JP2018516983A - Compositions and methods for treating viral infections - Google Patents

Abstract

The present invention provides methods and compositions for treating viral infections. Nucleases are used to cleave viral nucleic acids in infected cells. Nucleases cleave viral nucleic acids in a sequence-specific manner and therefore do not cleave infected host genes or other important genomic features derived from the infected host genome. In a preferred embodiment, the nuclease is a CRISPR-related protein such as Cas9 and is delivered to infected cells as a ribonucleoprotein comprising Cas9 and a guide RNA designed to target HSV or CMV nucleic acids. In addition or alternatively, the nuclease may be delivered encoded in a plasmid or as mRNA expressed in the target cell. The methods and compositions may be used to treat a patient or may be used to treat a tissue, cell, or organ ex vivo.

Description

(Cross-reference to related applications)
This application includes both US Provisional Application No. 62 / 168,259, filed May 29, 2015, and US Provisional Application No. 62 / 168,262, filed May 29, 2015. All of which are incorporated by reference.

(Technical field)
The present invention relates generally to the treatment of viral infections and the cleavage of foreign nucleic acids in cells.

(background)
Viral infection is a serious medical problem. For example, herpes simplex virus (HSV) may cause oral herpes that show symptoms of blisters around the face or mouth, or genital herpes that show symptoms of blisters that can rupture, resulting in small ulcers. Stinging or electric shock may occur before blisters appear. In addition, HSV has the ability to remain indefinitely in cells in a latently infected state and not to be completely eradicated after treatment. Latent infection can bypass immune surveillance and can re-activate the lysis cycle at any time, thus sustaining the incidence of infected individuals and the associated pain and distress.

  Worldwide, about 90% of the population is infected with one or both of HSV-1 and HSV-2, and HSV-1 infection is far more common than HSV-2 infection. About 65% of the US population has antibodies to HSV-1, and about 16% of Americans aged 14 to 49 are infected with HSV-2.

  Another annoying virus example is cytomegalovirus (CMV). Patients may be infected when undergoing an organ transplant. CMV infection can spread to the lungs, liver, pancreas, kidneys, stomach, intestines, brain, and parathyroid glands, causing organ failure and even death. When spread to the eye, the virus can cause retinal detachment and blindness. In addition, the fetus can be infected with cytomegalovirus (CMV) in the uterus, ultimately causing hearing loss, visual impairment, mental retardation, coordination problems, and death. Infants infected with CMV should be monitored for signs of hearing loss or vision loss or mental deficits over the years of growth, even if they are born healthy.

(Summary)
The present invention provides methods and compositions for treating viral infections. Nucleases are used to cleave viral nucleic acids in infected cells. Nucleases cleave viral nucleic acids in a sequence-specific manner and therefore do not cleave infected host genes or other important genomic features derived from the infected host genome. In a preferred embodiment, the nuclease is a CRISPR-related protein such as Cas9 and is delivered to infected cells as a ribonucleoprotein comprising Cas9 and a guide RNA designed to target HSV or CMV nucleic acids. . In addition or alternatively, the nuclease may be delivered encoded in a plasmid or as mRNA expressed in the target cell. The methods and compositions may be used to treat a patient or may be used to treat a tissue, cell, or organ ex vivo.

  Embodiments of the invention relate to HSV.

  The present invention provides compositions and methods for selectively treating HSV infection using a guided nuclease system that targets specific regions of the HSV genome, such as oriS, UL9, RL2, or LAT. To do. Using the methods of the present invention, even latent HSV genetic material can be removed from the host organism without disturbing the integrity of the host genetic material. It is possible to target nucleases to HSV nucleic acids to destroy the nucleic acids, thereby preventing viral replication or transcription, or even excise viral genetic material from the host genome. By using a sequence-specific targeting moiety directed to HSV, the nuclease can act on the host material regardless of whether the viral nucleic acid is present as a particle in the cell or integrated into the host genome. Can be specifically directed to the target so that only HSV nucleic acids can be removed. The sequence specific portion may include a guide RNA that targets the viral genomic material to be destroyed by the nuclease but does not target the host cell genome. In some embodiments, CRISPR / Cas9 nuclease and guide RNA (gRNA) are used that both target viral genomic material and selectively edit or destroy it. CRISPR (short palindromic repeats that form clusters and are regularly spaced) is a naturally occurring element of the bacterial immune system that protects bacteria from phage infection. The guide RNA localizes the CRISPR / Cas9 complex to HSV target sequences such as oriS, UL9, RL2, or LAT. By binding the complex, the Cas9 endonuclease is localized to the HSV genomic target sequence, causing cleavage of the viral genome.

  The guided nuclease system can be introduced transdermally into an infected host, for example, by applying a topical solution. The topical solution may be applied directly to the infected tissue.

  The sequence specific portion is used, for example, to degrade or interfere with HSV nucleic acids without interfering with the normal functioning of zinc finger nuclease, transcription activator-like effector nuclease (TALEN), meganuclease, or host genetic material. Other nuclease systems, including other systems that can be capable of targeting HSV nucleic acids.

  Aspects of the invention include compositions for treating herpes simplex virus (HSV) infections. The composition includes a vector encoding a nuclease and a sequence-specific targeting moiety that is complementary to the HSV nucleic acid and capable of directing the nuclease to the HSV nucleic acid.

  In certain embodiments, the HSV nucleic acid may comprise one or more of the following regions: origin of replication S (oriS), long unique region 9 (UL9, long unique region 9), or long repeat region 2 ( RL2, long repeat region 2). The composition may be configured to be administered transdermally, for example, with a topical solution. The nuclease may be a zinc finger nuclease, a transcriptional activator-like effector nuclease, and a meganuclease.

  In various embodiments, the nuclease may be a Cas9 endonuclease and the sequence specific binding module may include a guide RNA that specifically targets a portion of the viral genome. The composition of the present invention may be packaged for delivery to a human patient.

  In certain embodiments, the targeting sequence can be a guide RNA that does not have more than 60% identity in the human genome.

  Vectors include retrovirus, lentivirus, adenovirus, herpes virus, poxvirus, alphavirus, vaccinia virus, adeno-associated virus, plasmid, nanoparticle, cationic lipid, cationic polymer, metal nanoparticle, nanorod, liposome, micro Bubbles, cell penetrating peptides, or lipospheres may be included.

  In certain aspects, the present invention provides a method for treating HSV infection, wherein a vector encoding a nuclease and a sequence-specific targeting moiety complementary to an HSV nucleic acid is introduced into a host cell. Including a method comprising steps. Further steps include allowing the nuclease to target the HSV nucleic acid in order for the sequence specific targeting moiety to cleave the HSV nucleic acid.

  The HSV nucleic acid may comprise part or all of one or more genomic regions, including the origin of replication S (oriS), long unique region 9 (UL9), or long repeat region 2 (RL2). The methods of the invention may include transdermally administering the vector to a host, and transdermal administration may include applying a topical solution containing the vector.

  The nuclease may be a zinc finger nuclease, a transcriptional activator-like effector nuclease, or a meganuclease. In certain embodiments, the nuclease may comprise a Cas9 endonuclease and the sequence specific binding module may comprise a guide RNA that specifically targets a portion of the viral genome.

  In various methods of the invention, the host may be a living human subject and the steps may be performed in vivo. The targeting sequence may be a guide RNA and does not have more than 60% identity in the human genome.

  In certain methods, the vector is a retrovirus, lentivirus, adenovirus, herpes virus, poxvirus, alphavirus, vaccinia virus, adeno-associated virus, plasmid, nanoparticle, cationic lipid, cationic polymer, metal nanoparticle , Nanorods, liposomes, microbubbles, cell penetrating peptides, or lipospheres.

  The method allows HSV genome editing or disruption, thereby resulting in incompetence of HSV virus growth without adversely affecting the host's uninfected cells. Thus, the compositions and methods of the present invention target 1 to target sites for endonuclease action in the HSV genome, such as target disruption of HSV genome function or oriS, UL9, RL2, or LAT. It can be used to treat HSV infection by causing one or more cleavages and digesting the viral nucleic acid.

  In a preferred embodiment, the present invention provides a composition for treating herpes simplex virus (HSV) infection. The composition comprises a ribonucleoprotein comprising a nuclease and a sequence-specific targeting moiety that is complementary to the HSV nucleic acid and capable of directing the nuclease to the HSV nucleic acid. The HSV nucleic acid may include a replication origin S (oriS) region, a long unique region 9 (UL9), a long repeat region 2 (RL2), or a combination thereof. Preferably, the nuclease comprises a Cas9 endonuclease and the sequence specific binding module comprises a guide RNA that specifically targets a portion of the viral genome. Targeting sequences are guide RNAs and do not have more than 60% identity in the human genome.

  The composition may be configured for transdermal administration. In some embodiments, the composition comprises a pharmaceutically acceptable carrier for topical application to the infected tissue.

  Embodiments of the invention relate to CMV.

  The present invention treats tissue cytomegalovirus (CMV) infection such that the infected individual is not affected by CMV infection such as hearing loss / vision loss, mental deficits, multiple organ failure, and death. Provide a way. CMV infection is treated by using nucleases that target CMV nucleic acids. Nucleases cleave CMV nucleic acids and thus interfere with the function of CMV nucleic acids, thereby preventing infection of CMV in tissues or patients. Nucleases may be introduced into patients who are infected with CMV or may be introduced into organs prior to transplantation. For example, the heart required for transplantation to save lives may be exposed to a CMV targeted nuclease to neutralize the CMV genome prior to transplantation. Thus, the heart transplant recipient need not choose to accept an infected heart or to wait and wait in the hope that the next heart will be CMV free.

  In certain aspects, the present invention provides a composition for treating cytomegalovirus (CMV) infection. The composition includes a nuclease and a sequence-specific targeting moiety that is complementary to the CMV nucleic acid and capable of directing the nuclease to the CMV nucleic acid.

  In certain aspects, the invention provides a method for treating cytomegalovirus (CMV) infection of an organ. The method includes introducing a nuclease that cleaves a CMV nucleic acid into an organ and then causing the nuclease to target and cleave the CMV nucleic acid. The organ may be derived from a transplant donor or may be grown or generated from cells. In some aspects, the organ is the heart, liver, kidney, eye, lung, pancreas, intestine, thymus, or any other biological tissue that is transplanted into the transplant recipient. It should be understood that preparation of a delivery nuclease may include associating the nuclease with a viral or non-viral vector. For example, the nuclease may be prepared for delivery into an organ by encapsulating or binding the nuclease within a liposome.

  In some embodiments of the invention, the tissue (or organ, which are used interchangeably herein) can be the tissue used for transplantation. For example, the tissue may be an organ provided by a donor for transplantation into a recipient. In some embodiments, organ tissue is treated with nucleases to cleave and neutralize CMV nucleic acids.

  In some embodiments, the transplant donor is treated with or exposed to a nuclease. According to the method of the present invention, the donor receives nuclease as a therapeutic agent and removes CMV from tissue within the patient. It should be understood that any means for delivery to a patient can be used. For example, the nuclease may be delivered by oral, parenteral, inhalation, topical, rectal, or vaginal means.

  In some embodiments, the transplant recipient is treated with or exposed to a nuclease after transplantation. According to the method of the present invention, transplant recipients are treated with nucleases to prevent CMV from spreading within and across patients. It should be understood that the nuclease can be delivered by any known means in the art.

  In some embodiments, the tissue is located within the patient. According to the method of the present invention, a patient may be treated with a nuclease that targets CMV to prevent infection. For example, the patient may be a pregnant woman. Nucleases target the CMV genome and disrupt or destroy viral function. Thus, pregnant women do not transfer the virus to the fetus in the womb.

  In some aspects, the present invention provides a method for treating fetal cytomegalovirus (CMV) infection. The method includes providing a nuclease that cleaves a CMV nucleic acid and preparing the nuclease for delivery into a pregnant woman. It should be understood that the nuclease may be delivered into the pregnant woman, into the pregnant woman's uterus, or into the fetus of the uterus by techniques known in the art.

  It should also be understood that any type of nuclease can be used to cleave the CMV nucleic acid. The nuclease may be a zinc finger nuclease, a transcriptional activator-like effector nuclease, or a meganuclease. The nuclease may be a structure specific nuclease or a sequence specific nuclease. In some embodiments, the nuclease is a Cas9 endonuclease. The method of the invention may further comprise cleaving the CMV nucleic acid using a nuclease.

  In some embodiments, the method comprises delivering a guide RNA to a cell or tissue that directs the nuclease to a portion of the CMV nucleic acid. In some embodiments, for example, the guide RNA directs the Cas9 endonuclease to a portion of the CMV nucleic acid. In certain embodiments, the guide RNA is designed not to have a perfect match with the human genome. The guide RNA may direct nucleases to the regulatory elements of the CMV genome.

  Nucleases may be delivered to tissues by viral vectors. The viral vector may be a retrovirus, lentivirus, adenovirus, herpes virus, poxvirus, alphavirus, vaccinia virus, or adeno-associated virus. In some embodiments, nucleases may be delivered by plasmids, nanoparticles, cationic lipids, cationic polymers, metal nanoparticles, nanorods, liposomes, cell permeable peptides, lipospheres, and polyethylene glycol (PEG). .

