CN116803428A - Methods and pharmaceutical compositions for treating viral infections - Google Patents

Abstract

The present application provides a method of inhibiting a virus in a subject comprising administering to the subject a system that binds to a viral polypeptide or nucleic acid sequence and localizes the virus to a region of nuclear repressor gene expression, the system comprising a localization domain and a binding domain. The present application has found and tested that repositioning virus to specific cell nucleus site, such as the nucleus periphery, can inhibit the expression of virus gene effectively and control the replication and proliferation of virus. Thus, the present application further provides methods and medicaments for preventing or treating viral infections and related diseases.

Description

Methods and pharmaceutical compositions for treating viral infections

The present application claims priority from the following chinese patent applications: the application number 202210309785.5, entitled "methods and pharmaceutical compositions for treating viral infections" filed on 25/3/2022, the entire contents of which are incorporated herein by reference.

Technical Field

The application relates to the technical field of molecular biology, in particular to a method and a pharmaceutical composition for preventing or treating viral infection.

Background

Diseases caused by viruses are responsible for many of the pain and inconvenience that humans and animals typically endure, and in some cases, cause death. For example: influenza is a common disease in humans caused by viruses, with tremendous economic and public health effects.

A virus is a non-cellular organism that is structurally simple, parasitic in living cells and proliferates in a replicative manner. Viruses are generally composed of a long chain of nucleic acids and a protein coat, without their own metabolic machinery and without enzymatic systems. Replication, transcription, and translation of viruses are all performed in host cells, and when introduced into host cells, the materials and energy in the cells can be used to complete vital activities, producing a new generation of viruses based on the genetic information contained in their nucleic acids.

Viruses can be classified into two major categories, DNA viruses and RNA viruses, depending on the chemical composition of the viral nucleic acid. Viruses also include retroviruses, such as double-stranded DNA retroviruses and positive-stranded RNA retroviruses (e.g., lentiviruses, representative viruses being human immunodeficiency virus 1 (HIV-1)). Among them, DNA viruses can be further classified into double-stranded DNA viruses or single-stranded DNA viruses. The DNA virus types mainly include adenovirus, poxvirus, human papillomavirus (Human Papillomavirus, HPV), human herpesvirus, and the like.

Herpes viruses are typical DNA viruses, including human herpes simplex virus type 1 (HSV-1), human herpes simplex virus type 2 (HSV-2), cytomegalovirus (CMV), varicella Zoster Virus (VZV), human herpes virus-4 (Human herpesvirus, HHV-4) also known as Epstein-Barr virus (EBV) or EBV, human herpes virus-6 (HHV-6), human herpes virus-7 (HHV-7), human herpes virus-8 (HHV-8), pseudorabies virus (Pseudorabies virus, PRV), and the like. Herpes Simplex Virus (HSV) is divided into two types, HSV-1 and HSV-2.HSV-1 is transmitted through intimate contact of the respiratory tract, skin and mucous membranes, and mainly causes infections of the lips, pharynx, eyes and skin, and a small number also causes genital infections. HSV-2 is the main pathogen of genital herpes, and exists in exudates, semen, prostatic secretions, cervical and vaginal secretions damaged by skin and mucous membrane, and is mainly transmitted through intercourse to cause primary genital herpes. HSV-1 is a member of the herpesviridae family, a double stranded DNA virus, approximately 153kb in size, and has an infection rate of over 66.7% in the world population between 0 and 49 years of age.

Mature infectious virus of HSV-1 consists of a viral envelope, an envelope layer and an icosahedral envelope surrounding a viral genome. The viral coat interfaces with the Nuclear Pore Complex (NPC) and after release and entry of the viral genome into the nucleus, the three sets of viral genes are expressed in strict temporal order, i.e., immediate early (α), early (β) and late genes (γ1, γ2). Immediate events following viral genome entry into the nucleus include a strong competition between viral gene expression and the amount of suppression from the host cell to silence these exogenous DNA.

In recent years, innovation of intracellular structure mapping and operation technology has driven the scientific community to recognize the causal relationship between cellular gene transcription regulation and its relative nuclear location on a three-dimensional scale. It is recognized that the location of the cell chromosome in the nucleus is not random, and that the spatially and temporally dynamic organization of DNA within the nucleus plays an important role in the regulation of the cell transcriptome. The knowledge of the complexity of host genome tissues and key transcriptional regulation has great help to the pathogenic mechanism and treatment method of viruses.

There is a need in the art for new therapies and drugs that are provided based on new insights into the mechanism of action and replication routes of viruses within the nucleus.

Disclosure of Invention

The present application provides novel methods and medicaments for inhibiting viruses. In the application, typical DNA viruses such as HSV-1 and ADV are taken as examples, and the virus can be effectively inhibited from expressing by repositioning to specific cell nucleus positions (such as the periphery of the nucleus) through the first discovery and experiment, so that the replication and proliferation of the virus can be controlled. Thus, the present application provides methods and medicaments for preventing or treating viral infections and diseases related thereto.

