DNA polymerase, or a VZV/SW glycoprotein, ORF 63/70, ORF 62/71 , ORF6, ORF28, ORF55, ORF25, ORF26, ORF30, ORF34, ORF 42/45, ORF 43, ORF54, ORF4, ORF5, ORF9A, ORF9, ORF 17, ORF20, ORF21 , ORF22, ORF24, ORF27, ORF29, ORF 31, ORF33, ORF33.5, ORF37, ORF38, ORF39, ORF40, ORF41, ORF44, ORF46, ORF48, ORF50, ORF51, ORF52, ORF53, ORF56, ORF60, ORF61 , ORF62, ORF64, ORF65, ORF66, ORF67, ORF68, and/or ORF69.

[0063] The polynucleotide sequence encoding gRNAs may encode at least one gRNA targeted to a Herpes simplex virus type 1 (HSV-1) protein selected from DNA Polymerase (UL42), DNA Polymerase Catalytic Subunit (UL30), DNA Helicase (UL5), DNA Primase (UL52), 1CP4 (transcriptional regulator), US 1 (host range factor), UL49A (envelope protein), ICPO (transcriptional regulator), UL1, UL8, UL9, UL 14, UL15, UL17, UL36, UL37, UL38, UL48, UL49, UL49.5, UL53, UL54, RS I, and/or US6.

[0064] The polynucleotide sequence encoding gRNA may encode at least one gRNA targeted to a Herpes simplex virus type 2 (HSV-2) protein selected from DNA

Polymerase (UL42), DNA Polymerase Catalytic Subunit (UL30), DNA Helicase (UL5),

DNA Primase (UL52), ICP4 (transcriptional regulator), US1 (host range factor), UL49A

(envelope protein), ICPO (transcriptional regulator), UL 1, UL8, UL9, UL14, UL15, UL17,

UL18, UL19, UL22, UL25, U126, UL26.5, UL27, UL28, UL29 UL31 , UL34, UL35,

UL36, UL37, UL38, UL48, UL49, UL49.5, UL53, UL54, RS I, and/or US6.

[0065] The polynucleotide sequence encoding gRNA may encode at least one gRNA targeted to a Human immunodeficiency virus (HIV) protein selected from Gag, Pol, gp 160, Tat, Rev, Nef, Vpr, Vlf, and Vpu.

[0066] As described above, this disclosure provides polynucleotides or nucleic acid molecules comprising the various components employed in the recombinant Herpesviridae viral mutant constructs of this disclosure (i.e., any one of or any combination of nucleases, promoters, recognition sites, insert nucleic acids, polynucleotides of interest, targeting mutants, selection markers, and other components). One or more of the components of the recombinant viral mutants of this disclosure can be provided in an expression cassette for expression in a prokary otic cell, a eukaryotic cell, a bacterial, a yeast cell, or a mammalian cell or other organism or cell type of interest. The cassette can include 5' and 3' regulatory sequences operably linked to a polynucleotide provided herein. When used to refer to the joining of two protein coding regions, 'operably linked' means that the coding regions are in the same reading frame. A nucleic acid sequence encoding a protein may be Operably linked' to regulatory sequences (e.g., promoter, enhancer, silencer sequence, etc.) so as to retain proper transcriptional regulation.

[0067] These expression cassettes may include in the 5 -3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a recombinant polynucleotide provided herein, and a transcriptional and translational termination region (i.e., termination region) functional in mammalian cell or a host cell of interest. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or a polynucleotide provided herein may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or a polynucleotide provided herein may be heterologous to the host cell or to each other. For example, a the species from which the polynucleotide was derived, or, if from the same/analogous species, one or bom are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. Alternatively, the regulatory regions and/or a recombinant polynucleotide provided herein may be entirely synthetic. The termination region may be native with the transcriptional initiation region, may be native with the operably linked recombinant polynucleotide, may be native with the host cell, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the recombinant polynucleotide, the host cell, or any combination thereof. In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved. A number of promoters can be used in the expression cassettes provided herein. The promoters can be selected based on the desired outcome. It is recognized that different applications can be enhanced by the use of different promoters in the expression cassettes to modulate the timing, location and/or level of expression of the polynucleotide of interest. Such expression constructs may also contain, if desired, a promoter regulator}' region (e.g., one conferring inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal. The expression cassette containing the polynucleotides provided herein can also comprise a selection marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Where appropriate, the sequences employed in the methods and