  In some embodiments, the nuclease is a CRISPR / Cas9 endonuclease. Guide RNAs that specifically target one or more portions of the CMV genome within the cells or tissues of the graft may be used. The CRISPR / Cas9 complex binds to and alters the CMV genome. In other embodiments, the present invention can use CRISPR / Cas9 nuclease and guide RNA (gRNA) that both target CMV genomic material and selectively edit or destroy CMV genomic material. CRISPR (short palindromic repeats that form clusters and are regularly spaced) is an element of the bacterial immune system that protects bacteria from phage infection. Guide RNA localizes the CRISPR / Cas9 complex to the CMV target sequence. By binding the complex, the Cas9 endonuclease is localized to the CMV genome target sequence, causing CMV genome breakage. In a preferred embodiment, the guide RNA is designed to target multiple sites in the CMV genome in order to destroy the CMV nucleic acid and reduce the opportunity for it to functionally recombine.

  The method allows for CMV genome disruption, thereby leading to incompetence of virus growth without observing cytotoxicity to the cells. Aspects of the invention design a CRISPR / gRNA / Cas9 complex to selectively target CMV genomic material (DNA or RNA), and convert the CRISPR / gRNA / Cas9 complex into a cell or tissue containing the CMV genome. Cleaving the CMV genome to neutralize the virus. The method allows for targeted disruption of CMV genome function or, in a preferred embodiment, CMV by multiple cleavages caused by targeting multiple sites for nuclease action in the CMV genome. Makes it possible to digest nucleic acids. Embodiments of the present invention completely inhibit CMV proliferation by providing for transfection of a CRISPR / gRNA / Cas9 complex cocktail. Further aspects and advantages of the present invention will become apparent in view of the following detailed description thereof.

FIG. 1 schematically illustrates a method for targeting HSV infection. FIG. 2 is an HSV genome map. FIG. 3 shows amplification products (amplicons) of the HSV genomic region before and after digestion with cas9. FIG. 3 is a lane gel showing genomic DNA size bands of cells treated with RL2, LATi, LATp, UL9, OriS, and US12 guide sequences with or without Cas9. FIG. 4 shows the results of a quantitative PCR assay showing various levels of HSV DNA reduction in CRISPR treated samples. FIG. 5 shows that an EGFP marker enabling the selection of Cas9 positive cells is fused after the Cas9 protein. FIG. 6 shows that inclusion of ori-P promoted active plasmid replication inside the cell and increased transfection efficiency by more than 60%. FIG. 7 is a schematic diagram of the EBV genome showing candidate structure-related targets, transformation-related targets, and latency-related targets. FIG. 8 shows the genomic organization around the guide RNA sgEBV2 and PCR primer positions. FIG. 9 shows a large deletion induced by sgEBV2 (lanes 1-3 are before, 5 days, and 7 days after sgEBV2 treatment, respectively). FIG. 10 shows the genomic organization around the guide RNA sgEBV3 / 4/5 and PCR primer positions. FIG. 11 shows the large deletions induced by sgEBV3 / 5 and sgEBV4 / 5. Lanes 1 and 2 are 3F / 5R PCR amplification products before and 8 days after sgEBV3 / 5 treatment. Lanes 3 and 4 are 4F / 5R PCR amplification products before and 8 days after sgEBV4 / 5 treatment. FIG. 12 shows that genomic cleavage and repair ligation were confirmed 8 days after sgEBV3 / 5 by Sanger sequencing. FIG. 13 shows that the Sanger sequencing method confirmed genomic cleavage and repair ligation 8 days after sgEBV4 / 5. FIG. 14 shows relative cell growth after targeting with various combinations of guide RNAs in the EBV genomic region. FIG. 15 shows the flow cytometric scatter signal before treatment with sgEBV1-7. FIG. 16 shows the flow cytometric scatter signal after 5 days of treatment with sgEBV1-7. FIG. 17 shows the flow cytometric scatter signal after 8 days of treatment with sgEBV1-7. FIG. 18 shows annexin V Alexa647 and DAPI staining results before treatment with sgEBV1-7. FIG. 19 shows annexin V Alexa647 and DAPI staining results after 5 days of treatment with sgEBV1-7. FIG. 20 shows annexin V Alexa647 and DAPI staining results after 8 days of treatment with sgEBV1-7. Figures 21 and 22 show that microscopic observation revealed apoptotic cell morphology after treatment with sgEBV1-7. Figures 21 and 22 show that microscopic observation revealed apoptotic cell morphology after treatment with sgEBV1-7. FIG. 23 shows the nuclear morphology prior to treatment with sgEBV1-7. FIGS. 24-26 show nuclear morphology after treatment with sgEBV1-7. FIGS. 24-26 show nuclear morphology after treatment with sgEBV1-7. FIGS. 24-26 show nuclear morphology after treatment with sgEBV1-7. FIG. 27 shows EBV loading after various CRISPR treatments by digital PCR. Cas9 and Cas9-oriP had 2 replicates and sgEBV1-7 had 5 replicates. FIG. 28 shows a single Raji cell trapped in a microfluidic chip. FIG. 29 shows a single sgEBV1-7 treated cell trapped on the chip. FIG. 30 is a histogram of single cell EBV quantitative PCR Ct values prior to treatment. FIG. 31 is a histogram of EBV quantitative PCR Ct values of single viable cells after 7 days of treatment with sgEBV1-7. FIG. 32 shows the SURVEYOR assay for EBV CRISPR (lanes are numbered from left to right: lane 1: NEB 100 bp ladder; lane 2: sgEBV1 control; lane 3: sgEBV1; lane 4: sgEBV5 control; lane 5: sgEBV5; lane 6: sgEBV7 control; lane 7: sgEBV7; lane 8: sgEBV4). FIG. 33 shows that CRISPR treatment was not cytotoxic to the EBV negative Burkitt lymphoma cell line DG-75. FIG. 34 shows that CRISPR treatment was not cytotoxic to primary human lung fibroblast IMR90. FIG. 35 shows ZFNs that have been used to cleave viral nucleic acids. FIG. 36 shows a composition for treating viral infection. FIG. 37 shows a flowchart of an embodiment of the present invention for targeting CMV. FIG. 38 is a map of the CMV genome. FIG. 39 shows the results from the in vitro CRISPR endonuclease assay, showing PCR amplification products from the CMV genome and corresponding products from in vitro CRISPR digestion (shown in pairs). FIG. 40 shows the results from the in vitro CRISPR endonuclease assay, showing PCR amplification products from the CMV genome and corresponding products from in vitro CRISPR digestion (shown in pairs). FIG. 41 shows the results from the in vitro CRISPR endonuclease assay, showing PCR amplification products from the CMV genome and corresponding products from in vitro CRISPR digestion (shown in pairs). FIG. 42 shows a method 3201 for treating cells 3237 to remove foreign nucleic acids.

(Detailed explanation)
Embodiments of the present invention use guided nuclease systems that target specific regions of the HSV genome, such as RL2, LAT, UL9, or OriS, to detect HSV infections, including HSV-1 and HSV-2 infections. It relates to compositions and methods for treatment. The composition of the present invention destroys the HSV viral nucleic acid of the host cell by nuclease activity without affecting the host genome. The methods and compositions of the present invention can eradicate even latent HSV infection by disrupting the HSV genome of the infected host cell.

  In addition or alternatively, embodiments of the invention relate to methods for treating cytomegalovirus (CMV). The method of the present invention systematically causes large or repetitive insertions or deletions in the CMV genome, thereby neutralizing or destroying CMV in cells, tissues, or patients, and restructuring the complete genome. Used to reduce the likelihood of being done. Insertion or deletion into the genome neutralizes or destroys the virus, thus treating CMV. In some embodiments, the nuclease may be a CRISPR / Cas9 complex. In some embodiments, the nuclease is induced by a sequence such as a guide RNA. In some embodiments, tissues such as organs are treated with nucleases prior to transplantation. In some embodiments, the transplant donor or transplant recipient is treated before and after transplant surgery. In some embodiments, a patient infected with CMV is treated with a nuclease.

  FIG. 1 illustrates a method for targeting HSV infection. This method involves obtaining a nuclease (eg, as a nuclease protein or gene) that can be targeted. Any suitable nuclease can be used, such as ZFN, TALEN, or meganuclease. In a preferred embodiment, the nuclease is Cas9. Sequences are provided that direct the nuclease to a specific target of the HSV genome, such as oriS, UL9, RL2, or LAT. The nuclease gene and the encoded gRNA may be provided in a DNA vector, such as a plasmid or adenovirus-based vector, and the vector may optionally further comprise a promoter. The composition is then introduced into HSV infected cells. Any suitable transfection or delivery method can be used. When introduced into a cell, the gene is expressed and the Cas9 enzyme uses the gRNA to target the HSV genome and cleave it. Since gRNA is specific for the HSV genome and does not match the human genome, the methods and criteria described herein keep the host genome intact and do not interfere with normal human gene function. .

  A discussion of HSV and targeted regions can be found in the following literature: Summers et al., 2002, Herpes Simplex Virus Type 1 Origins of DNA Replication Play No Role in the Regulation of Flanking Promoters, J Virol., 76 Eom et al., 2003, Replication-initiator protein (UL9) of the herpes simplex virus 1 binds NFB42 and is degraded via the ubiquitin-proteasome pathway, Proc Natl Acad Sci USA, 100 (17): 9803 ˜9807; McGeoch et al., 1991, Comparative sequence analysis of the long repeat regions and adjoining parts of the long unique regions in the genomes of herpes simplex viruses types 1 and 2, J Gen Virol., 72 (Pt12): 3057 ~ 75 pages. The contents of each of these documents are incorporated by reference in their entirety for all purposes.

  FIG. 2 shows the HSV genome and HSV oriS palindromic dimer (oriS), RL2-ICP0 (RL2), LAT intron (LATi), LAT promoter (LATp), UL9, which can be targeted by CRISPR guide RNA. , And the US12 gene is shown.

  FIG. 3 shows amplification products of the HSV genomic region before and after digestion with cas9 and the corresponding guide RNA. T7 in vitro transcription was used to generate a complete guide RNA with scaffolds for RL2, LATi, LATp, UL9, oriS, and US12. The flanking region of the genomic target was amplified by PCR from HSV2 G strain genomic DNA (obtained from ATCC). Cas9 protein (obtained from PNA Bio), guide RNA, and target DNA were mixed and incubated for in vitro endonuclease assay. RL2, LATi, LATP, UL9, oriS, and US12 PCR amplification products before and after digestion with cas9 and guide RNA targeting each individual genomic region are shown in FIG. Thus, DNA gel electrophoresis of the digested DNA revealed high endonuclease activity.

  FIG. 37 shows a flowchart of an embodiment of the present invention for targeting CMV. In general, the method 100 includes introducing a nuclease 105 that targets the CMV genome. Nucleases can be introduced into organs or pregnant women. The organ or fetus can be suspected of having CMV infection. The method further includes 110 causing the nuclease to target and cleave the CMV nucleic acid. Nucleases can bind to and alter the CMV genome.

(I. Treatment of infected cells)
The cells may be treated (eg, delivered as mRNA or plasmid) with a nuclease encoded in the nucleic acid, or delivered in an active form. If the nuclease is a CRISPR-related protein such as Cas9, active ribonucleoprotein (RNP) may be delivered to the cell.

  FIG. 42 shows a method 3201 for treating cells 3237 to remove foreign nucleic acids such as viral nucleic acid 3251 derived from herpes simplex virus. Method 3201 may be used for use to treat an infection, or method 3201 may be used in vitro for research and development to remove foreign nucleic acids from a subject cell, such as a cell derived from a human. May be.

  Method 3201 forms ribonucleoprotein (RNP) 3231 comprising nuclease 3205 and RNA 3213; obtaining cell 3237 from a donor; delivering RNP 3231 to cell 3237 (preferably in vitro) step 3245; and Cleaving viral nucleic acid 3251 in cell 3237 with RNP3231. Method 3201 may include providing cells 3237 for implantation in a patient.

  Delivery step 3245 may include electroporation, or the RNP may be packaged in liposomes for delivery step 3245. In some embodiments, viral nucleic acid 3251 may exist as an episomal virus genome, ie, as a viral episome or episomal vector. RNA 3213 has a portion that is substantially complementary to a target in viral nucleic acid 3251 and preferably not substantially complementary to any position in the human genome. In a preferred embodiment, the virus is a herpes family virus, such as those selected from the group consisting of HSV-1, HSV-2, varicella-zoster virus, Epstein-Barr virus, and cytomegalovirus. The virus may be in the latent stage in the cell.

  In a preferred embodiment, nuclease 3205 is preferably a crisper-related protein such as Cas9. RNA 3213 may be a single guide RNA (sgRNA) (providing the functionality of crRNA and tracrRNA). In a preferred embodiment, nuclease 3205 and RNA 3213 are delivered to the cell as RNP3231.

  In a preferred embodiment, cell 3237 has viral nucleic acid 3251 therein, and the method further comprises cleaving the viral nucleic acid using a nuclease.

  In some embodiments, RNP may prove to be preferred (eg, compared to plasmid DNA) for clinical applications, particularly for parenteral delivery. RNP is an active preformed drug that provides advantages over DNA (AAV) or mRNA. The drug component need not be transcribed, translated, or assembled within the cell. Delivery of RNP3231 can result in improved drug properties, such as earlier initiating activity and clearance (toxicity) control.

  FIG. 36 illustrates a composition for treating a viral infection according to certain embodiments. The composition preferably targets a nuclease and a targeting moiety (eg gRNA) that targets the nuclease to HSV and may be complementary to a genomic region of HSV such as RL2, LAT, UL9, or OriS And a vector (which may be a plasmid, linear DNA, or viral vector). The composition may optionally include one or more of a promoter, origin of replication, other elements, or combinations thereof, as further described herein.