In particular, the application provides a method of inhibiting a virus in a subject comprising administering to the subject a system that binds to a viral polypeptide or nucleic acid sequence and localizes the virus to a region in the nucleus that inhibits gene expression. The methods of the application can inhibit viruses by inhibiting their gene expression, replication and proliferation.

In one aspect of the application, the aforementioned system for administration in a method of inhibiting a virus in a subject has:

a chimeric polypeptide comprising a localization domain (localization domain) which is a polypeptide that localizes to a region within the nucleus that inhibits gene expression and a polypeptide binding domain (binding domain) which is a binding moiety that specifically recognizes and binds to a viral protein (e.g., capsid protein). In general, the polypeptide binding domain can recognize and bind to a specific protein of a virus and not to a host protein.

In one aspect of the application, the aforementioned system for administration in a method of inhibiting a virus in a subject has:

a chimeric polypeptide comprising a localization domain (localization domain) and a nucleic acid binding domain (binding domain), wherein the localization domain is a polypeptide that localizes to a region within the nucleus that inhibits gene expression, and the nucleic acid binding domain is a binding moiety that specifically recognizes and binds to a viral nucleic acid sequence. In general, the nucleic acid binding domain can recognize and bind to a specific nucleic acid sequence of a virus and not recognize and bind to a nucleic acid sequence of a host.

As used herein, "polypeptide" includes proteins, protein fragments, and peptides, whether isolated from natural sources, produced by recombinant techniques, or chemically synthesized. The polypeptide may have one or more modifications, such as post-translational modifications (e.g., glycosylation, etc.), or any other modification (e.g., pegylation, etc.). The peptide or protein also includes active fragments thereof, i.e., portions or fragments of the protein, which comprise portions of the protein that maintain full or partial activity, or fragments, e.g., domain fragments, of the protein that have some or several of the various activities of the protein. The proteins also include derivatives or variants or modifications thereof, including proteins (polypeptides) having a primary and/or tertiary structure similar to the native form of the protein, but differing from the native form by one or more amino acid residues (e.g., one or more amino acid substitutions, insertions and/or deletions), or having/additionally having other chemical groups or protein (polypeptide) groups that can be bound thereto; these derivatives have or retain all or part of the activity of the protein. The conjugated chemical groups include, for example, post-translational derivatization or modification of polypeptides, such as pegylation and/or sulfhydryl groups. The bondable protein (polypeptide) groups include, for example, his tags or IgG proteins, and the like.

"nucleic acid", "polynucleotide" or "nucleotide" as used interchangeably herein refers to a polymer of nucleotides of any length, including DNA and RNA. The polynucleotide or nucleotide sequence may be double-stranded or single-stranded. When a polynucleotide or nucleotide sequence is single stranded, it may refer to either of the two complementary strands. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base and/or analogue thereof, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. Modification of the nucleotide structure, if present, may be performed before or after assembly of the polymer.

The nuclear envelope makes the nucleus a relatively independent system in the cell, allowing a relatively stable environment to be formed within the nucleus. Recent studies based on sequencing methods and microscopy methods have shown that within the nucleus, there are two chromatin regions in which gene transcriptional activity differs greatly: one is a chromatin region rich in genes and active in transcription, and the other is a chromatin region with fewer genes and suppressed transcription (Abigail Buchwalter et al, coaching from the sidelines: the nuclear periphery in genome regulation. Nature Reviews, 2018).

The nuclear envelope (nuclear envelope) is wrapped on the surface of the core and consists of two layers of substantially parallel inner and outer membranes. The gap between the two membranes is the perinuclear gap (perinuclear cisterna), also known as the perinuclear cavity. The nuclear membrane inner membrane has nuclear fiber layer with thickness of 20-80 nm, and its main component is nuclear fiber layer protein (lamin).

It has been found that genomic sequences associated with the periphery of the nucleus (nuclear periphery) generally exhibit low transcriptional activity, whereas genomic sequences located inside the nucleus generally exhibit relatively high activity. The nuclear periphery generally includes the layer portion of the nuclear membrane facing the nuclear space and peripheral regions, particularly the peripheral heterochromatin enrichment region of the nucleus. The perinuclear heterochromatin enrichment region is generally considered to belong to the chromatin region in which gene transcription is inhibited. In one aspect of the application, the core perimeter includes a layer portion of the core membrane facing the core interior space, and includes a nuclear lablab (nuclear lablab).

In one embodiment of the present application, the region in which gene expression is inhibited in the nucleus is the periphery of the nucleus. In yet another aspect of the present application, wherein the region of the nuclear repressor gene expression is the nuclear envelope (or referred to as the endonucleomembrane), particularly the layer of the endonucleomembrane facing the nuclear space (nucleoplasm).