compositions (i.e., the polynucleotide of interest, the nuclease agent, etc.) may be optimized for increased expression in the host cell. That is, the genes can be synthesized using codons preferred in a given cell of interest including, for example, mammalian-preferred codons, human-preferred codons, rodent-preferred codons, non-rat-rodent-preferred codons, mouse- preferred codons, rat-preferred codons, hamster-preferred codons, etc. for improved expression. One cassette of this disclosure may include a polynucleotide encoding a Cas9 cassette may be expressed to produce a recombinant fusion protein comprising a Cas9 nuclease fused to at least one destabilization domain. In this fusion protein, the

destabilization domain may be an E. coli dihydrofolate reductase. Thus, this disclosure also provides a fusion protein comprising a Cas9 nuclease fused to at least one destabilization domain, wherein the destabilization domain an E. coli dihydrofolate reductase protein.

[0068] A bacterial artificial chromosome (BAC) clone may be produced to include the recombinant Herpesviridae mutant constructs of this disclosure, or any portions thereof.

[0069] Similarly, this disclosure provides a cell, including a host cell, or a mammalian cell, that has been transfected with a recombinant Herpesviridae mutant construct described above. This may include an in vitro cell culture comprising at least one mammalian cell transfected with a recombinant Herpesviridae mutant construct of this disclosure.

[0070] This disclosure also provides pharmaceutical compositions containing the recombinant Herpesviridae mutants described above, and a pharmaceutically acceptable carrier or excipient. These may include conventional carriers, stabilizers, and/or excipients, useful to stabilize a recombinant virus mutant of this disclosure. These may also include conventional carriers useful in formulations for administering the recombinant viral mutant constructs of this disclosure to a mammal, including parenteral routes, nasal and/or oral routes, and by inhalation. Such excipients may include lipids, such as cationic lipids.

[0071] These pharmaceutical compositions are administered to a subject, for example, to a human subject, in order to effect a targeted genomic modification such as excision of a specific viral gene, within the subject. Cells may be obtained from the subject and contacted with a pharmaceutical composition ex vivo. The cells removed from a subject and infected ex vivo with a recombinant virus mutant of this disclosure may be reintroduced into the subject, optionally after the desired genomic modification has been effected or detected in the cells. Methods of delivering pharmaceutical compositions comprising viral mutants are known, and are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled pharmaceutical scientist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions are contemplated include, but are not limited to, humans and/or other primates; mammals, domesticated animals, pets, and commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.

[0072] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the pharmaceutical arts. In general, such preparatory methods include the step of bringing the virus mutants into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.

[0073] These pharmaceutical formulations may include pharmaceutically acceptable excipients, which includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21.sup.st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated in its entirety herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. See also PCT application PCT/US2010/055131 (Publication number WO2011053982 A8, filed Nov. 2, 2010), incorporated in its entirety herein by reference, for additional suitable methods, reagents, excipients and solvents for producing pharmaceutical compositions comprising a viral mutant. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure. These pharmaceutical compositions may optionally include one or more additional therapeutically active substances, including, for example, trimethoprim. [0074] This disclosure also provides methods of treating an infection by a T-cell tropic DNA virus by administering a recombinant Herpesviridae mutant of this disclosure to a patient in need of such treatment. As described above, administration of an agent that stabilizes the destabilization domain, such as trimethoprim, to the patient treated (i.e., infected) with these recombinant Herpesviridae mutant of mis disclosure will stabilize the Cas9 nuclease expressed by the recombinant virus. The stabilized Cas9 nuclease will act in concert with the encoded and expressed gRNA to cut DNA at the specific target site defined and directed by the gRNA sequence. As described above, the targeted site may include a component of a T-cell tropic DNA virus. Thus, the infection treated by these therapeutic methods may include a herpes virus type 1 infection, a herpes virus type 2 infection, a varicella virus infection (chicken pox or shingles), a human or simian immunodeficiency virus (HIV or SIV) infection, and a latent HIV or varicella zoster virus (VZV) infection.