(Ii. Nuclease)
The method of the present invention involves specifically destroying a viral nucleic acid using a nuclease that is programmable or can be directed to a target. For example, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), short palindromic repeat (CRISPR) nuclease, meganuclease, other endonuclease or exonuclease with clustering and regular spacing, or Any suitable targeting nuclease can be used, including combinations of: See Schiffer, 2012, Targeted DNA mutagenesis for the cure of chronic viral infections, J Virol, 88 (17): 8920-8936. This document is incorporated by reference.

  In the CRISPR methodology, a CRISPR-related nuclease that forms a complex with a small RNA (gRNA) as a guide to cleave DNA upstream of a protospacer adjacent motif (PAM) at any genomic location in a sequence-specific manner ( Cas9) is used. CRISPR can use separate guide RNAs known as crRNA and tracrRNA. Combining these two separate RNAs into a single RNA allows site-specific mammalian genome cleavage by short guide RNA design. Cas9 and guide RNA (gRNA) can be synthesized by known methods. Cas9 / guide RNA (gRNA) uses the non-specific DNA cleavage protein Cas9 and RNA oligos that hybridize to target and mobilize the Cas9 / gRNA complex. Chang et al., 2013, Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos, Cell Res, 23: 465-472; Hwang et al., 2013, Efficient genome editing in zebrafish using a CRISPR-Cas system, Nat. Biotechnol 31: 227-229; see Xiao et al., 2013, Chromosomal deletions and inversions mediated by TALENS and CRISPR / Cas in zebrafish, Nucl Acids Res, 1-11.

  CRISPR (short palindromic repeats that form clusters and regularly spaced) are found in bacteria and are thought to protect bacteria from phage infection. CRISPR has recently been used as a means to alter gene expression of eukaryotic DNA, but has not been proposed as an antiviral therapy or more widely as a method of destroying genomic material. Rather, CRISPR has been used to introduce insertions or deletions as a means to increase or decrease transcription of DNA in a targeted cell or cell population. For example, Horvath et al., Science (2010) 327: 167-170; Terns et al., Current Opinion in Microbiology (2011) 14: 321-327; Bhaya et al., Annu Rev Genet (2011) 45: 273-297; Wiedenheft et al., Nature (2012) 482: 331-338; Jinek M et al., Science (2012) 337: 816-821; Cong L et al., Science (2013) 339: 819-823; Jinek M et al. (2013), eLife 2: e00471; Mali P et al. (2013) Science 339: 823-826; Qi LS et al. (2013) Cell 152: Gilbert LA et al. (2013) Cell, 154: 442-451; Yang H et al. (2013) Cell, 154: 1370-1379; and Wang H et al. (2013). Ce ll, 153: 910-918). The contents of each of these documents are incorporated by reference in their entirety for all purposes.

  In aspects of the invention, the Cas9 endonuclease causes a double-strand break at at least two positions in the genome. These two double strand breaks cause deletion of the genomic fragment. Even if the two ends are annealed by the viral repair pathway, the genome may still remain deleted. One or more deletions using this mechanism will neutralize the viral genome. As a result, virus infection is eliminated from the host cell.

  In embodiments of the invention, the nuclease cleaves the genome of the target virus. A nuclease is an enzyme capable of cleaving a phosphodiester bond between nucleotide subunits of a nucleic acid. An endonuclease is an enzyme that cleaves a phosphodiester bond in a polynucleotide chain. Some, such as deoxyribonuclease I, cleave DNA relatively non-specifically (regardless of sequence), but many are typically referred to as restriction endonucleases or restriction enzymes, Cleaves only very specifically. In a preferred embodiment of the present invention, Cas9 nuclease is incorporated into the compositions and methods of the present invention, but it should be understood that any nuclease can be used.

  In a preferred embodiment of the invention, Cas9 nuclease is used to cleave the genome. Cas9 nuclease can generate double-strand breaks in the genome. Cas9 nuclease has two functional domains, RuvC and HNH, each cleaving a different strand. If both of these domains are active, Cas9 causes a double-strand break in the genome.

  In some embodiments of the invention, insertions into the genome can be designed to cause neutralization or alter genome expression. In addition, insertions / deletions terminate prematurely by either generating a premature stop codon for double-strand breaks or by shifting the reading frame to generate a premature stop codon downstream of the double-strand break. Also used to introduce cordons. Any of these results of the NHEJ repair pathway can be exploited to destroy the target gene. Changes introduced through the use of the CRISPR / gRNA / Cas9 system persist permanently in the genome.

  In some embodiments of the invention, at least one insertion is caused by a CRISPR / gRNA / Cas9 complex. In a preferred embodiment, the virus is neutralized by causing multiple insertions in the genome. In aspects of the invention, the number of insertions reduces the probability that the genome can be repaired.

  In some embodiments of the invention, at least one deletion is caused by the CRISPR / gRNA / Cas9 complex. In a preferred embodiment, the virus is neutralized by causing multiple deletions in the genome. In aspects of the invention, the number of deletions reduces the probability that the genome can be repaired. In a highly preferred embodiment, the CRISPR / Cas9 / gRNA system of the present invention causes significant genomic damage while keeping the host genome intact, resulting in effective destruction of the viral genome.

  TALEN uses a non-specific DNA-cleaving nuclease fused to a DNA binding domain that can target essentially any sequence. In the case of TALEN technology, a target site is identified and an expression vector is produced. A linearized expression vector (eg, with Not1) can be used as a template for mRNA synthesis. Commercially available kits such as the mMESSAGE mMACHINE SP6 transcription kit from Life Technologies (Carlsbad, CA) may be used. See Joung and Sander, 2013, TALENs: a widely applicable technology for targeted genome editing, Nat Rev Mol Cell Bio, 14: 49-55.

  The TALEN and CRISPR methods provide a one-to-one relationship with the target site. That is, one unit of tandem repeat of the TALE domain recognizes one nucleotide of the target site, and the CRISPR / Cas system crRNA, gRNA, or sgRNA hybridizes to the complementary sequence of the DNA target. These methods can include using a Cas9 protein with a pair of TALENs or one gRNA to generate a double stranded break in the target. The cleavage site is then repaired by non-homologous end joining or homologous recombination (HR).

  FIG. 35 shows the ZFN being used to cleave the viral nucleic acid. Briefly, the ZFN method involves introducing into an infected host cell at least one vector (eg, an RNA molecule) encoding a targeted ZFN 305 and optionally at least one accessory polynucleotide. See, for example, U.S. Patent Application Publication No. 2011/0023144 to Weinstein. This document is incorporated by reference. The cell contains the target sequence 311. Cells are incubated to express ZFN305 and double-strand breaks 317 are introduced into the targeted chromosomal sequence 311 by ZFN305. In some embodiments, a donor polynucleotide or exchange polynucleotide 321 is introduced. By exchanging parts of the viral nucleic acid with unrelated sequences, transcription or replication of the viral nucleic acid can be completely prevented. The target DNA 311 can be repaired with the exchange polynucleotide 321 by an error-prone non-homologous end-joining DNA repair process or a DNA repair process guided by homology.

  Typically, ZFNs include a DNA binding domain (ie, zinc finger) and a cleavage domain (ie, nuclease), and this gene is mRNA (eg, 5 ′ capped, polyadenylated, or Both) can be introduced. The zinc finger binding domain can be engineered to recognize and bind to any selected nucleic acid sequence. See, for example, Qu et al., 2013, Zinc-finger-nucleases mediate specific and efficient excision of HIV-1 proviral DAN from infected and latently infected human T cells, Nucl Ac Res, 41 (16): 7771-7782. I want to be. This document is incorporated by reference. Engineered zinc finger binding domains may have novel binding specificities as compared to naturally occurring zinc finger proteins. The operating methods include, but are not limited to, rational design and various types of selection. A zinc finger binding domain can be designed to recognize a target DNA sequence via a zinc finger recognition region (ie, a zinc finger). See, for example, US Pat. Nos. 6,607,882; 6,534,261; and 6,453,242. These documents are incorporated by reference. Exemplary methods for selecting a zinc finger recognition region can include phage display and two-hybrid systems, US Pat. No. 5,789,538; US Pat. No. 5,925,523; US Pat. US Pat. No. 6,007,988; US Pat. No. 6,013,453; US Pat. No. 6,410,248; US Pat. No. 6,140,466; US Pat. No. 6,200,759; This is disclosed in Japanese Patent No. 6,242,568. Each of these documents is incorporated by reference.

  ZFN also contains a cleavage domain. The cleavage domain portion of ZFN can be obtained from any suitable endonuclease or exonuclease such as restriction endonucleases and homing endonucleases. See, for example, Belfort and Roberts, 1997, Homing endonucleases: keeping the house in order, Nucleic Acids Res, 25 (17): 3379-3388. The cleavage domain may be derived from an enzyme that requires dimerization for cleavage activity. If each nuclease contains an active enzyme dimer monomer, two ZFNs may be required for cleavage. Alternatively, a single ZFN may contain both monomers for generating active enzyme dimers. An existing restriction endonuclease may be capable of sequence-specific binding and DNA cleavage at or near the binding site. Certain restriction enzymes (eg, type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the type IIS enzyme FokI is active as a dimer and is 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. Catalyze cleavage. The FokI enzyme used for ZFN can be regarded as a cleavage monomer. Thus, in the case of targeted double-strand breaks using the FokI cleavage domain, two ZFNs each containing a FokI cleavage monomer can be used to reconstitute the active enzyme dimer. Wah et al., 1998, Structure of FokI has implications for DNA cleavage, PNAS, 95: 10564-10569; US Pat. No. 5,356,802; US Pat. No. 5,436,150; US Pat. No. 487,994; U.S. Patent Application Publication No. 2005/0064474; U.S. Patent Application Publication No. 2006/0188987; U.S. Patent Application Publication No. 2008/0131962. Each of these documents is incorporated by reference.

  Viral targeting using ZFN may involve introducing at least one donor polynucleotide comprising a sequence into the cell. The donor polynucleotide preferably comprises upstream and downstream sequences that share sequence similarity with either side of the chromosomal integration site and sequences to be introduced. The upstream and downstream sequences of the donor polynucleotide are selected to facilitate recombination of the chromosomal sequence of interest with the donor polynucleotide. Typically, the donor polynucleotide can be DNA. The donor polynucleotide may be a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, or a naked nucleic acid, using a delivery vehicle such as a liposome. May be. The sequence of the donor polynucleotide may include exons, introns, regulatory sequences, or combinations thereof. Double-strand breaks are repaired by homologous recombination with the donor polynucleotide, and the desired sequence is integrated into the chromosome. In the case of a ZFN-mediated process, double-strand breaks introduced into the target sequence by ZFN can be repaired by homologous recombination with the exchange polynucleotide, exchanging the sequence of the exchange polynucleotide with a portion of the target sequence. . The presence of double-strand breaks promotes homologous recombination and repair of the breaks. The exchange polynucleotide may be physically incorporated, or the exchange polynucleotide sequence information is exchanged with the sequence information of that portion of the target sequence, using the exchange polynucleotide as a template to repair the cleavage. Result. Thus, a portion of the viral nucleic acid can be converted to the sequence of the exchange polynucleotide. The ZFN method can include using a vector to deliver a nucleic acid molecule encoding ZFN and optionally at least one exchange polynucleotide or at least one donor polynucleotide to an infected cell.

  A meganuclease is an endodeoxyribonuclease that is characterized by a large recognition site (a 12-40 base pair double stranded DNA sequence), so that there is generally only one such site in any given genome. . For example, an 18 base pair sequence recognized by an I-SceI meganuclease will require an average 20 times the size of the genome of the human genome, even if one is found by chance (sequences with a single mismatch are , Which is found approximately three times per human-sized genome). Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes. Meganucleases can be classified into five families based on sequence and structural motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD- (D / E) XK. The most well-studied family is the family of LAGLIDADG proteins found all over the life world, generally encoded within introns or inteins, but there are also independent members. The sequence motif LAGLIDADG is an essential element of enzyme activity. Some proteins contain only one such motif, while other proteins contain two such motifs. In any case, this motif was followed by about 75-200 amino acid residues with little or no sequence similarity to other family members. The crystal structure shows the following sequence of LAGLIDADG family sequence specificity and cleavage mechanism: (i) Specific contacts occur when the extended β chain is buried in the major groove of DNA, and the DNA binding saddle With a pitch and contour that mimics the helix twist of (ii) the complete hydrogen bonding ability between protein and DNA is never fully realized; (iii) the characteristic 4-nt Cleavage to produce a 3′-OH overhang occurs across the minor groove, and due to DNA distortion in the central “4-base” region, the phosphate bond undergoing cleavage is closer to the protein catalytic core; (iv) a unique “metal” The proposed bimetallic mechanism, which may involve a “shared” paradigm, results in cleavage; and (v) Finally, “adaptation” of the outer region of the core α / β folding From Sera were (Adapted) "scaffold, further affinity and / or specific contact may occur. See Silva et al., 2011, Meganucleases and other tools for targeted genome engineering, Curr Gene Ther, 11 (1): 11-27. This document is incorporated by reference.

  In some embodiments of the invention, the template sequence is inserted into the genome. In order to introduce nucleotide modifications into genomic DNA, there must be a DNA repair template containing the desired sequence during homology-guided repair (HDR). The DNA template is usually transfected into cells with gRNA / Cas9. The length and binding position of each homology arm depends on the size of the change introduced. In the presence of a suitable template, HDR can introduce significant changes in Cas9-induced double strand breaks.

  In some embodiments of the invention, modified nucleases may be used. A modified Cas9 enzyme containing a single inactive catalytic domain of either RuvC- or HNH- is called "nickase". With only one active nuclease domain, Cas9 nickase cleaves only one strand of the target DNA, producing a single strand break or “nick”. Like inactive dCas9 (RuvC- and HNH-), Cas9 nickase can still bind to DNA based on gRNA specificity, but nickase will only cleave one of the DNA strands. The majority of CRISPR plasmids are From Pyogenes, the RuvC domain can be inactivated by the D10A mutation and the HNH domain can be inactivated by the H840A mutation.