In one embodiment of the application, the nucleic acid binding domain of the chimeric polypeptide recognizes and binds to RNA of a virus. The nucleic acid binding domain may include a protein capable of recognizing a specific RNA sequence, such as dCAS13a, and the like. In yet another aspect of the application, the viral nucleic acid is genomic RNA of an RNA virus.

In one embodiment of the application, the nucleic acid binding domain of the chimeric polypeptide recognizes and binds to viral DNA. In yet another aspect of the application, the viral nucleic acid is genomic DNA of a virus, for example, genomic DNA of a DNA virus. In yet another aspect of the application, the viral nucleic acid is DNA present or present in the viral replication cycle, for example, double stranded DNA synthesized by reverse transcriptase of RNA of a retrovirus.

The method provided by the application is suitable for inhibiting various viruses. Viruses can be classified into two major categories, DNA viruses and RNA viruses, depending on the chemical composition of the viral nucleic acid. Viruses also include retroviruses, such as double-stranded DNA retroviruses and positive-stranded RNA retroviruses (e.g., lentiviruses, representative viruses being human immunodeficiency virus 1 (HIV-1)). In one of its aspects, the present application provides a method particularly suitable for use with viruses whose viral genomes have DNA or DNA that occurs during the replication cycle phase thereof, such as double stranded DNA synthesized by reverse transcriptase of the RNA of a retrovirus.

In one aspect of the application, the virus is a DNA virus, such as an adenovirus, poxvirus, papovavirus or herpes virus. In one aspect of the application, the virus is a retrovirus, such as lentivirus, and the representative virus is human immunodeficiency virus 1 (HIV-1). In one aspect of the application, the virus is an RNA virus, such as rotavirus, picornavirus (e.g., enterovirus), calicivirus, and the like.

In yet another aspect of the present application, the virus is a virus that replicates in the nucleus. In yet another aspect of the application, the virus comprises a virus that proceeds in the nucleus at some or all stages of the replication cycle.

In the present application, the virus may be human herpes simplex virus type 1 (HSV-1), human herpes simplex virus type 2 (HSV-2), cytomegalovirus (CMV), varicella-zoster virus (VZV), human herpes virus-4 (Human herpesvirus, HHV-4) also known as Epstein-Barr virus (EBV) or EBV, human herpes virus-6 (HHV-6), human herpes virus-7 (HHV-7), human herpes virus-8 (HHV-8), pseudorabies virus (Pseudorabies virus, PRV), hepatitis B Virus (HBV), human immunodeficiency virus 1 (HIV-1), or the like.

In one embodiment of the application, the nucleic acid binding domain of the chimeric polypeptide comprises a Cas protein, a Zinc Finger Nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a Argonaute (Ago) protein.

In one embodiment of the application, the nucleic acid binding domain comprises a Cas protein. Cas proteins, also known as CRISPR-associated (Cas) proteins or Cas nucleases, function in non-naturally occurring CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) systems. CRISPR/Cas systems have been widely used as genome engineering tools in a variety of organisms including different mammals, animals, plants and yeasts. Cas proteins useful in the present application may refer to any modified (e.g., shortened, mutated, lengthened) polypeptide sequence or homolog of a Cas protein. Cas proteins may be enzymatically inactive, partially active, constitutively active, fully active, inducible active, and/or more active (e.g., more active than wild type homologs of the protein or polypeptide). In some embodiments, the Cas protein may be a type II Cas protein. In some embodiments, the Cas protein may be Cas9. In some embodiments, the Cas protein may be a V-type Cas protein. In some embodiments, the Cas protein may be Cpf1 or Cas12a. In some embodiments, the Cas protein may be C2C1. In some embodiments, the Cas protein may be C2C3. In some embodiments, the Cas protein may be a type VI Cas protein. In some embodiments, the Cas protein may be C2 or Cas13a. In some embodiments, the Cas protein may be Cas13b, cas13c, or Cas13d. Cas proteins (e.g., variant, mutant, enzymatically inactive, and/or conditionally-enzymatically inactive site-directed polypeptides) can bind to a target nucleic acid.