[0075] In these therapeutic methods, the infection may be a latent VZV infection and the recombinant Herpesviridae mutant includes at least one polynucleotide encoding a gRNA targeted to a VZV viral protein.

[0076] In these therapeutic methods, the infection may be a latent HIV infection and the recombinant Herpesviridae mutant includes at least one polynucleotide encoding a gRNA targeted to an HTV viral protein.

[0077] In these therapeutic methods, the infection may be a herpes virus type 1 infection and the recombinant Herpesviridae mutant includes at least one polynucleotide encoding a gRNA targeted to a herpes virus type 1 viral protein.

[0078] In these therapeutic methods, the infection may be a herpes virus type 2 infection and the recombinant Herpesviridae mutant includes at least one polynucleotide encoding a gRNA targeted to a herpes virus type 2 viral protein.

[0079] In these therapeutic methods, trimethoprim may be administered to the patient for a period between 1 and 20 days, and may be administered to the patient for a period of about 10 days. The trimethoprim may be administered to the patient at a dose between about S mg/kg/day and about 40 mg/kg/day, and may be administered to the patient at a dose between about 10 mg/kg/day and about 20 mg/kg/day. The trimethoprim may be administered to the patient in combination with a sulfur drug, such as sulfamethoxazole (e.g. administration within the combination antibiotic drug product BACTRIM™). This antibiotic may be administered shortly before the administration of a recombinant administered concurrently or shortly after (within several hours or a day) after the recombinant Herpesviridae mutant is administered.

[0080] Hie recombinant Herpesviridae mutant of this disclosure and this antibiotic may be in the same or separate pharmaceutical compositions. They may be separate as the mode of administration of these moieties may be different, e.g., the antibiotic may be administered orally and the virus by injection, including intravenous, intramuscular, subcutaneous, or topically, or intranasally.

[0081] In these methods, the patient may be an immunocompromised patient. The patient may also be 60 years old, or older, and/or may be immune deficient secondary to an infection or cancer, or as a result of disease or therapeutic regimen such as drug therapy, chemo or radiotherapy or drugs used during organ or tissue transplantation.

[0082] The recombinant Herpesviridae mutant of this disclosure may be used in infants, children, teens and adolescents, or in adults of all ages to prevent

activation/reactivation of latent zoster or shingles or HIV. Thus, these recombinant mutant virus constructs and antibiotic may be administered to adults prone to developing zoster or shingles.

[0083] Additionally, this disclosure provides methods of using trimethoprim to function as a trigger to stabilize the Cas9 nuclease, thereby allowing the precise excision or cutting of the target DNA, many days, weeks or even years after the initial administration of the recombinant viral mutant construct to the patient.

[0084] Another embodiment of the disclosure relates to the use of any of the recombinant Herpesviridae mutants or pharmaceutical compositions described herein in the preparation of a medicament for the treatment of an infection with a T-cell tropic DNA virus, including a herpes virus type 1 infection, a herpes virus type 2 infection, a varicella virus infection (chicken pox or shingles), a human or simian immunodeficiency virus (HIV or SIV) infection, and a latent HTV or varicella zoster virus (VZV) infection.

[0085] Each publication or patent cited herein is incorporated herein by reference in its entirety.

[0086] The constructs and methods of this disclosure having been generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present disclosure. The examples are not intended to limit the disclosure, as one of skill techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed disclosure.