  Single-strand breaks or nicks are usually quickly repaired by the HDR pathway using an intact complementary DNA strand as a template. However, the nicks of two adjacent opposite strands introduced by Cas9 nickase are treated as double-strand breaks and are often referred to as “double nick” or “double nickase” CRISPR systems. Double nick-induced double strand breaks can be repaired by either NHEJ or HDR, depending on the desired effect on the gene target. Such double strand breaks cause insertions and deletions by the CRISPR / Cas9 complex. In aspects of the invention, the deletion is caused by placing two double-strand breaks in close proximity to each other, thereby causing a deletion of a genomic fragment.

(Iii. Target-directed part)
Nucleases can use the target specificity of guide RNA (gRNA). As discussed below, guide RNAs or single guide RNAs are specifically designed to target the viral genome.

  The CRISPR / Cas9 gene editing complex of the present invention works optimally with guide RNA targeting the viral genome. Guide RNA (gRNA) (including single guide RNA (sgRNA), crisprRNA (crRNA), trans-activated RNA (tracrRNA), any other targeting oligo, or any combination thereof) In order to cause destruction, the CRISPR / Cas9 complex is led into the viral genome. In aspects of the invention, the CRISPR / Cas9 / gRNA complex is designed to target a specific virus in the cell. It should be understood that the compositions of the present invention can be used to target any virus. Identifying specific regions of the viral genome aids the development and design of CRISPR / Cas9 / gRNA complexes.

  In aspects of the invention, the CRISPR / Cas9 / gRNA complex is designed to target intracellular latent viruses. When transfected intracellularly, the CRISPR / Cas9 / gRNA complex causes neutralization of the genome by repeated insertions or deletions, or significantly increases the potential for repair due to the number of insertions or deletions. Reduce.

  As an example, Epstein-Barr virus (EBV), also called human herpesvirus 4 (HHV-4), is inactivated in cells by the CRISPR / Cas9 / gRNA complex of the present invention. EBV is a herpes family of viruses and is one of the most common viruses in humans. Viruses are approximately 122 nm to 180 nm in diameter, and are composed of double helix DNA wrapped in a protein capsid. In this example, it is the Raji cell line that serves as a suitable in vitro model. The Raji cell line is the first serially passaged human cell line from hematopoietic origin, which produces an unusual strain of Epstein-Barr virus, but is one of the most widely studied EBV models. is there. To target the EBV genome in Raji cells, a CRISPR / Cas9 complex with specificity for EBV is required. The design of the CRISPR / Cas9 plasmid targeting EBV is described in Addgene, Inc. From a U6 promoter-driven chimeric guide RNA (sgRNA) and a ubiquitous promoter-driven Cas9. In the present invention, commercially available guide RNA and Cas9 nuclease can be used. The EGFP marker fused after the Cas9 protein allowed the selection of Cas9 positive cells.

  In aspects of the invention, the guide RNA is designed to target a specific viral genome, whether or not purchased commercially. Viral genomes are identified and guide RNAs targeting selected portions of the viral genome are developed and incorporated into the compositions of the present invention. In aspects of the invention, a reference genome of a particular virus strain is selected for guide RNA design.

  For example, guide RNA targeting the EBV genome is a component of the system in this example. For EBV, for example, a reference genome derived from B95-8 strain was used as a design guide. A selected region or gene within the genome of interest, such as EBV, is targeted. For example, six regions can be targeted with seven guide RNA designs with different genomic editing objectives.

  FIG. 7 is a schematic diagram of the EBV genome showing candidate structure-related targets, transformation-related targets, and latency-related targets. Further, FIG. 7 shows the locations where sgEBV1, sgEBV2, sgEBV3, sgEBV4 / 5, sgEBV6, and sgEBV7 target the EBV genome.

  For EBV, the only nuclear Epstein-Barr virus (EBV) protein that is expressed in both latent and lytic modes of infection is EBNA1. Although EBNA1 is known to play several important roles in latent infection, EBNA1 is important 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 to excise this entire region of the genome. These “structural” targets allow the EBV genome to be digested systematically into smaller pieces. EBNA3C and LMP1 are essential for host cell transformation, and guide RNAs sgEBV3 and sgEBV7 were designed to target the 5 'exons of these two proteins, respectively.

(Iv. Introduction into cells)
The methods of the invention involve introducing nucleases and sequence-specific targeting moieties into cells. Nucleases target viral nucleic acids with sequence-specific targeting moieties and then cleave the viral nucleic acids without interfering with the host genome. Any suitable method can be used to deliver the nuclease to infected cells or tissues. For example, a nuclease or gene encoding a nuclease can be delivered by injection, orally, or by hydrodynamic delivery. The nuclease or gene encoding the nuclease may be delivered to the systemic circulation, or may be delivered to a particular tissue type, or may be otherwise localized. Modify or program a nuclease or nuclease-encoding gene to use a tissue-specific promoter so that the encoded nuclease is preferentially transcribed in certain tissue types or only in certain tissue types It may be activated only under certain conditions, such as by chance.

  In some embodiments, a specific CRISPR / Cas9 / gRNA complex is introduced into the cell. Guide RNAs are designed to target at least one category of sequence in the viral genome. In addition to latent infection, the present invention can also be used to actively control viral replication by targeting the viral genome before packaging or after release.

  In some embodiments, a cocktail of guide RNAs may be introduced into the cell. Guide RNAs are designed to target multiple categories of sequences in the viral genome. By targeting several regions along the genome, double-strand breaks at multiple positions will fragment the genome and reduce the likelihood of repair. Even if the repair mechanism works, such a large deletion neutralizes the virus.

  In some embodiments, several guide RNAs are added to generate a cocktail that targets different categories of sequences. For example, 2, 5, 7, or 11 guide RNAs may be present in a CRISPR cocktail that targets three different categories of sequences. However, any number of gRNAs can be introduced into the cocktail to target the category of sequence. In preferred embodiments, the sequence categories are important for genomic structure, host cell transformation, and latent infectivity, respectively.

  In some aspects of the invention, it is possible to determine the most essential target within the viral genome by in vitro experiments. For example, to understand the most essential target for effectively neutralizing the genome, a subset of guide RNAs are transfected into model cells. The assay can determine which guide RNA or which cocktail is most effective in targeting the essential categories of sequences.

  For example, when targeting the EBV genome, three different categories of sequences in which the seven guide RNAs in the CRISPR cocktail are each identified as important for EBV genome structure, host cell transformation, and latent infectivity Was targeted. In order to understand the most essential targets for effective EBV treatment, a subset of guide RNA was transfected into Raji cells.

  FIG. 14 shows the relative cell growth after targeting with various combinations of guide RNAs in the region of the EBV genome. sgEBV4 / 5 reduced the EBV genome by 85% but failed to suppress cell proliferation as effectively as the full cocktail (FIG. 14). Guide RNA targeting the structural sequence (sgEBV1 / 2/6) did not completely eliminate EBV loading (26% reduction) but was able to completely stop cell growth. In view of the high efficiency of genome editing and the cessation of growth, the residual EBV genomic signature of sgEBV1 / 2/6 is not attributed to the intact genome, but free floating DNA digested from the EBV genome, That is, it was suspected to be a false positive.

  Once the CRISPR / Cas9 / gRNA complex is constructed, the complex is introduced into the cell. It should be understood that the complex can be introduced into cells of an in vitro model or an in vivo model. In aspects of the invention, the CRISPR / Cas9 / gRNA complex is designed to leave no intact genome of the virus after transfection, and the complex is designed for efficient transfection.

  In aspects of the invention, CRISPR / Cas9 / gRNA can be transfected into cells by a variety of methods including viral and non-viral vectors. Viral vectors can include retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. It should be understood that any viral vector can be incorporated into the present invention to achieve delivery of the CRISPR / Cas9 / gRNA complex into the cell. Depending on the CRISPR / Cas9 / gRNA complex designed for digestion or neutralization, some viral vectors may be more effective than others. In aspects of the invention, the vector includes essential components such as an origin of replication necessary to replicate and maintain the vector in a host cell.

  In aspects of the invention, the viral vector is used as a delivery vector to deliver the complex into the cell. The use of viral vectors as delivery vectors is known in the art. See, for example, US Patent Application Publication No. 2009/0017543 to Wilkes et al. The contents of this document are incorporated by reference.

  Retroviruses are single-stranded RNA viruses that store their nucleic acids in the form of mRNA genomes (including 5 'caps and 3' PolyA tails) and target host cells as obligate parasites. In some methods in the art, retroviruses have been used to introduce nucleic acids into cells. Once inside the cytoplasm of the host cell, the virus uses its own reverse transcriptase to produce DNA from its RNA genome. This is the reverse of the normal pattern and is therefore retro (reverse). This new DNA is then integrated into the host cell genome by the integrase enzyme, at which point the retroviral DNA is called a provirus. For example, recombinant retroviruses such as Moloney murine leukemia virus have the ability to integrate into the host genome in a stable manner. They contain reverse transcriptases that allow integration into the host genome. Retroviral vectors can be either replicable or replication defective. In some embodiments of the invention, retroviruses are incorporated to achieve transfection into cells, but the CRISPR / Cas9 / gRNA complex is designed to target the viral genome.

  In some embodiments of the invention, lentivirus, a subclass of retrovirus, is used as a viral vector. Lentiviruses can be adapted as delivery vehicles (vectors) given their ability to integrate into the genome of non-dividing cells. This is an inherent feature of lentiviruses because other retroviruses can only infect dividing cells. The viral genome in the form of RNA is reverse transcribed as the virus enters the cell and produces DNA, which is then inserted into the genome at random locations by the viral integrase enzyme. At this point, a vector called a provirus remains in the genome and is inherited by the cell's progeny when the cell divides.

  In contrast to lentiviruses, adenoviral DNA is not integrated into the genome and is not replicated during cell division. Adenovirus and related AAV are promising delivery vectors because they do not integrate into the host genome. In some aspects of the invention, only the targeted viral genome is affected by the CRISPR / Cas9 / gRNA complex and the host cells are not affected. Adeno-associated virus (AAV) is a small virus that infects humans and several other primate species. AAV can infect both dividing and non-dividing cells and can integrate its genome into the genome of the host cell. For example, researchers have generated modified AAVs called self-complementing adeno-associated viruses (scAAV) because they are promising for use as gene therapy vectors. AAV packages a single strand of DNA and requires a second strand synthesis process, whereas scAAV packages both strands and they both anneal to yield double stranded DNA. Form. By omitting the synthesis of the second strand, scAAV can be rapidly expressed in cells. Otherwise, scAAV has many features of its corresponding AAV. The methods of the invention can incorporate herpes virus, pox virus, alpha virus, or vaccinia virus as a delivery vector means.

  In certain embodiments of the invention, non-viral vectors can be used to achieve transfection. Non-viral delivery methods for nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations, or lipid: nucleic acid conjugates, naked DNA, artificial virions, and enhanced agent of DNA Sex uptake may be mentioned. Lipofection is described, for example, in US Pat. Nos. 5,049,386, 4,946,787, and 4,897,355 (the contents of each of these references are all in their entirety) Lipofection reagents are commercially available (eg, Transfectam and Lipofectin). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides are described in Jessee US Pat. No. 7,166,298 or Jesse US Pat. No. 6,890,554. And the contents of each of these documents are incorporated by reference. Delivery may be to cells (eg, in vitro or ex vivo administration), or to target tissue (eg, in vivo administration).

  Synthetic vectors are typically based on cationic lipids or polymers that can complex with negatively charged nucleic acids to form particles having a diameter on the order of 100 nm. The complex protects the nucleic acid from degradation by nucleases. Furthermore, cellular and local delivery strategies must address the need for internalization, release, and distribution in the appropriate intracellular compartment. Systemic delivery strategies encounter additional hurdles such as strong interactions between cationic delivery vehicles and blood components, uptake by the reticuloendothelial system, renal filtration, carrier toxicity to the cells of interest and targeting ability. By modifying the surface of the cationic non-viral to minimize their interaction with blood components, reduce reticuloendothelial system uptake, reduce their toxicity, and their binding affinity to target cells Sex can be increased. Binding to plasma proteins (also called opsonization) is the primary mechanism by which RES recognizes circulating nanoparticles. For example, macrophages such as liver Kupffer cells recognize nanoparticles that are opsonized via scavenger receptors.

  In some embodiments of the invention, the non-viral vector is modified to achieve targeted delivery and transfection. PEGylation (ie, modifying the surface with polyethylene glycol) reduces opsonization and aggregation of non-viral vectors, minimizes reticuloendothelial clearance, and is administered intravenously (iv). It is the primary method used to provide later circulation life extension. Thus, PEGylated nanoparticles are often referred to as “stealth” nanoparticles. Nanoparticles that are not cleared rapidly from the circulation will have the opportunity to encounter infected cells.

  However, surface PEG may reduce uptake by target cells and reduce biological activity. Therefore, it is necessary to add a targeting ligand to the distal end of the pegylation component and project the ligand beyond the PEG “shield” to allow binding to receptors on the target cell surface Let When cationic liposomes are used as gene carriers, neutral helper lipids are useful for nucleic acid release, in addition to promoting hexagonal phase formation and enabling endosome escape. In some embodiments of the invention, neutral or anionic liposomes are developed for systemic delivery of nucleic acids and for obtaining therapeutic effects in experimental animal models. The design and synthesis of novel cationic lipids and polymers, and genes, targeting ligands, polymers, or environmentally sensitive moieties that bind covalently or non-covalently to peptides also solve the problems encountered by non-viral vectors. Has been noticed for. Prepare inorganic vectors (eg, metal nanoparticles, iron oxide, calcium phosphate, magnesium phosphate, manganese phosphate, double hydroxide, carbon nanotubes, and quantum dots) on delivery vectors in many different ways Can be surface functionalized.