In one aspect of the application, the Cas protein employed in the nucleic acid-binding domain of the chimeric polypeptide substantially lacks DNA cleavage activity. When the Cas protein is a modified form without nucleic acid cleavage activity, it may be referred to as enzymatically inactive and/or "dead" (abbreviated as "d", i.e., dCas). In some cases, the Cas protein is mutated and/or modified to produce a nuclease-deficient protein or a protein having reduced nuclease activity relative to the wild-type Cas protein. Nuclease deficient proteins may retain the ability to bind DNA but lack or have reduced nucleic acid cleavage activity. For example, one or more nuclease domains (e.g., ruvC, HNH) of the Cas protein may be deleted or mutated such that they are no longer functional or comprise reduced nuclease activity. If all nuclease domains of the Cas protein (e.g., ruvC and HNH nuclease domains in Cas9 protein; ruvC nuclease domains in Cpf1 protein) are deleted or mutated, the resulting Cas protein may have a reduced or no ability to cleave both strands of double-stranded DNA. An example of a mutation that can convert Cas9 protein to a nickase is a D10A (aspartic acid to alanine at position 10 of Cas 9) mutation in the RuvC domain of Cas9 from streptococcus pyogenes. H939A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) in the HNH domain of Cas9 from streptococcus pyogenes can convert Cas9 to a nickase. Examples of mutations that can convert Cas9 protein into dead Cas9 (i.e., cas9 lacking nucleic acid cleavage activity) are the D10A (aspartic acid to alanine at position 10 of Cas 9) mutation in the RuvC domain of Cas9 from streptococcus pyogenes and H939A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) in the HNH domain.

In the present application, cas9 polypeptide or dCas9 polypeptide can bind to small guide RNAs (sometimes also referred to as "guide RNAs") and then bind to a specific nucleic acid sequence in a nucleic acid of a virus of interest in a sequence-specific manner. Wherein the dCas9 polypeptide is capable of binding to a nucleic acid sequence in the polynucleotide in a sequence-specific manner, but is incapable of cleaving a polypeptide of the target polynucleotide.

In one aspect of the application, the system further comprises a small guide RNA (sgRNA) complexed with the Cas protein. The small guide RNA hybridizes to a viral nucleic acid sequence of interest. The small guide RNA can be administered to the subject along with the Cas protein of the binding moiety, or can be administered separately to the subject.

In one embodiment of the application, the nucleic acid binding domain comprises a zinc finger nuclease. "Zinc finger nuclease" or "ZFN" refers to a chimeric cleavage domain (e.g., the cleavage domain of FokI) to at least one zinc finger motif (e.g., at least 2, 3, 4, or 5 zinc finger motifs) capable of binding to polynucleotides (e.g., DNA and RNA) that is capable of specifically recognizing and binding to a zinc finger motif (motif) of a triplet DNA fragment, the zinc finger nuclease having the zinc finger motif as a basic unit for recognizing a particular DNA sequence, rather than a base. In some embodiments of the application, the nuclease domain of the ZFN comprises a modified version of the wild-type nuclease domain. Modified forms of the nuclease domain may comprise amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the nuclease domain.

In one embodiment of the application, the nucleic acid binding domain comprises a TALEN. TALENs (transcription activator-like (TAL) effector nucleases), a transcriptional activator-like effector nuclease, can target modification of specific DNA sequences. It recognizes specific DNA base pairs by means of TAL effectors. Such TALENs can be engineered to bind any desired DNA sequence. In some embodiments of the application, the nuclease domain of a TALEN comprises a modified form of a wild-type nuclease domain. Modified forms of the nuclease domain may comprise amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the nuclease domain.

In one embodiment of the application, the nucleic acid binding domain comprises a Argonaute (Ago) protein. Ago proteins can be prokaryotic Argonaute (pAgo), archaebacteria Argonaute (aAgo), and eukaryotic Argonaute (eAgo).

In one aspect of the application, the localization domain of the chimeric polypeptide is a polypeptide that localizes to a region within the nucleus that inhibits gene expression. The polypeptide is positioned at a desired spatial location in the cell after expression, whereby the chimeric polypeptide can be "repositioned".

In one embodiment of the application, the localization domain of the chimeric polypeptide comprises a perinuclear specific protein. In the present application, the perinuclear specific proteins include proteins present in the perinuclear region including the nuclear membrane, particularly the nuclear membrane layer portion, and the nuclear fiber layer portion, particularly proteins expressed on the subcellular structure of the nucleus. In one embodiment of the application, the perinuclear specific protein is a nuclear membrane protein, such as SUN1-SUN5, emerin, INM protein transmembrane protein 201 (TMEM 201), and the like, as well as active fragments thereof. In one embodiment of the application, the perinuclear specific protein is lamin, and active fragments thereof. In one embodiment of the application, the perinuclear specific protein is selected from Emerin, lap2β, lamin a or B. Emerin protein is fixed on the nuclear membrane/nuclear fiber layer, and plays a role in fixing and maintaining the functions of the cell nucleus.

In one embodiment of the application, the localization domain of the chimeric polypeptide comprises a nucleopore complex-associated protein such as nucleoporins (Nups).