EXAMPLES

Example 1 : Preparation of a CRISPR-Cas 9 expression mutant in which the sequences encoding Cas9 protein are fused to destabilization domains

[0087] Figure 1 shows the production scheme for the preparation of a VZV genome containing gRNAs and a Cas9-coding sequence. A portion of the CRISPR cas9 mutant (available commercially from Santa Cruz Biotechnology: 'Control CRISPR/Cas9 Plasmid' catalog: sc-418922) comprising the Nuclear localization signal - SpCas9 nuclease - Nuclear localization signal sequences (boxed portion of Figure 1, upper left comer) is modified to contain degradation domains on either the amino terminus (DDN) or carboxy terminus (DDC) of the protein (Figure 1, upper right comer). The destabilization domains will be the dihydrofolate reductase gene/protein:

DHFR nucleotide sequence:

[0088] The inventors' well-established red recombination technology (Brazeau, E, et al., Simian varicella virus open reading frame 63/70 expression is required for efficient Varicella Viruses Inhibit Interferon-Stimulated JAKSTAT Signaling through Multiple Mechanisms. 2015 PLoS. Pathog. Il:el004901) is used to confirm mat the expression of cas9 protein is completely dependent on low levels of trimethoprim (TMP). These cassettes are linked to gRNA guide sequences. Transcription of gRNA and Cas9 will be driven by human U6 promoter, and chicken β-actin (CBh) promoter, respectively.

[0089] These destabilization domain-cas9 cassettes (NLS-Cas9-NLS-DD_frgRNA-

U6 and DD_c-NLS-Cas9-NLS-gRNA-U6) are individually inserted into the VZV genome (Figure 1, middle portion) using red recombination in E. coli. As shown in Figure 1 (middle portion), the VZV genome contains unique long (UL) and unique short (US) segments flanked by inverted repeat (TRL, IRL, IRS and TRS) sequences. These cassettes containing the modified Cas9 and gRNA insertion sites are inserted within the VZV genome (in the commercially available recombinant bacterial artificial chromosome (BAC) clone) (Figure 1, bottom portion) in a region of the VZV genome that is not essential for virus replication (Yoshii, H., et al., Cloning of full length genome of varicella-zoster virus vaccine strain into a bacterial artificial chromosome and reconslitution of infectious virus. 2007 Vaccine 25:5006-5012) using red recombination in K coli. A VZV IE63 promoter is inserted upstream of Cas9 (replacing the CBh promoter) because the IE63 promoter is active during productive VZV replication in culture as well as during latency in human ganglia

(Mahalingam et al., Expression of protein encoded by varicella-zoster virus open reading frame 63 in latently infected human ganglionic neurons. 1996 Proc. Natl. Acad. Sci. U.S.A. 93:2122-2124).

Example 2: The VZV IE63 protein is required for virus replication

[0090] The inventors fused a degradation domain (DD) to the C-terminus of the

VZV open reading frame 63 (ORF63) in the BAC and generated a mutant VZV. This VZV BAC mutant in which the DD was fused to the C-terminus of ORF63/70 was prepared and transfected into Vero cells to generate mutant virus. The Vero cells infected with this mutant VZV were cultured in the presence of varying amounts of trimethoprim (TMP) and infected cell lysates were analyzed by Western blot using antibodies specific for VZV IE63 and glycoprotein E. B-actin was used as a cellular control. Western blot analysis of protein extracts from cells infected in the presence of TMP in a concentration range of 0.0 - 0.1 uM revealed that the VZV IE63 protein is degraded in the absence of TMP (Figure 2) in a dose- confirming that the VZV IE63 protein is required for virus replication/reactivation.

Example 3: Testing the efficiency of editing the VZV genome in human skin cells (MeWo) infected with mutant VZV expressing VZV-specific guide RNA.

[0091] Using the red recombination protocol, several VZV BAC mutants are prepared by inserting different gRN A sequences (in the form of cDN A) that are specific for the VZV IE63 gene. The target VZV IE63 gene sequences are identified using Benchling bioinformatics software to minimize off-target effects. Multiple gRNAs specific for VZV 1E63 are selected since the inventors have recently shown that IE63 is required for virus replication (see Example 2). Mutant VZV BACs containing VZV virus-specific gRNAs and regulatable Cas9 expression are transfected into MeWo human skin cells, in the absence of trimethoprim, to generate infectious mutant viruses. The efficiency of cas9 inactivation of VZV replication by the gRN A guide sequences specific for VZV 1E63 is tested using 00.1- 0.001 μΜ TMP, and VZV genome editing is confirmed by PCR followed by sequence analysis.