  The guided nuclease system of the present invention may be administered alone or in combination with pharmaceutically acceptable carriers, diluents, adjuvants, and vehicles as active ingredients. The vector may be incorporated into a topical or intravenous formulation that may contain liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, from one organ or body part to another. Or it may be involved in transporting or transporting the agent of interest to a body part. Each carrier should be compatible with the other ingredients of the formulation.

  In certain embodiments, the compositions of the present invention may be encapsulated in a hydrogel. In another embodiment, the compositions disclosed herein may comprise a lipid based formulation. Any of the known lipid-based drug delivery systems can be used in the practice of the present invention. For example, multivesicular liposomes, multilamellar liposomes, and unilamellar liposomes can all be used as long as it is possible to establish a sustained release rate of the encapsulated active compound. Such formulations can be used to alter the release profile of the guided nuclease composition. Methods for making controlled release multivesicular liposomal drug delivery systems are described in PCT application publication numbers: WO9703652, WO9513796, and WO9423697. The contents of these documents are incorporated herein by reference.

  Synthetic membrane vesicles may contain a combination of phospholipids usually combined with steroids, especially cholesterol. Other phospholipids or other lipids can also be used. Examples of lipids useful for the production of synthetic membrane vesicles include phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides, and in preferred embodiments egg phosphatidylcholine, dipalmitoylphosphatidylcholine, Distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol.

  When preparing lipid-based vesicles containing the composition of the present invention, compound encapsulation efficiency, compound labiality, homogeneity and size of the resulting vesicle population, active compound to lipid ratio, permeation Variables such as sex, instability of the preparation, and pharmacological tolerance of the formulation should be considered.

  In another embodiment, the guided nuclease system of the present invention can be delivered in vesicles, particularly liposomes (see Langer (1990) Science, 249: 1527-1533). The contents of this document are incorporated by reference in their entirety for all purposes. In yet another embodiment, the guided nuclease system can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer (1990) supra). In another embodiment, polymeric materials can be used (see Howard et al., (1989), J. Neurosurg., 71: 105, the contents of which are incorporated by reference in their entirety) Incorporated for purposes). In another embodiment, the vectors of the invention are used, for example, by using retroviral vectors (see, eg, US Pat. No. 4,980,286), or by direct injection or using microparticle bombardment. (Eg, gene gun; Biolistic, Dupont) or by coating with a lipid or cell surface receptor or transfection agent or linked to a homeobox-like peptide known to enter the nucleus (See Joliot et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 1864-1868). The contents of these documents are incorporated by reference in their entirety for all purposes. Alternatively, a nucleic acid may be introduced into a cell and expressed by being incorporated into host cell DNA by homologous recombination.

  In some embodiments of the invention, a targeted controlled release system is used that is responsive to the native environment of the tissue and external stimuli. Gold nanorods have a strong absorption band in the near-infrared region, and thus absorbed light energy is converted into heat by the gold nanorods. This is the so-called “photothermal effect”. Since near-infrared light can penetrate to the deep part of the tissue, the surface of the gold nanorod can be modified with a nucleic acid and controlled release. When the modified gold nanorods are irradiated with near infrared light, nucleic acids are released by thermal denaturation induced by the photothermal effect. The amount of nucleic acid released depends on the output of light radiation and the exposure time.

  In some embodiments of the invention, liposomes can be used to achieve transfection into cells or tissues. Most of the pharmacology of nucleic acid liposome formulations is determined by the extent to which the nucleic acid is encapsulated within the liposome bilayer. Encapsulated nucleic acids are protected from nuclease degradation, but those that are simply associated with the liposome surface are not protected. Encapsulated nucleic acids share long circulation life and in vivo dispersion with intact liposomes, but those associated with the surface follow the pharmacology of naked nucleic acids when detached from the liposomes.

  In some embodiments, the complexes of the invention are encapsulated in liposomes. Unlike small molecule drugs, nucleic acids cannot pass through intact lipid bilayers due to their large size and hydrophilic nature. Thus, nucleic acids can be captured in liposomes using conventional passive loading techniques such as the ethanol droplet method (such as SALP), the reverse phase evaporation method, and the ethanol dilution method (such as SNALP).

  In some embodiments, linear polyethyleneimine (L-PEI) is used as a non-viral vector because of its versatility and relatively high transfection efficiency. L-PEI has been used to efficiently deliver genes in vivo into a wide range of organs such as the lung, brain, pancreas, retina, and bladder, as well as into tumors. L-PEI can efficiently concentrate, stabilize and deliver nucleic acids in vitro and in vivo.

  Low intensity ultrasound combined with microbubbles has recently attracted attention as a safe gene delivery method. Ultrasound exhibits a tissue penetration effect. Low intensity ultrasound in combination with microbubbles can be non-invasive, site specific, and can destroy tumor cells after systemic delivery without affecting non-target organs. Ultrasound-mediated microbubble destruction has been proposed as an innovative method for non-invasive delivery of drugs and nucleic acids to various tissues. Using microbubbles to carry drugs or genes until they reach a specific area of interest, and then rupturing the microbubbles using ultrasound results in site-specific delivery of the bioactive agent . Furthermore, the ability of albumin-coated microbubbles to attach to vascular areas with glycocalix damage or endothelial dysfunction is another mechanism that can deliver drugs even in the absence of ultrasound. is there. See Tsutsui et al., 2004, The use of microbubbles to target drug delivery, Cardiovasc Ultrasound, 2:23. The contents of this document are incorporated by reference. For ultrasound-induced drug delivery, tissue penetration effects can be enhanced using ultrasound contrast agents, gas-filled microbubbles. The use of microbubbles to deliver nucleic acids breaks down the DNA-carrying microbubbles by focusing the ultrasound beam while they travel through the target area and through the microvessels. It is based on the hypothesis that local transduction is caused upon shell failure, but not in non-target areas.

  In addition to ultrasound-mediated delivery, magnetic targeting delivery can be used for delivery. Magnetic nanoparticles are usually captured in a gene vector to image nucleic acid delivery. The nucleic acid carrier can respond to both ultrasound and magnetic fields, ie it can be magnetic and acoustically active lipospheres (MAAL). The basic premise is that the therapeutic agent is attached to or encapsulated in the magnetic micro or nanoparticle. Such particles may have a magnetic core with a polymer or metal coating that can be functionalized, or may be composed of a porous polymer that includes magnetic nanoparticles precipitated within the pores. . By functionalizing the polymer or metal coating, it is possible to add, for example, cytotoxic drugs for targeted chemotherapy or therapeutic DNA to correct gene defects. Once bound, the particle / therapeutic agent complex is positioned at the injection site near the target, often using a catheter, and injected into the bloodstream. Generally, a magnetic field from a rare earth magnet with a strong magnetic field and high gradient is concentrated on the target site, and when the particle enters the magnetic field, the particle can be trapped and overflowed by the target. Become.

  Nanoparticles (about 100 nm in diameter) based on synthetic cationic polymers that have enhanced transfection efficiency combined with reduced cytotoxicity compared to conventional liposomes have been developed. Incorporation of a separate layer composed of lipid molecules with various physical and chemical characteristics into the polymer nanoparticle formulation provides better fusion with the cell membrane and entry into the cell, allowing it to enter the cell interior. Increased efficiency results because enhanced molecular release and reduced intracellular degradation of the nanoparticle complex.

  In some embodiments, the conjugates are conjugated to nanosystems for systemic therapy such as liposomes, albumin based particles, PEGylated proteins, biodegradable polymer drug composites, polymer micelles, dendrimers, among others. See Davis et al., 2008, Nanotherapeutic particles: an emerging treatment modality for cancer, Nat Rev Drug Discov., 7 (9): 771-782. This document is incorporated by reference. Long-circulating macromolecular carriers such as liposomes can take advantage of enhanced permeability and retention effects on preferential extravasation from tumor blood vessels. In certain embodiments, the complexes of the invention are conjugated or encapsulated in liposomes or polymersomes for delivery to cells. For example, liposomal anthracycline has achieved very efficient encapsulation, and examples thereof include ultralong-term circulation types such as liposomal daunorubicin and pegylated liposomal doxorubicin. See Krishna et al., Carboxymethylcellulose-sodium based transdermal drug delivery system for propranolol, J Pharm Pharmacol., April 1996; 48 (4): 367-70.

  Liposome delivery systems provide stable formulations, provide improved pharmacokinetics, and some “passive” or “physiological” targeting to tissues. Encapsulation of hydrophilic and hydrophobic substances such as promising chemotherapeutic agents is known. See, for example, the following literature: Schneider US Pat. No. 5,466,468, which discloses parenterally administrable liposomal formulations containing synthetic lipids; Hostetler et al. US Pat. No. 5,580,571. Nucleoside analogs conjugated to phospholipids are disclosed; US Pat. No. 5,626,869 to Nyqvist, wherein a pharmaceutically active compound comprises at least one amphiphilic component and a polar lipid component and at least Disclosed is a pharmaceutical composition that is heparin or a fragment thereof contained in a limited lipid system comprising one non-polar lipid component.

  Liposomes and polymersomes may contain multiple solutions and compounds. In certain embodiments, the complexes of the invention are coupled to or encapsulated in polymersomes. A polymersome as a kind of artificial vesicle is a very small hollow sphere surrounding a solution, and is produced by forming a vesicle membrane using an amphiphilic synthetic block copolymer. Common polymersomes contain aqueous solutions in their cores and are useful for encapsulating and protecting sensitive molecules such as drugs, enzymes, other proteins and peptides, and DNA and RNA fragments. Polymersome membranes provide a physical barrier that sequesters encapsulated material from external materials, such as those found in biological systems. Polymersomes can be produced from double emulsions by known techniques. See Lorenceau et al., 2005, Generation of Polymerosomes from Double-Emulsions, Langmuir, 21 (20): 9183-6. This document is incorporated by reference.

  Some embodiments of the present invention provide a gene gun or biolistic gun particle delivery system. A gene gun is a device for injecting genetic information into cells, and the payload may be heavy metal element particles coated with plasmid DNA. This technique is sometimes referred to as bioballistic or biolistic. Gene guns are also used to deliver DNA vaccines. The gene gun can transfect cells with a wide variety of organic and non-organic species such as DNA plasmids, fluorescent proteins, dyes and the like.

  Aspects of the invention provide a number of uses for delivery vectors. The choice of delivery vector is based on the targeted cell or tissue and the specific composition of the CRISPR / Cas9 / gRNA. For example, in the EBV example discussed above, lymphocytes are known to be resistant to lipofection, so DNA delivery into Raji cells is generated by a device called Nucleofector (Nucleofector). In combination with cell type specific reagents that transfer the substrate directly into the cell nucleus and cytoplasm). The Lonza pmax promoter drives Cas9 expression. This is because this promoter resulted in strong expression in Raji cells. 24 hours after nucleofection, a clear EGFP signal was observed from a small number of cells on a fluorescence microscope.

  FIG. 6 shows that when the plasmid contains ori-P, active plasmid replication inside the cell was promoted and transfection efficiency increased by more than 60%. The left panel shows that untreated cells and cells treated with Cas9 had a similarly low number of cells with fluorescent markers. The right panel shows that oriP promoted higher transfection.

  However, the EGFP positive cell population decreased dramatically and transfection efficiencies of less than 10% were measured 48 hours after nucleofection (FIG. 6). The CRISPR plasmid, which contained the EBV origin of replication sequence oriP, resulted in transfection efficiencies greater than 60% (FIG. 6).

  Embodiments of the invention use CRISPR / Cas9 / gRNA complexes for targeted delivery. Common and known routes include the transdermal route, the transmucal route, the nasal route, the ocular route, and the transpulmonary route. Examples of drug delivery systems include liposomes, proliposomes, microspheres, gels, prodrugs, cyclodextrins and the like. In embodiments of the invention, nanoparticles composed of biodegradable polymers are used that will be transferred to aerosols to target specific regions or cell populations of the lung, and drug release in a predetermined manner. And degradation within an acceptable period of time is provided. Transdermal and mucosal controlled release delivery systems, nasal and buccal aerosol sprays, drug-impregnated lozenges, encapsulated cells, oral soft gels, iontophoretic devices for administering drugs through the skin, and various programmable Targeted controlled delivery is achieved using controlled release technology (CRT), such as an implanted drug delivery device, with the conjugates of the present invention.

  It should be understood that a CMV targeting nuclease can be delivered into a cell, organ, patient, or fetus by the techniques described herein and by techniques known in the art. The CMV targeting nucleases of the invention can be prepared for delivery by associating (binding, encapsulating, etc.) with a vector and / or guide RNA.

  Aspects of the invention provide for delivery of a CMV targeting nuclease across the placenta, ie transplacental. The placenta as a conduit to the fetus is both a drug target and a drug barrier. Alternatively, the nuclease can be introduced into the amniotic sac or into the fetus. For example, the nuclease can be introduced into the amniotic sac or fetus by injection. Alternatively, the fetus can use any technique known in the art for delivering therapeutic agents to infants and treat the CMV infection using the methods of the invention after delivery.