In one aspect of the application, the chimeric polypeptide further comprises a nuclear localization sequence (Nuclear Localization Sequence, NLS). The nuclear localization sequence is typically a short amino acid sequence that provides a signal in the protein to enter the nucleus so that the protein can be transported into the nucleus without being excised during the introduction into the nucleus. In the present application, the chimeric polypeptide is aimed at binding and repositioning viral nucleic acid within the nucleus to a target spatial location within the nucleus, e.g., repositioning viral nucleic acid bound by the chimeric polypeptide to the periphery of the nucleus. In such cases, it may be advantageous to direct the chimeric polypeptide into the nucleus, i.e., to allow the chimeric polypeptide to enter the nucleus in order to exert its effect of binding and repositioning (via the localization domain of the chimeric polypeptide, not the nuclear localization sequence) of viral nucleic acid within the nucleus. Various types of NLS known in the art can be used in the present application. Known NLS include alkaline NLS or classical NLS (cNLS), as well as non-classical NLS such as proline-tyrosine NLS (PY-NLS). Classical NLS is in turn divided into two types, namely haplotype (monopartite type) and bipartite (bipartite type). The former is a basic sequence of 4-8 residues, such as the earliest found SV40 large T antigen NLS (PKKKRKV); the latter is a two-piece basic sequence, separated by about 10-12 residues.

The subject of the methods of inhibiting viruses provided herein encompasses hosts of a variety of viruses, including a variety of eukaryotes. The method of the application can be used to inhibit viral infection of natural or artificially cultured eukaryotic cells (plant cells, animal cells, bacteria, etc.). In one aspect of the application, the "subject" to be treated (to inhibit viral replication or infection, or to treat a related disease) is a mammal, including but not limited to pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, and the like.

In one aspect of the application, a system is also provided that specifically recognizes and binds a viral polypeptide or nucleic acid sequence and localizes the virus to a region within the nucleus that inhibits gene expression. The system is as described hereinbefore, having: a chimeric polypeptide comprising a localization domain and a nucleic acid binding domain, wherein the localization domain is a polypeptide that localizes to a region within the nucleus that inhibits gene expression, and the nucleic acid binding domain is a binding moiety that specifically recognizes and binds a viral nucleic acid sequence. In one aspect of the application, the system is a system for inhibiting viral replication in a subject.

In one aspect of the application, there is also provided an isolated nucleic acid encoding a system that specifically recognizes and binds to a viral nucleic acid sequence and localizes the viral nucleic acid to a region of nuclear repressor gene expression, wherein the system is as described previously, having: a chimeric polypeptide comprising a localization domain and a nucleic acid binding domain, wherein the localization domain is a polypeptide that localizes to a region within the nucleus that inhibits gene expression, and the nucleic acid binding domain is a binding moiety that specifically recognizes and binds a viral nucleic acid sequence.

In one aspect of the application, the nucleic acid comprises a nucleotide sequence encoding a chimeric polypeptide comprising a localization domain, which is a polypeptide localized in a region of the nucleus that inhibits gene expression, and a nucleic acid binding domain, which is a binding moiety that specifically recognizes and binds to a viral nucleic acid sequence.

In one embodiment of the application, the binding portion of the chimeric polypeptide encoded by the nucleic acid comprises a Cas protein, a Zinc Finger Nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a Argonaute (Ago) protein, which nucleic acid further comprises a nucleotide sequence encoding the protein.

In one embodiment of the application, the nucleic acid comprises a nucleotide sequence encoding a Cas protein, and a small guide RNA (sgRNA) complexed with the Cas protein. The small guide RNA hybridizes to a viral nucleic acid sequence of interest. In one aspect of the application, the Cas protein employed in the nucleic acid binding domain of the chimeric polypeptide encoded by the nucleic acid is substantially devoid of DNA cleavage activity.

In one embodiment of the application, the localization domain of the chimeric polypeptide encoded by the nucleic acid comprises a perinuclear specific protein, such as Emerin, lap2β, lamin a or lamin B, and the nucleic acid further comprises a nucleotide sequence encoding the protein.

In one aspect of the application, the chimeric polypeptide encoded by the nucleic acid further comprises a nuclear localization sequence (Nuclear Localization Sequence, NLS).

In one aspect of the application there is also provided an expression vector for a system which specifically recognizes and binds viral nucleic acid sequences and localizes viral nucleic acids to a region of nuclear repressor gene expression comprising a nucleic acid as defined previously.

In one embodiment of the application, the expression vector is a viral vector, such as a lentiviral vector or an adenoviral vector.

In one aspect of the application, there is also provided a pharmaceutical composition comprising a system as described hereinbefore that specifically recognizes and binds to viral nucleic acid sequences and localizes viral nucleic acids to regions of nuclear repression of gene expression, or a nucleic acid as described hereinbefore, or an expression vector as described hereinbefore. The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient that delivers the system or nucleic acid or expression vector to a subject. The pharmaceutical composition is for inhibiting viral replication in a subject.