[0092] The foregoing examples of the present disclosure have been presented for purposes of illustration and description. Furthermore, these examples are not intended to limit the disclosure to the form disclosed herein. Consequently, variations and modifications commensurate with the teachings of the description of the disclosure, and the skill or knowledge of the relevant art, are within the scope of the present disclosure. The specific embodiments described in the examples provided herein are intended to further explain the best mode known for practicing the disclosure and to enable others skilled in the art to utilize the disclosure in such, or other, embodiments and with various modifications required by the particular applications or uses of the present disclosure. It is intended mat the appended claims be construed to include alternative embodiments to the extent permitted by the prior art.

Claims

1. A recombinant Herpesviridae mutant comprising:

a a Herpesviridae virus genome recombinant^' engineered to include:

i. a polynucleotide sequence encoding a Cas9 nuclease fused to a polynucleotide sequence encoding at least one destabilization domain; and, ii. at least one polynucleotide sequence encoding at least one guide RNA (gRNA).

2. The recombinant Herpesviridae mutant of claim 1 , wherein the Herpesviridae virus genome is a strain selected from Varicella-zoster virus (VZV), Simian varicella virus, HSV- 1, HSV-2, HHV-5 or Cytomegalovirus, HHV5 or Epstein Barr virus, HHV6A and HHV6B or herpes lymphotropic virus, HHV7 or Pityriasis Rosacea, and SHV/HHV8.

3. The recombinant Herpesviridae mutant of claim 1, wherein the polynucleotide sequence encoding a Cas9 nuclease, and the at least one polynucleotide sequence encoding at least one gRNA, are inserted into a region of the viral genome that is not essential for virus replication.

4. The recombinant Herpesviridae mutant of claim 1, wherein at least one of the polynucleotide sequence encoding a Cas9 nuclease, and the at least one polynucleotide sequence encoding at least one gRNA, are inserted into the unique long (UL) region of the VZV viral genome.

5. The recombinant Herpesviridae mutant of claim 1, wherein at least one of the polynucleotide sequence encoding a Cas9 nuclease, and the at least one polynucleotide sequence encoding at least one gRNA, are inserted into the unique short (US) region of the VZV viral genome.

6. The recombinant Herpesviridae mutant of claim 1, wherein the polynucleotide sequence encoding a Cas9 nuclease is fused to at least one polynucleotide sequence encoding a nuclear localization signal protein (NLS). sequence encoding a Cas9 nuclease is fused at each end to at least one polynucleotide sequence encoding a nuclear localization signal protein (NLS).

8. The recombinant Herpesviridae mutant of claim 1, wherein the at least one destabilization domain is the Escherichia dihydrofolate reductase (DHFR) destabilization domain.

9. The recombinant Herpesviridae mutant of claim 1 , wherein the destabilization domain is located upstream of the polynucleotide sequence encoding the Cas9 nuclease, such that expression of the polynucleotides produces a protein destabilization domain fused to the amino-terminus of the Cas9 nuclease.

10. The recombinant Herpesviridae mutant of claim 1 , wherein the destabilization domain is located downstream of the polynucleotide sequence encoding the Cas9 nuclease, such that expression of the polynucleotides produces a protein destabilization domain fused to the carboxy terminus of the Cas9 nuclease.

11. The recombinant Herpesviridae mutant of claim 1 , further comprising an expression control element that regulates expression of the at least one polynucleotide sequence encoding at least one gRNA.

12. The recombinant Herpesviridae mutant of claim 11, wherein the expression control element is a polymerase II promoter or a polymerase III promoter.