(V. Viral nucleic acid cleavage)
Once inside the cell, the CRISPR / Cas9 / gRNA complex targets the viral genome. In aspects of the invention, the complex is targeted to the viral genome. In addition to latent infection, the present invention can also be used to actively control viral replication by targeting the viral genome before being packaged or released. In some embodiments, the methods and compositions of the invention use nucleases such as Cas9 to target latent viral genomes, thereby reducing the chance of growth. Nucleases can form complexes with gRNA (eg, crRNA + tracrRNA or sgRNA). The complex cleaves the viral nucleic acid in a targeted manner and neutralizes the viral genome. As discussed above, Cas9 endonuclease causes double-strand breaks in the viral genome. By targeting several locations along the viral genome and causing double strand breaks rather than single strand breaks, the genome is effectively cleaved at several locations along the genome. In preferred embodiments, double-strand breaks cause small deletions or small fragments are removed from the genome so that the genome is neutralized even when the natural repair machinery joins the genome together. Designed to be removed.

  After introduction into the cell, the CRISPR / Cas9 / gRNA complex acts on the viral genome, gene, transcript, or other viral nucleic acid. Double-stranded DNA breaks generated by CRISPR are repaired resulting in small deletions. Such a deletion disrupts the protein code and thus exerts a knockout effect.

  Nucleases or genes encoding nucleases can be delivered into infected cells by transfection. For example, infected cells can be transfected (in a single piece or separate pieces) with DNA encoding Cas9 and gRNA. The gRNA is designed to localize the Cas9 endonuclease at one or several positions along the viral genome. Cas9 endonuclease causes double-strand breaks in the genome and deletes small fragments from the viral genome. Deletion neutralizes the viral genome, even if the repair mechanism works.

  Thereafter, nuclease treated cells and tissues according to the methods of the invention are provided for transplantation. In some embodiments, the organ is treated with a nuclease that renders the tissue CMV-free prior to transplantation.

  In some embodiments of the invention, the nuclease is prepared for use in a transplanted organ. Organ transplantation moves an organ from one body to another, or from a donor site to another location on the individual's own body, replacing a damaged or missing organ in the recipient That is. An organ can also be generated or repopulated from its own cells (stem cells or cells extracted from a defective organ) or from cells of another individual. The organ may be from either a biological source or a cadaver source. Organs that can be transplanted are the heart, kidney, liver, lung, pancreas, intestine, and thymus. Tissue includes bone, tendons (both called musculoskeletal grafts), cornea, skin, heart valves, nerves, and veins. Corneal and musculoskeletal grafts are the tissues or organs that are generally transplanted.

(Vi. Host genome)
It will be appreciated that the methods and compositions of the invention can be used to target viral nucleic acids without interfering with host genetic material. The methods and compositions of the invention use a targeting moiety such as a guide RNA having a sequence that hybridizes to a target within the viral sequence. The methods and compositions of the present invention use a gRNA to bind to the viral genome exclusively and to remove a viral sequence from the host by cleaving the duplex, or a nuclease directed to a target such as the cas9 enzyme or the like A vector encoding a specific nuclease may also be used.

  If the targeting moiety comprises a guide RNA, the sequence of the gRNA or guide sequence is examined in the viral sequence and does not appear in the host genome adjacent to the protospacer adjacent motif (PAM) and adjacent to the protospacer motif It can be determined by finding a region of about 20 nucleotides.

  Preferably, the guide sequence is such that the gRNA / cas9 complex produced according to the guide sequence will bind to and digest the specified feature or target of the viral sequence without interfering with the host genome. Satisfy certain similarity criteria (eg, be at least 60% identical to an identity biased towards a region closer to the PAM). Preferably, the guide RNA corresponds to the next nucleotide string of the protospacer flanking motif (PAM) (eg, NGG, where N is any nucleotide) in the viral sequence. Preferably, the host genome (1) matches a string of nucleotides according to a given similarity criterion and (2) also lacks any region adjacent to the PAM. A given similarity criterion may include, for example, the requirement that there are at least 12 matching nucleotides within the 20 nucleotides 5 'to the PAM, and the 10 nucleotides 5' to the PAM. It may include the requirement that there be at least 7 matching nucleotides within. An annotated viral genome (eg, from GenBank) is used to identify the characteristics of viral sequences and select characteristics of viral sequences (eg, viral origin of replication, terminal repeats, replication factor binding site, promoter, coding sequence, Alternatively, the next nucleotide character of the protospacer adjacent motif (PAM) of the viral sequence within the repeat region) may be found. Viral sequences and annotations can be obtained from genomic databases.

  If multiple candidate gRNA targets are found in the viral genome, the selection of the sequence that is the template for the guide RNA may favor the candidate target that is closest or most 5 'to the target feature as the guide sequence. Selection may preferably preferentially select sequences having an intermediate (eg 40% to 60%) GC content. Further background on RNA-directed targeting by endonucleases is discussed in the following literature: US Patent Application Publication No. 2015/0050699; US Patent Application Publication No. 20140356958; US Patent Application Publication No. 2014/0349400; U.S. Patent Application Publication No. 2014/0342457; U.S. Patent Application Publication No. 2014/0295556; and U.S. Patent Application Publication No. 2014/0273037. The contents of each of these documents are incorporated by reference for all purposes. Since there is a human genome background in the infected cells, a set of steps is provided to ensure high efficiency against the viral genome and low off-target effects on the human genome. These steps include (1) selecting a target within the viral genome, (2) circumventing the PAM + target sequence of the host genome, (3) methodologically selecting a viral target that is conserved among strains, (4) select targets with appropriate GC content, (5) control nuclease expression in cells, (6) design vectors, (7) validate with assays, etc. and Various combinations may be included. Target-directing moieties (such as guide RNA) preferably bind to targets within a particular category, such as (i) latency-related targets, (ii) infection and symptom-related targets, and (iii) structure-related targets. .

  The first category of targets of gRNA includes latency related targets. The viral genome requires certain features to maintain latency. Such features include, but are not limited to, master transcriptional regulators, latency specific promoters, signaling proteins that exchange information with host cells, and the like. If the host cell is dividing during incubation, the viral genome requires the replication system to maintain a copy level of the genome. Viral origins of replication, terminal repeats, and replication factors that bind to the origin of replication are good targets. When the function of these features is disturbed, the virus reactivates and it can be treated with conventional antiviral therapy.

  The second category of targets for gRNA includes infection-related and symptom-related targets. Viruses produce a variety of molecules to promote infection. When the virus enters the host cell, it can initiate a lysis cycle, thereby causing cell death and tissue damage (HBV). In certain cases, such as HPV16, cell products (E6 and E7 proteins) can transform host cells and cause cancer. Interfering with the important genomic sequences (promoter, coding sequence, etc.) that produce such molecules can prevent further infection and / or alleviate if the disease symptoms are not cured.

  The third category of targets of gRNA includes structure related targets. The viral genome may contain repetitive regions that support genome integration, replication, or other functions. Targeting the repeat region allows the viral genome to be cut into multiple pieces, which physically destroys the genome.

  When the nuclease is a cas protein, the targeting moiety is a guide RNA. Each cas protein requires a specific PAM (not a guide RNA) next to the targeted sequence. This is the same as in the case of human genome editing. In the current understanding, the guide RNA / nuclease complex first binds to PAM and then searches for homology between the guide RNA and the target genome. Sternberg et al., 2014, DNA interrogation by the CRISPR RNA-guided endonuclease Cas9, Nature, 507 (7490): 62-67. Once recognized, the DNA is digested 3-nt upstream of PAM. These results suggest that for off-target digestion to occur, PAM must be present in the host DNA and the affinity between the guide RNA and the host genome immediately before PAM must be high.

  It may be preferable to use a targeting portion that targets a highly conserved portion of the viral genome. The viral genome is much more mutated than the human genome. In order to target different strains, the guide RNA will preferably target a conserved region. Since PAM is important for initial sequence recognition, it is also essential to have a PAM in the conserved region.

  In preferred embodiments, the methods of the invention are used to deliver nucleic acids to cells. The nucleic acid delivered to the cell may comprise a gRNA having a determined guide sequence, or the nucleic acid comprises a vector such as a plasmid encoding an enzyme that will act on the target genetic material. Also good. Expression of the enzyme allows for degradation or otherwise disruption of the target genetic material. The enzyme may be a nuclease such as Cas9 endonuclease and the nucleic acid may encode one or more gRNAs having a determined guide sequence.

  With gRNA, nucleases target target genetic material. If the target genetic material contains a viral genome, a gRNA complementary to a portion of that genome can induce degredation of the genome by nucleases, thereby preventing any further replication of the intact viral genome. Or completely removed from the cells. By such means, it is possible to eradicate the latent virus infection as a target.

  Host cells may grow at different rates depending on the particular cell type. Rapid cell replication requires high nuclease expression, but lower expression helps to avoid off-target cleavage of uninfected cells. Control of nuclease expression can be achieved in several ways. If the nuclease is expressed from a vector, the vector's copy origin can be dramatically increased only in infected cells by having a viral origin of replication. Each promoter has a different activity in different tissues. Gene transcription can be regulated by selecting different promoters. Transcripts and protein stability can also be adjusted by incorporating stabilizing or destabilizing motifs (such as ubiquitin targeting sequences) into the sequence.

  Specific promoters for gRNA sequences, nucleases (eg, cas9), other elements, or combinations thereof may be used. For example, in some embodiments, the gRNA is driven by a U6 promoter. Vectors containing promoters for protein expression may be designed (eg, using the promoters described in the vector sold under the trade name PMAXCLONING by Lonza Group Ltd (Basel, Switzerland)). The vector may be a plasmid (eg, generated with a synthesizer 255 and a recombinant DNA laboratory). In certain embodiments, the plasmid comprises U6 promoter-driven gRNA or chimeric guide RNA (sgRNA) and ubiquitous promoter-driven cas9. Optionally, the vector may include a marker such as EGFP fused after the cas9 protein to allow subsequent selection of cas9 + cells. cas9 uses gRNA (similar to the original bacterial CRISPR RNA (crRNA)) with a complementary trans-activated crRNA (tracrRNA) to target viral sequences complementary to gRNA It is understood that It has also been shown that cas9 can be programmed with a single RNA molecule, a chimer of gRNA and tracrRNA. A single guide RNA (sgRNA) can be encoded in the plasmid, and transcription of sgRNA can provide cas9 programming and tracrRNA functions. For background, see Jinek, 2012, A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Science, 337: 816-821, and particularly FIG. 5A of this document.

  Using the above principles, the methods and compositions of the present invention can be used to target viral nucleic acids of infected hosts without adversely affecting the host genome.

  Further background is Hsu, 2013, DNA targeting specificity of RNA-guided Cas9 nucleases, Nature Biotechnology, 31 (9): 827-832; and Jinek, 2012, A programmable dual-RNA-guided DNA endonuclease in See adaptive bacterial immunity, Science 337: 816-821. The contents of each of these documents are incorporated by reference. Cleavage these sequences because target locations are selected that are within a certain category, such as (i) latency-related targets, (ii) infection and symptom-related targets, or (iii) structure-related targets. This inactivates the virus and removes the virus from the host. Targeted RNA (gRNA or sgRNA) is designed to meet the similarity criterion that matches the target of the viral gene sequence but does not cause any off-target that matches the host genome, thus The material is removed from the host without interfering with the host genome at all.

(Ii. Apoptotic pathway)
If only a small number of cells are infected and it is sufficient to remove the whole cell (as well as the CMV genome), embodiments of the invention include a promoter that is active in the latent viral state and that drives the cell killing gene. Administration of the vector is contemplated. For this approach, a particularly interesting target is HSV. This is because it is estimated that only thousands to tens of thousands of neurons are latently infected. See Hoshino et al., 2008, The number of herpes simplex virus-infected neurons and the number of viral genome copies per neuron correlate with latent viral load in ganglia, Virology, 372 (1): 56-63. This document is incorporated by reference. Examples of cytocidal genes include (1) targetable nucleases that target the cell genome; and (2) disrupt the integrity of cell or mitochondrial membranes such as apoptotic effectors such as BAX and BAK and alpha hemolysin Both proteins are mentioned (Bayles, “Bacterial programmed cell death: making sense of a paradox”, Nature Reviews Microbiology, 12, 63-69 (2014)). Promoters that are only active in latently infected cells can be used not only in this situation, but can also be used to increase the selectivity of nuclease therapy by making the activity specific to infected cells. An example of such a promoter is latency-related promoter 1 or “LAP1” (Preston and Efstathiou, “Molecular Basis of HSV Latency and Reactivation”, Human Herpesviruses: Biology, Therapy and Immunoprophylaxis, 2007). In some embodiments, the present invention provides methods and therapeutics that can be used to cause the killing of only infected cells that are host cells. For example, treatment may include delivering a gene for a protein that causes cell death, wherein the gene is under the control of a viral regulatory element, such as a promoter derived from the genome of the infecting virus, or the gene Is encoded by a vector containing the viral origin of replication. If the virus is present, the gene is expressed and the gene product will cause the cell to die. The gene can encode a protein important for apoptosis, or the gene can encode a nuclease that digests the host genome.

  Apoptotic embodiments can be used to remove infected cells from within a sample containing a mixture of infected and uninfected cells. A targetable nuclease can be used to provide a composition comprising a virus-driven promoter, a targetable nuclease, and a guide RNA that targets a cellular (eg, human) genome. In the presence of the virus, the nuclease will kill the cell. Only uninfected cells will be left in the sample.