The system of the application that specifically recognizes and binds viral nucleic acid sequences and localizes viral nucleic acids to regions of nuclear repression of gene expression, or nucleic acids may be delivered to a subject in need thereof by a vector, e.g., by plasmid, virus. Suitable vectors include, for example, adeno-associated virus (AAV), lentivirus, adenovirus, or other viral vector types, or combinations thereof. The carrier may be delivered, for example, by intramuscular injection into the tissue, or via intravenous, transdermal, intranasal, oral, mucosal, and other delivery methods. Such delivery may be via single or multiple doses.

Non-viral delivery methods of nucleic acids include lipofection, microinjection, gene gun, viral particles, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, and the like.

It will be appreciated by those skilled in the art that the actual dosage delivered will vary depending upon a variety of factors, such as the carrier, the target cell or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the route of administration, the mode of administration, the type of transformation/modification sought, and the like.

The application also provides methods and pharmaceutical compositions for preventing or treating a virus-related disease, wherein a system is administered to the subject that specifically recognizes and binds viral nucleic acid sequences and localizes viral nucleic acids to regions of nuclear repressor gene expression. The system is as described previously.

The virus-related disease which can be prevented or treated by the present application is selected from hepatitis B, oral herpes, herpetic keratitis, herpetic febrile, traumatic herpes, herpetic eczema, neonatal herpes, genital herpes, atypical genital herpes, herpetic cervicitis, herpetic proctitis, herpetic encephalitis, herpetic meningitis, disseminated herpes simplex infection, alzheimer's disease, dementia, infectious mononucleosis, chronic active EB virus infection, EB virus-related nasopharyngeal carcinoma, EB virus-related lymphoma, AIDS and HPV-related cervical cancer.

In the present application, treatment includes modifying, alleviating, reducing or preventing symptoms associated with a viral infection; normalizing body function in a condition where viral infection causes impairment of specific body function; or one or more clinically measurable parameters that ameliorate a disease caused by a viral infection. In one embodiment, the therapeutic purpose is to prevent or slow down an undesired physiological condition, disorder or disease, or to obtain a beneficial or desired result. In the present application, treatment may be prophylaxis (prevention), cure, or improvement of a clinical condition in a patient, including reduction in the course of disease or severity of disease, or extension of survival of a patient.

The conditions required to eliminate different types of viral infections may vary greatly. For acute diseases, treatment has a clear goal, slowing down the infection of the virus, giving the immune system time to eliminate the virus. For latent viruses, the main problem is the elimination of cells that are latently infected. For chronic viruses, treatment may be aimed at preventing the spread of infection, to eliminate infection in all or in many cells, or at least to keep the replication of the virus under some control. In general, the treatment of chronic infections is a long-term process. The methods and medicaments of the present application are useful for eliminating different types of viral infections.

In addition, the methods and medicaments of the present application have been found to be particularly useful in the early stages of infection of cells by viruses. Thus, the methods and medicaments of the present application may be used as a method or pharmaceutical composition for prophylactic or emergency treatment in situations where a viral infection may be or may have been suffered.

One of the advantages of the present application is that the use of Cas proteins without DNA cleavage activity as part of binding to viral nucleic acid sequences in a system that binds to viral nucleic acid sequences and localizes the virus to regions of nuclear repression of gene expression can avoid the potential safety hazard of editing host genomes by CRISPR active nucleic acid cleavage techniques while increasing the effect on viral repression. The technical basis of CRISPR editing is double-stranded cleavage of the genome, the direct consequences of which do not represent inactivation of the viral genome: 1. the sheared viral genome can be directly religated; 2. often, a large number of viral genomes are arranged in a single cell nucleus, so that homologous recombination repair can be carried out; 3. the cleavage efficiency and the virus replication efficiency compete, and only a small amount of virus genome is finally wrapped and secreted in the virus cleavage replication to form mature virus particles; 4. the effect on viruses in high titer infections may be limited, with competing effects prior to nuclease activity and viral replication efficiency. 5. There are a large number of repeat sequences in the viral genome, some indels are not directly viral fire, are tolerant to editing and may even promote the emergence of highly virulent strains. DNA editing enzymes employed in CRISPR-active nucleic acid cleavage techniques, which are off-target or otherwise themselves nuclease active, are threatening to host/individual genome integrity and function, present a safety risk.