13. The recombinant Herpesviridae mutant of claim 11, wherein the promoter is a U6 or an HI RNA polymerase III promoter, a simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), human Ubiquitin C promoter (UBC), human elongation factor la promoter (EF1 A), mouse phosphogly cerate kinase 1 promoter (PGK), and chicken β-Actin promoter coupled with CMV early enhancer (CAGG), or a varicella zoster virus (VZV) IE63 promoter. control element that regulates expression of the polynucleotide sequence encoding a Cas9 nuclease.

15. The recombinant Herpesviridae mutant of claim 14, wherein the expression control element is a U6 or an HI RNA polymerase III promoter, a simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), human Ubiquitin C promoter (UBC), human elongation factor la promoter (EF1 A), mouse phosphoglycerate kinase 1 promoter (PGK), and chicken β-Actin promoter coupled with CMV early enhancer (CAGG), or a varicella zoster virus (VZV) IE63 promoter.

16. The recombinant Herpesviridae mutant of claim 1, further comprising an expression control element that regulates expression of both the at least one polynucleotide sequence encoding at least one gRNA, and the polynucleotide sequence encoding a Cas9 nuclease.

17. The recombinant Herpesviridae mutant of claim 1 , wherein the at least one polynucleotide sequence encodes two distinct gRNA sequences.

18. The recombinant Herpesviridae mutant of claim 1 , wherein the at least one polynucleotide sequence encodes at least one gRNA targeted to a protein of a T-cell tropic DNA virus.

19. The recombinant Herpesviridae mutant of claim 1 , wherein the at least one polynucleotide sequence encodes at least one gRNA targeted to a polynucleotide sequence encoding viral components of a virus selected from a VZV virus, a Herpes simplex virus type 1, a Herpes simplex virus type 2, an HIV virus, and a Simian varicella virus (SW).

20. The recombinant Herpesviridae mutant of claim 1, wherein the at least one polynucleotide sequence encodes at least one gRNA targeted to a varicella zoster virus (VZV) or a simian varicella virus (SW) protein selected from VZV glycoprotein E, VZV viral kinase ORF47, VZV viral kinase ORF66, VZV IE62 protein, VZV IE63 protein, VZV IE70 protein, VZV IE71 protein, VZV DNA polymerase, and a VZV glycoprotein, ORF 63/70, ORF 62/71 , ORF6, ORF28, ORF55, ORF25, ORF26, ORF30, ORF34, ORF 42/45, ORF24, ORF27, ORF29, ORF 31, ORF33, ORF33.5, ORF37, ORF38, ORF39, ORF40, ORF41, ORF44, ORF46, ORF48, ORF50, ORF51, ORF52, ORF53, ORF56, ORF60, ORF61 , ORF62, ORF64, ORF65, ORF66, ORF67, ORF68, and/or ORF69.

21. The recombinant Herpesviridae mutant of claim 1 , wherein the at least one polynucleotide sequence encodes at least one gRNA targeted to a Herpes simplex virus type

1 (HSV-1) protein selected from DNA Polymerase (UL42), DNA Polymerase Catalytic Subunit (UL30), DNA Helicase (UL5), DNA Primase (UL52), 1CP4 (transcriptional regulator), US 1 (host range factor), UL49A (envelope protein), ICPO (transcriptional regulator), UL1, UL8, UL9, UL 14, UL15, UL17, UL18, UL19, UL22, UL25, U126, UL26.5, UL27, UL28, UL29 UL31 , UL34, UL35, UL36, UL37, UL38, UL48, UL49, UL49.5, UL53, UL54, RS I, and/or US6.

22. The recombinant Herpesviridae mutant of claim 1 , wherein the at least one polynucleotide sequence encodes at least one gRNA targeted to a Herpes simplex virus type

2 (HSV-2) protein selected from DNA Polymerase (UL42), DNA Polymerase Catalytic Subunit (UL30), DNA Helicase (UL5), DNA Primase (UL52), ICP4 (transcriptional regulator), US1 (host range factor), UL49A (envelope protein), ICPO (transcriptional regulator), UL 1, UL8, UL9, UL14, UL15, UL17, UL18, UL19, UL22, UL25, U126, UL26.5, UL27, UL28, UL29 UL31 , UL34, UL35, UL36, UL37, UL38, UL48, UL49, UL49.5, UL53, UL54, RS I, and/or US6.