  Apoptotic proteins can be used as therapeutic agents. A therapeutic agent encoded in the vector may be provided, and the vector also encodes a sequence that allows the therapeutic agent to be expressed in cells infected with the virus. The sequence may be regulatory elements (eg, promoter and origin of replication) derived from the viral genome. The therapeutic agent can provide a mechanism that selectively causes the death of virus-infected cells. For example, a protein that restores a defect in the apoptotic pathway of cells may be used. The gene may be, for example, BAX, BAK, BCL-2, or alpha hemolysin. Preferably, the therapeutic agent induces apoptosis in cells infected with the virus and does not induce apoptosis in uninfected cells.

  In some embodiments, the invention provides a composition comprising a viral vector, a plasmid, or other encoding nucleic acid encoding at least one gene that promotes apoptosis, and at least one promoter associated with the viral genome. . Apoptosis regulator Bcl-2 governs mitochondrial outer membrane permeability (MOMP) and is a protein that includes pro-apoptotic proteins such as Bax, BAD, Bak, Bok, Bcl-rambo, Bcl-xs, and BOK / Mtd It is a family.

  Apoptosis regulator BAX is also known as bcl-2-like protein 4, and is a protein encoded by the BAX gene in humans. BAX is a member of the Bcl-2 gene family. This protein forms a heterodimer with BCL2 and functions as an apoptosis activator. This protein has been reported to interact with the mitochondrial membrane voltage-dependent anion channel (VDAC), increasing its opening, leading to loss of membrane potential and release of cytochrome c.

  A Bcl-2 homologous antagonist / killer is a protein encoded by the BAK1 gene on chromosome 6 in humans. This protein is localized to the mitochondria and functions to induce apoptosis. It interacts with the mitochondrial membrane voltage-dependent anion channel, accelerating its opening, leading to loss of membrane potential and the release of cytochrome c.

  Human genes encoding proteins belonging to this family include the following: BAK1, BAX, BCL2, BCL2A1, BCL2L1, BCL2L2, BCL2L10, BCL2L13, BCL2L14, BOK, and MCL1.

(Incorporation by reference)
Throughout this disclosure, other references such as patents, patent applications, patent publications, journals, books, papers, web content, etc. are referenced and cited. All such documents are hereby incorporated by reference in their entirety for all purposes.

(Equivalent)
In addition to what is shown and described herein, various modifications to the present invention and its numerous further embodiments will occur to those skilled in the art to the scientific and patent literature cited herein. It will be clear from the complete contents of this document, including references to The subject matter of this specification includes important information, examples, and guidance that can be applied to implement the invention in various embodiments and equivalents thereof.

Example 1
Target HSV
T7 in vitro transcription produced a complete guide RNA with a scaffold. The flanking region of the genomic target was amplified by PCR from HSV2 G strain genomic DNA (obtained from ATCC). Cas9 protein (obtained from PNA Bio), guide RNA, and target DNA were mixed and incubated for in vitro endonuclease assay.

  In order to further test the efficiency against intracellular HSV, we subcloned each of the HSV2 amplification products described above into an expression vector. The same guide RNA sequence (RL2, LATi, LATp, UL9, OriS, and US12) was also cloned into a CRISPR plasmid containing CMV promoter driven cas9 and U6 promoter driven sgRNA scaffolds. We transfected 293T cells with Lipofectamine 2000, both HSV2 amplification product clones and anti-HSV CRISPR plasmids. Cells were harvested and genomic DNA was isolated 72 hours after transfection.

  FIG. 3 is a lane gel showing genomic DNA size bands of cells treated with RL2, LATi, LATp, UL9, OriS, and US12 guide sequences with or without Cas9.

  FIG. 4 shows the results of a quantitative PCR assay showing various levels of HSV DNA reduction in CRISPR treated samples. For in vivo anti-HSV treatment using a transient cell model, we used a CRISPR plasmid with a scrambled sgRNA sequence as a control. DNA sample input was normalized with each control sample. OriS showed the highest viral DNA removal activity, followed by RL2, UL9, and LATi.

(Example 2)
Target EBV
Burkitt lymphoma cell lines Raji, Namalwa, and DG-75 were obtained from ATCC and cultured in RPMI 1640 supplemented with 10% FBS and PSA according to ATCC recommendations. Human primary lung fibroblast IMR-90 was obtained from Coriell and cultured in Advanced DMEM / F-12 supplemented with 10% FBS and PSA.

  Cong L et al. (2013), Multiplex Genome Engineering Using CRISPR / Cas Systems., Science, 339: 819-823, U6 promoter driven chimeric guide RNA (sgRNA) and ubiquitous promoter driven A plasmid consisting of sex Cas9 was obtained from addgene. The EGFP marker fused after the Cas9 protein allowed selection of Cas9 positive cells (FIG. 6). We adopted a modified chimeric guide RNA design for more efficient Pol-III transcription and a more stable stem-loop structure (Chen B et al. (2013), Dynamic Imaging of Genomic Loci. in Living Human Cells by an Optimized CRISPR / Cas System. Cell, 155: 1479-1491).

  We have identified pX458 in Addgene, Inc. Obtained from. A modified CMV promoter with a synthetic intron (pmax) was amplified by PCR from the Lonza control plasmid pmax-GFP. Modified guide RNA sgRNA (F + E) was ordered from IDT. The EBV origin of replication oriP was amplified by PCR from the B95-8 transformed lymphoblastoid cell line GM12891. We cloned pmax, sgRNA (F + E), and oriP into pX458 using standard cloning protocols, replacing the original CAG promoter, sgRNA, and the f1 origin. We designed EBV sgRNA based on the B95-8 standard and ordered DNAT from IDT. The original sgRNA placeholder of pX458 serves as a negative control.

  Lymphocytes are known to be resistant to lipofection and therefore we used nucleofection for DNA delivery into Raji cells. We select the Lonza pmax promoter to drive Cas9 expression. This is because this promoter resulted in strong expression in Raji cells. We used Lonza Nucleofector II for DNA delivery. Five million Raji cells or DG-75 cells were each transfected with 5 ug plasmid in a 100 ul reaction. Cell line kit V and program M-013 were used according to Lonza's recommendations. In the case of IMR-90, 1 million cells were transfected with program T-030 or X-005 using 5 ug plasmid in 100 ul Solution V. We observed a clear EGFP signal from a small number of cells with a fluorescence microscope 24 hours after nucleofection. However, the EGFP positive cell population subsequently decreased dramatically and we measured transfection efficiencies of less than 10% 48 hours after nucleofection (FIG. 6). We thought that this decrease in transfection efficiency was due to plasmid dilution due to cell division. In order to maintain the plasmid level in the host cell active, we redesigned the CRISPR plasmid to include the EBV origin of replication sequence, oriP. Due to active plasmid replication inside the cells, transfection efficiency increased to over 60% (FIG. 6).

  In order to design a guide RNA targeting the EBV genome, we relied on an EBV reference genome derived from the B95-8 strain. We targeted six regions with seven guide RNA designs with different genome editing objectives.

  For more information on primer design, etc., see Wang and Quake, 2014, RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection, PNAS, 111 (36): 13157-13162, and online at the PNAS website It is shown in the supplementary information of this paper published in The contents of both of these documents are incorporated by reference for all purposes.

  EBNA1 is important for many EBV functions, including gene regulation and latent genome replication. To excise this entire region of the genome, guide RNAs sgEBV4 and sgEBV5 to both ends of the EBNA1 coding region were used. Since guide RNA sgEBV1, 2, and 6 are within the repeat region, the success rate of at least one CRISPR cleavage is doubled. These “structural” targets allow the EBV genome to be digested systematically into smaller pieces. EBNA3C and LMP1 are essential for host cell transformation, we designed guide RNA sgEBV3 and sgEBV7, respectively, to target the 5 'exons of these two proteins.

  EBV genome editing. Double-stranded DNA breaks generated by CRISPR are repaired resulting in small deletions. Figures 8-13 show that CRISPR / Cas9 induced a large deletion.

  FIG. 8 shows the genomic organization around the guide RNA sgEBV2 and PCR primer positions.

  FIG. 9 shows a large deletion induced by sgEBV2. Lanes 1-3 are before, 5 days, and 7 days after sgEBV2 treatment, respectively.

  FIG. 10 shows the genomic organization around the guide RNA sgEBV3 / 4/5 and PCR primer positions.

  FIG. 11 shows the large deletions induced by sgEBV3 / 5 and sgEBV4 / 5. Lanes 1 and 2 are 3F / 5R PCR amplification products before and 8 days after sgEBV3 / 5 treatment. Lanes 3 and 4 are 4F / 5R PCR amplification products before and 8 days after sgEBV4 / 5 treatment.

  FIGS. 12 and 13 show that Sanger sequencing confirmed genomic cleavage and repair ligation 8 days after sgEBV3 / 5 treatment (FIG. 12) and sgEBV4 / 5 treatment (FIG. 13). . Regions 690 and 700 (FIG. 12) and regions 690 and 700 (FIG. 13) show the two ends prior to repair ligation.

  Such a deletion disrupts the protein code and thus exerts a knockout effect. SURVEYOR assay confirmed efficient editing of individual sites.

  FIG. 32 shows the SURVEYOR assay for EBV CRISPR (lanes are numbered from left to right: lane 1: NEB 100 bp ladder; lane 2: sgEBV1 control; lane 3: sgEBV1; lane 4: sgEBV5 control; lane 5: sgEBV5; lane 6: sgEBV7 control; lane 7: sgEBV7; lane 8: sgEBV4).

  Large deletions between target sites beyond the independent small deletions induced by each guide RNA can systematically disrupt the EBV genome. Guide RNA sgEBV2 targets a region with 12 125 bp repeat units (FIG. 8). The PCR amplification product of the entire repeat region showed a band of about 1.8 kb (FIG. 9). After 5 or 7 days after sgEBV2 transfection, we obtained an approximately 0.4 kb band derived from the same PCR amplification (FIG. 9). The approximately 1.4 kb deletion is the expected product of repair ligation between the cleavage of the first repeat unit and the last repeat unit (FIG. 8).

  The DNA sequence flanking the sgRNA target was PCR amplified using Phusion DNA polymerase. The SURVEYOR assay was performed according to the manufacturer's instructions. DNA amplification products with large deletions were TOPO cloned and Sanger sequencing was performed using a single colony. EBV loading was measured by Taqman digital PCR at Fluidigm BioMark. A Taqman assay targeting conserved human loci was used for human DNA normalization. 1 ng of single cell whole genome amplification product derived from Fluidigm C1 was used for EBV quantitative PCR.

  It is possible to delete regions between unique targets (FIG. 10). Six days after sgEBV4-5 transfection, PCR amplification of the entire flanking region (using primers EBV4F and 5R) resulted in a shorter amplification product, while the expected 2 kb band was much faint. (FIG. 11). Sanger sequencing of the amplified product clones confirmed that the two expected cleavage sites were directly connected (FIG. 13). A similar experiment using sgEBV3-5 also revealed a larger deletion from EBNA3C to EBNA1 (FIG. 11).

  For more information on primer design, etc., see Wang and Quake, 2014, RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection, PNAS, 111 (36): 13157-13162, and online at the PNAS website It is shown in the supplementary information of this paper published in The contents of both of these documents are incorporated by reference for all purposes.

  Cell growth arrest due to EBV genome disruption. Two days after CRISPR transfection, EGFP positive cells were flow sorted for further culture and viable cells were counted daily. 14 to 26 show cell growth arrest due to EBV genome disruption. FIG. 14 shows cell growth curves after different CRISPR treatments. Here, five independent sgEBV1-7 treatments are shown. FIGS. 15-20 show flow cytometry scatter signals before (FIG. 15), 5 days (FIG. 16) and 8 days (FIG. 14) before sgEBV1-7 treatment. 18-20 show the annexin V Alexa 647 and DAPI staining results before (FIG. 18), 5 days (FIG. 19), and 8 days (FIG. 20) of sgEBV1-7 treatment. Regions 300 and 200 correspond to subpopulations P3 and P4 (FIGS. 15-17).

  FIG. 21 and FIG. 22 show that microscopic observation revealed apoptotic cell morphology after sgEBV1-7 treatment.

  FIG. 23 shows the nuclear morphology prior to treatment with sgEBV1-7.

  FIGS. 24-26 show the nuclear morphology after treatment with sgEBV1-7.

  As expected, cells treated with Cas9 plasmid lacking oriP or sgEBV lost EGFP expression within a few days and grew at a similar rate as the untreated control group (FIG. 14). The plasmid with Cas9-oriP and scrambled guide RNA maintained EGFP expression after 8 days, but the cell growth rate was not reduced. Treatment with mixed cocktail sgEBV1-7 did not result in measurable cell proliferation and the total cell number either remained constant or decreased (FIG. 14).

  In FIG. 15, the flow cytometric scatter signal clearly reveals that the cell morphology has changed after sgEBV1-7 treatment, with the majority of cells shrinking in size and increasing granulation (from P4 to P3). Shift).

  FIG. 19 shows the results of DAPI staining showing that the cells of population P3 also showed a membrane permeability disorder. The same treatment was performed on two other samples: EBV negative Burkitt lymphoma cell line DG-75 and primary human lung fibroblast IMR90, specifically to eliminate the possibility of CRISPR cytotoxicity by multiple guide RNAs. .

  FIG. 33 shows that CRISPR treatment was not cytotoxic to the EBV negative Burkitt lymphoma cell line DG-75.

  FIG. 34 shows that CRISPR treatment was not cytotoxic to primary human lung fibroblast IMR90.

  At 8 and 9 days after transfection, the cell growth rate was unchanged from the untreated control group, suggesting that the cytotoxicity was negligible.