Drawings

FIG. 1 shows the structure and validation of a two-component CRISPR-nuPin system. Schematic of the crispr-nuPin system. Subcellular localization of NLS-dCS 9-NLS-emerin-Flag-T2A-GFP and emerin-Flag in transiently transfected HEK293T cells. Immunoblots were performed using anti-Flag, anti-GAPDH and anti-histone antibodies. C and D.dCAs9-emery cells were either doxycycline treated (+DOX) or non-doxycycline treated (-DOX), immunofluorescent stained with anti-Flag and anti-dCAs9 (NUP) antibodies, and intracellular distribution of dCAs 9-emery protein was examined. dCS 9-emery cells (+DOX) containing 170kb BAC plasmid were electroporated with BAC-targeted sgRNA (E, F), or dCS 9-emery cells (+DOX) were electroporated with telomere-targeted sgRNA (F, H). At the indicated time point after electroporation, cells were fixed, BACs or telomeres were stained with FISH and scanned under Nikon microscope (N-STORM). The upper layer of E and G is a representative Z-series image, and the lower layer of E and G is a three-dimensional reconstructed image. The percentage of BAC staining (F) or telomere staining (H) located at the periphery of the nucleus was calculated and plotted against the total amount of nuclear staining at each time point. The total count (n) for each time point is given at the bottom of the plot of F (BAC) and H (telomeres).

FIG. 2 shows that transcription of the HSV-1 genome located at the periphery of the nucleus is strongly inhibited. After 24 hours of transfection of the A-F.dCAs9-emerin cells with plasmids expressing HSV-1sgRNA or Ctr sgRNA, HSV-1 infection was performed with MOI values of 1 (A), as shown in the figure, MOI (B) or MOI values of 5 (C to F). A. The kinetics of single cycle growth of HSV-1 was studied by the virus plaque assay. B. Intracellular HSV-1 virus was titrated by the virus plaque assay over a period of 12 hours. C. Total RNA was extracted at 0, 0.17, 0.51, 2, 3, 5 and 9hpi and mRNA levels of ICP27, TK, ICP0 and VP16 were measured by qPCR (0.17 and 0.5hpi are not labeled). D. Viral protein levels of ICP0, ICP8, TK, VP16 at the indicated time points post infection were detected by immunoblotting. Beta-actin served as a loading control. E. ChIP detection was performed on dCas9-emerin cells expressing HSV-1sgRNA or Ctr sgRNA using antibodies specific for H3K4me3, H3K9ac, H3K9me3, H4K20me3, and H3K27me3 on the ICP27, ICP8, and VP16 promoters of 0.5hpi after infection with HSV-1. F. The ChIP assay was performed at indicated time points using antibodies against H3K9me3 on the heterochromosomes on the ICP27, ICP8 and VP16 promoters of HSV-1-or Ctr-sgRNA-expressing dCAS 9-emin cells infected with HSV-1. For E and F, the results are expressed in terms of enrichment (fold), i.e., comparing the portion of the viral promoter immunoprecipitated by a given antibody to the portion of GAPDH immunoprecipitated in the same reaction. G. dCAS9-emer cells expressing HSV-1sgRNA or Ctr sgRNA were treated with SBt for 5 hours and HSV-1 infection was performed with MOI value of 1, and mRNA levels of ICP27 were measured at the time points using qPCR.

Fig. 3 shows the general characteristics of the CRISPR-nuPin system. Schematic representation of DOX-induced CRISPR-nuPin expression cassette in dCAS9-emerin cells. The percentage of cells expressing dCas 9-emery in the dCas 9-emery cell line. Dmas 9-emerin cells were treated with DOX (+DOX) or DMSO (-DOX) and stained with antibodies to Flag (green) and dCas9 (red). C. dAS 9-emerin cells were treated with DOX at the indicated concentrations for 48 hours and examined for expression of dAS 9-emerin by immunoblotting. D. 1000 HEp-2 and dCAS9-emerin cells treated with DOX (+DOX) or DMSO (-DOX) were cultured in 96-well plates. Cell viability was tested using CCK-8 reagent for 96 hours. E, F. Expression of dCas9 protein in transiently transfected HEp-2 cells was confirmed by immunofluorescent staining (E) and immunoblotting of subcellular fraction (F) with anti-dCas 9 antibody. G, H. dmas 9-emery cells (+DOX) transfected with a 5kb plasmid were electroporated with plasmid-specific sgRNA. At the indicated time point after electroporation, cells were fixed, 5kb plasmids were stained using FISH technique and scanned under Nikon microscope (N-STORM). The upper layer of G is a representative Z-series image and the lower layer is a three-dimensional reconstructed image. The relationship between the plasmid staining at the periphery of the nucleus and the total number of nuclear staining at each time point was calculated and plotted. The total count (n) for each time point is given at the bottom of the plot of H.