23. The recombinant Herpesviridae mutant of claim 1 , wherein the at least one polynucleotide sequence encodes at least one gRNA targeted to a Human

immunodeficiency virus (HIV) protein selected from Gag, Pol, gp 160, Tat, Rev, Nef, Vpr, Vlf, and Vpu.

24. A bacterial artificial chromosome (BAC) clone comprising the recombinant Herpesviridae mutant of any one of claims 1-23.

25. A mammalian cell that has been infected with a recombinant Herpesviridae mutant of any one of claims 1-23.

26. An in vitro cell culture comprising at least one mammalian cell infected with a recombinant Herpes viridae mutant of any one of claims 1-23.

27. A polynucleotide encoding a Cas9 nuclease fused to a polynucleotide encoding at least one destabilization domain.

28. A recombinant fusion protein comprising a Cas9 nuclease fused to at least one destabilization domain.

29. The polynucleotide of claim 27, or the recombinant fusion protein of claim 28, wherein the destabilization domain is the K coli dihydrofolate reductase.

30. A pharmaceutical composition comprising an effective amount of the recombinant Herpes viridae mutant of any one of claims 1-23, and a pharmaceutically acceptable carrier or excipient.

31. A method of treating an infection by a T-cell tropic DN A virus comprising:

administering a recombinant Herpes viridae mutant of claim 1 to a patient in need of such treatment;

administering an amount of trimethoprim to the patient effective to stabilize the cas9 protein expressed by the recombinant Herpesviridae mutant of claim 1.

32. The method of claim 31 , wherein the infection is selected from the group consisting of

a. a herpes virus type 1 infection

b. a herpes virus type 2 infection

c. a varicella virus infection

d. a human or simian immunodeficiency virus (HIV or SIV) infection, and e. a latent HIV or varicella zoster virus (VZV) infection.

33. The method of claim 31 , wherein the infection is a varicella virus infection causing chicken pox.

34. The method of claim 31 , wherein the infection is a latent varicella virus infection causing varicella zoster (shingles).

35. The method of claim 31 , wherein the infection is a latent VZV infection and the recombinant Herpesviridae mutant of claim 1 comprises a polynucleotide encoding a gRNA targeted to a VZV viral protein.

36. The method of claim 31 , wherein the infection is a latent HIV infection and the recombinant Herpesviridae mutant of claim 1 comprises a polynucleotide encoding a gRNA targeted to an HIV viral protein.

37. The method of claim 31, wherein the infection is a herpes simplex virus type 1 infection and the recombinant Herpesviridae mutant of claim 1 comprises a polynucleotide encoding a gRNA targeted to a herpes simplex virus type 1 viral protein.

38. The method of claim 31 , wherein the infection is a herpes simplex virus type 2 infection and the recombinant Herpesviridae mutant of claim 1 comprises a polynucleotide encoding a gRNA targeted to a herpes simplex virus type 2 viral protein.

39. The method of any one of claims 31-38, wherein the trimethoprim is administered to the patient for a period between 1 and 20 days.

40. The method of any one of claims 31-38, wherein the trimethoprim is administered to the patient for a period of about 10 days.

41. The method of any one of claims 31-38, wherein the trimethoprim is administered to the patient at a dose between about 5 mg/kg/day and about 40 mg/kg/day.

42. The method of any one of claims 31-38, wherein the trimethoprim is administered to the patient at a dose between about 10 mg/kg/day and about 20 mg/kg/day. the patient in combination with sulfamethoxazole.

44. The method of any one of claims 31-43 wherein the patient is immunocompromised.

45. The method of any one of claims 31-43 wherein the patient is 60 year-old or older, and/or is immune deficient secondary to an infection or cancer.