  According to previous studies, EBV tumorigenicity is believed to be due to interfering with host cell apoptosis (Ruf IK et al. (1999), Epstein-Barr Virus Regulates c-MYC, Apoptosis, and Tumorigenicity in Burkitt Lymphoma., Molecular and Cellular Biology, 19: 1651-1660). Thus, suppression of EBV activity can restore the apoptotic process. In view of this, it can be explained that cell death was observed in our experiments. Annexin V staining revealed a characteristic subpopulation of cells with intact cell membranes but exposed phosphatidylserine. This suggests cell death due to apoptosis (FIG. 18). Bright field microscopy showed clear apoptotic cell morphology (FIG. 21), and fluorescent staining showed dramatic DNA fragmentation (FIG. 23). Taken together, these evidences suggest that the normal host cell apoptotic pathway has been restored following EBV genome disruption.

  27 to 31 show the EBV load after the CRISPR treatment.

  FIG. 27 shows EBV loading after various CRISPR treatments by digital PCR. Cas9 and Cas9-oriP had 2 replicates and sgEBV1-7 had 5 replicates.

(Complete clearance of EBV in subpopulation)
To study the possible link between cell growth arrest and EBV genome editing, digital PCR targeting EBNA1 was used to quantify EBV loading in different samples. Another Taqman assay targeting a conserved human somatic locus served as an internal control for human DNA normalization. Each untreated Raji cell has an average of 42 copies of the EBV genome (FIG. 27). Cells treated with Cas9 plasmid lacking oriP or sgEBV did not show EBV loading differences that were clearly different from untreated controls. Cells treated with the Cas9 plasmid with oriP but without sgEBV showed about 50% reduced EBV loading. In combination with the above observation that the cells in this experiment did not show any growth rate differences, we believe that this is likely due to competition for EBNA1 binding during plasmid replication. Interpret. Adding guide RNA cocktail sgEBV1-7 to the transfection dramatically reduced EBV loading. Both viable and dead cells show a EBV reduction of greater than 60% compared to untreated controls.

  Although seven guide RNAs were provided at the same molar ratio, the plasmid transfection and replication process is likely to be very stochastic. Some cells will inevitably receive different subsets or mixtures of guide RNA cocktails, which may affect treatment efficiency. To control such effects, EBV loading was measured at the single cell level by using single cell whole genome amplification with an automated microfluidic system.

  Freshly cultured Raji cells were mounted on a microfluidic chip and 81 single cells were captured.

  For sgEBV1-7 treated cells, viable cells were flow sorted 8 days after transfection to capture 91 single cells.

  FIG. 28 shows a single Raji cell trapped in a microfluidic chip.

  FIG. 29 shows a single sgEBV1-7 treated cell trapped on the chip.

  Approximately 150 ng of amplified DNA was obtained from each single cell reaction chamber according to the manufacturer's instructions. For quality control purposes, we performed 4 loci human somatic DNA quantitative PCR on each single cell amplification product (Wang J, Fan HC, Behr B, Quake SR, (2012). ) Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm., Cell, 150: 402-412), provided that a positive amplification product was obtained from at least one locus.

  FIG. 30 is a histogram of single cell EBV quantitative PCR Ct values prior to treatment. The dotted line represents the Ct value of one EBV genome per cell. A lognormal distribution of EBV load was shown by 69 untreated single cell products that passed quality control, with almost all cells showing significant amounts of EBV genomic DNA.

  We calibrated the quantitative PCR assay using a subclone of Namalwa Burkitt lymphoma cells containing a single integrated EBV genome. According to single copy EBV measurement, Ct was 29.8. This allowed us to determine that the average Ct of 69 Raji single cell samples corresponded to 42 copies of EBV per cell by bulk digital PCR measurements. For sgEBV1-7 treated samples, 71 single cell products passed quality control and the EBV load distribution was dramatically wider.

  FIG. 31 is a histogram of EBV quantitative PCR Ct values of single viable cells after 7 days of treatment with sgEBV1-7. The dotted line represents the Ct value of one EBV genome per cell.

  Twenty-two cells had the same EBV load as untreated cells, but 19 cells had no detectable EBV and the remaining 30 cells were compared to untreated samples. Showed a dramatic decrease in EBV load.

  FIG. 32 shows the URV CRISPR SURVEYOR assay. Lane 1 (lanes are numbered from left to right): NEB 100 bp ladder; Lane 2: sgEBV1 control; Lane 3: sgEBV1; Lane 4: sgEBV5 control; Lane 5: sgEBV5; Lane 6: sgEBV7 control; Lane 7: sgEBV7; Lane 8: sgEBV4. FIG. 33 shows a CRISPR cytotoxicity test using EBV negative Burkitt lymphoma DG-75. FIG. 34 shows a CRISPR cytotoxicity test using primary human lung fibroblast IMR-90.

  An essential target for EBV treatment. The seven guide RNAs of our CRISPR cocktail target three different categories of sequences that are important for EBV genomic structure, host cell transformation, and latent infectivity, respectively. In order to understand the most essential targets for effective EBV treatment, we transfected a subset of guide RNA into Raji cells. sgEBV4 / 5 reduced the EBV genome by 85% but failed to suppress cell proliferation as effectively as the full cocktail (FIG. 14). Guide RNA targeting the structural sequence (sgEBV1 / 2/6) did not completely eliminate EBV loading (26% reduction) but was able to completely stop cell proliferation. In view of the high efficiency of genome editing and the cessation of growth (FIG. 2), the present inventors found that the residual EBV genome trace of sgEBV1 / 2/6 was not due to an intact genome, but an EBV genome. It is assumed that the free floating DNA digested from the above, that is, false positive. We conclude that EBV treatment is more effective for systematically disrupting the EBV genomic structure than targeting specific critical proteins.

Claims (53)

A composition for the treatment of herpes simplex virus (HSV) infection, comprising:
Nuclease,
A composition comprising a vector that is complementary to an HSV nucleic acid and encodes a sequence-specific targeting moiety capable of directing the nuclease to the HSV nucleic acid.   The composition according to claim 1, wherein the HSV nucleic acid is a replication origin S (oriS) region.   The composition according to claim 1, wherein the HSV nucleic acid is a long unique region 9 (UL9) or a long repeat region 2 (RL2).   The composition of claim 1, wherein the composition is configured to be administered transdermally.   The composition of claim 1 comprising a topical solution.   2. The composition of claim 1, wherein the nuclease is a nuclease selected from the group consisting of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases.   2. The composition of claim 1, wherein the nuclease comprises a Cas9 endonuclease and the sequence specific binding module comprises a guide RNA that specifically targets a portion of the viral genome.   The composition of claim 1 further packaged for delivery to a human patient.   The composition of claim 1, wherein the targeting sequence is a guide RNA and does not have more than 60% identity in the human genome.   The vector is a retrovirus, lentivirus, adenovirus, herpesvirus, poxvirus, alphavirus, vaccinia virus, adeno-associated virus, plasmid, nanoparticle, cationic lipid, cationic polymer, metal nanoparticle, nanorod, liposome, 2. The composition of claim 1 comprising a vector selected from the group consisting of microbubbles, cell penetrating peptides, and lipospheres. A method for treating a herpes simplex virus (HSV) infection comprising:
Introducing a vector encoding a nuclease and a sequence-specific targeting moiety complementary to an HSV nucleic acid, or a ribonucleoprotein comprising the nuclease and the sequence-specific targeting moiety into a host cell;
The method comprising the step of causing the sequence-specific targeting moiety to direct the nuclease to the HSV nucleic acid, and causing the nuclease to cleave the HSV nucleic acid.   The method according to claim 11, wherein the HSV nucleic acid is a replication origin S (oriS) region.   The method according to claim 11, wherein the HSV nucleic acid is a long unique region 9 (UL9) or a long repeat region 2 (RL2).   12. The method of claim 11, further comprising the step of transdermally administering the vector to the host.   15. The method of claim 14, wherein the transdermal administration further comprises applying a topical solution containing the vector.   12. The method of claim 11, wherein the nuclease is a vector selected from the group consisting of a zinc finger nuclease, a transcriptional activator-like effector nuclease, and a meganuclease.   12. The method of claim 11, wherein the nuclease comprises a Cas9 endonuclease and the sequence specific binding module comprises a guide RNA that specifically targets a portion of the viral genome.   12. The method of claim 11, wherein the host is a living human subject and the step is performed in vivo.   12. The method of claim 11, wherein the targeting sequence is a guide RNA and has no more than 60% identity in the human genome.   The vector is a retrovirus, lentivirus, adenovirus, herpesvirus, poxvirus, alphavirus, vaccinia virus, adeno-associated virus, plasmid, nanoparticle, cationic lipid, cationic polymer, metal nanoparticle, nanorod, liposome, 12. The method of claim 11, comprising a vector selected from the group consisting of microbubbles, cell penetrating peptides, and lipospheres. A composition for the treatment of herpes simplex virus (HSV) infection, comprising:
Nuclease,
A composition comprising a ribonucleoprotein that is complementary to an HSV nucleic acid and comprises a sequence-specific targeting moiety capable of directing the nuclease to the HSV nucleic acid.   The composition according to claim 21, wherein the HSV nucleic acid is a replication origin S (oriS) region.   The composition according to claim 21, wherein the HSV nucleic acid is a long unique region 9 (UL9) or a long repeat region 2 (RL2).   24. The composition of claim 21, wherein the composition is configured to be administered transdermally.   24. The composition of claim 21, further comprising a pharmaceutically acceptable carrier for topical application to the infected tissue.   23. The composition of claim 21, wherein the nuclease comprises a Cas9 endonuclease and the sequence specific binding module comprises a guide RNA that specifically targets a portion of the viral genome.   23. The composition of claim 21, wherein the targeting sequence is a guide RNA and has no more than 60% identity in the human genome. A composition for the treatment of cytomegalovirus (CMV) infection, comprising:
Nuclease,
A sequence-specific targeting moiety that is complementary to a CMV nucleic acid and capable of directing the nuclease to the CMV nucleic acid.   32. The composition of claim 30, wherein the nuclease comprises a nuclease selected from the group consisting of a zinc finger nuclease, a transcriptional activator-like effector nuclease, and a meganuclease.   32. The composition of claim 30, wherein the nuclease is a Cas9 endonuclease.   33. The composition of claim 32, wherein the nuclease further comprises a guide RNA that targets the Cas9 endonuclease to a portion of the CMV nucleic acid.   32. The composition of claim 30, wherein the nuclease causes at least one double-strand break in the CMV nucleic acid.   32. The composition of claim 30, wherein the nuclease causes at least one insertion in the CMV nucleic acid. A method for treating a cytomegalovirus (CMV) infection of an organ comprising:
Introducing into the organ a composition comprising a nuclease and a sequence-specific targeting moiety that is complementary to a CMV nucleic acid and capable of directing the nuclease to the CMV nucleic acid; and A method comprising the steps of targeting and cleaving a nucleic acid.   40. The method of claim 36, wherein the organ is derived from a transplant donor.   40. The method of claim 36, wherein the organ is selected from the group consisting of heart, liver, kidney, eye, lung, pancreas, intestine, and thymus.   38. The method of claim 36, wherein the nuclease comprises a nuclease selected from the group consisting of a zinc finger nuclease, a transcriptional activator-like effector nuclease, and a meganuclease.   38. The method of claim 36, wherein the nuclease is a Cas9 endonuclease.   41. The method of claim 40, wherein the nuclease further comprises a guide RNA that targets the Cas9 endonuclease to a portion of the CMV nucleic acid.   38. The method of claim 36, wherein the introducing step comprises delivering the nuclease with a delivery viral vector.   43. The method of claim 42, wherein the viral vector is selected from the group consisting of retrovirus, lentivirus, adenovirus, herpes virus, pox virus, alphavirus, vaccinia virus, and adeno-associated virus.   The introducing step includes those selected from the group consisting of plasmids, nanoparticles, cationic lipids, cationic polymers, metal nanoparticles, nanorods, liposomes, cell-permeable peptides, lipospheres, and polyethylene glycol (PEG). 38. The method of claim 36, comprising delivering the nuclease with a vector.   40. The method of claim 36, wherein the nuclease causes at least one double-strand break in the CMV nucleic acid.   40. The method of claim 36, wherein the nuclease causes at least one insertion in the CMV nucleic acid. A method for treating fetal cytomegalovirus (CMV) infection comprising:
Introducing into a pregnant woman a composition comprising a nuclease and a sequence-specific targeting moiety that is complementary to a CMV nucleic acid and capable of directing the nuclease to the CMV nucleic acid; and Targeting and cleaving.   48. The method of claim 47, wherein the nuclease comprises a nuclease selected from the group consisting of a zinc finger nuclease, a transcriptional activator-like effector nuclease, and a meganuclease.   48. The method of claim 47, wherein the nuclease is a Cas9 endonuclease.   48. The method of claim 47, wherein the nuclease is associated with a guide RNA that targets the Cas9 endonuclease to a portion of the CMV nucleic acid.   48. The method of claim 47, wherein the introducing step comprises delivering the nuclease in a viral vector.   52. The method of claim 51, wherein the viral vector is selected from the group consisting of retroviruses, lentiviruses, adenoviruses, herpesviruses, poxviruses, alphaviruses, vaccinia viruses, and adeno-associated viruses.   The introducing step includes those selected from the group consisting of plasmids, nanoparticles, cationic lipids, cationic polymers, metal nanoparticles, nanorods, liposomes, cell-permeable peptides, lipospheres, and polyethylene glycol (PEG). 48. The method of claim 47, comprising delivering the nuclease with a vector.   48. The method of claim 47, wherein the nuclease causes at least one double-strand break in the CMV nucleic acid.   48. The method of claim 47, wherein the nuclease causes at least one insertion in the CMV nucleic acid.