FIG. 4 shows that nuclear peripherization of the HSV-1 viral genome inhibits viral replication. A. HEp-2 cells were infected with HSV-1 with a MOI value of 5. Cells were fixed at 0.5, 1, 1.5 and 2hpi, and the intranuclear HSV-1 genome was subjected to FISH staining and scanned under a Nikon microscope (N-STORM). The left panel is a representative Z-series image of 2hpi and its three-dimensional reconstruction showing only the surface of the DAPI stained area. On the right is a comparison of the statistical peri-nuclear HSV-1 viral genome to the total number of intra-nuclear viral genomes. N represents the number of counted viral genomes. B.5 HSV-1sgRNAs in the viral genome at a target sequence and nucleotide position. C. HSV-1 infection was performed on dCAS 9-emin cells expressing Ctr sgRNA or 5 HSV-1sgRNAs at MOI value of 5. The viral yield of 24hpi was quantified by the virus plaque assay. D. Stable GFP-expressing HEp-2 cell lines (GFP), dCAS9 and dCAS9-emerin cells were transfected with plasmids expressing HSV-1sgRNA2 or Ctr sgRNA for 24 hours and HSV-1 infection was performed with MOI value 1, and 24hpi of the cytovirus was titrated by plaque assay. E. dCAS9 and dCAS9-emer cells were transfected with plasmids expressing HSV-1sgRNA1, sgRNA2 or Ctr sgRNA for 24 hours and HSV-1 infection was performed with MOI value 1, and 24hpi of cytovirus was titrated by plaque assay. dCAS9 and dCAS9-emer cells were transfected with plasmid expressing HSV-1sgRNA2 for 24 hours and infected with HSV-1 virus (R8515) expressing recombinant GFP at MOI value 1. Cells were photographed at 24hpi, providing representative images. G. dCAS9-emerin cells were transfected with plasmid expressing Ctr sgRNA or HSV-1sgRNA2 for 24 hours, HSV-1 was infected with different MOI values, and mRNA levels of ICP27 were measured at the indicated time points after infection with qPCR.

FIG. 5 shows that HSV-1 viral genome in dCAS9-emerin cells is localized to the nuclear border after entry into the cell and replication is affected. HSV-1 infection was performed at MOI value 5 24 hours after transfection of HSV-1sgRNA or Ctr sgRNA expressing plasmids into A-B.dCAs9-emerin cells. At the indicated time points after infection, cells were fixed and stained with anti-ICP 8 (green) and anti-Flag (red) antibodies. A. Two representative images at each time point. White (light) arrows indicate cells with ICP8 diffusion (inefficient center of replication formation), red (dark) arrows indicate cells with ICP8 aggregation (center of replication formation). B. More than 200 nuclei per sample were counted and the formation efficiency of HSV-1 replicative centers (total number of nuclei aggregating ICP8 and ICP8 positive nuclei) was quantified and plotted. C. The fraction of viral DNA (ICP 27) (upper panel) or host genomic DNA (GAPDH) (lower panel) immunoprecipitated with the indicated antibodies was compared and mapped to the respective non-specific antibody (IgG) immunoprecipitated fraction.

FIG. 6 shows that ADV infection was performed at MOI value 5 24 hours after dCAS 9-emiin cells were transfected with plasmid expressing ADV sgRNA or Ctr sgRNA. After addition of ADV sgRNA to dCas9-emerin cells, replication of ADV virus was inhibited.

Detailed Description

The spirit and advantages of the present application will be further illustrated by the following examples, which are only intended to illustrate the application and not to limit it.

It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.

Example 1: materials and methods

(1) Plasmid and transfection

Emerin coding sequences were amplified from cDNA of HEp-2 cells. dCAS9 (D10A/N863A) was amplified from the lentismv 2 (Addgene, # 75112) backbone. The coding sequences for dCas9 and emerin were inserted into the pcdna3.1 vector for transient transfection. The elements in the expression vector included NLS (nuclear localization signal ) -dCS 9-NLS-emerin-Flag-T2A-GFP in order, using the CMV promoter.

Wherein, the primer sequences for amplifying Emerin are shown as SEQ ID No.1 and SEQ ID No. 2; the nucleotide sequence of Emerin is shown as SEQ ID No. 3; the primer sequences for amplifying dmas 9 are shown in SEQ ID No.4 and SEQ ID No. 5; the nucleotide sequence of dCAS9 is shown in SEQ ID No. 6.

NLS-dCS 9-NLS-emerin-Flag-T2A-GFP was cloned into the doxycycline-inducible lentiviral vector pCW57-MCS1-2A-MCS2 (from Deyin Guo professor laboratories, university of Zhongshan). Lentiviral packaging and transduction was performed in HEp-2 cells. The 170kb plasmid used in the experiment (BAC-HSV-1) was a Bacterial Artificial Chromosome (BAC) comprising HSV-1 (strain F) genomic sequence. The 5kb plasmid used in the experiment was a modified pcDNA3.1 vector. All constructs were verified by sequencing.

Wherein the nucleotide sequence of NLS-dCS 9-NLS-emerin-Flag-T2A-GFP is shown as SEQ ID No. 7.

The N-terminal nucleotide sequence of the NLS is shown as SEQ ID No. 8; the C-terminal nucleotide sequence is shown as SEQ ID No. 9.

Transient transfection of HEK293T cells used standard polyethylene glycol (PEI) (Sigma, # 408727). For transfection of HEp-2 cells and other mammalian cell lines, jetprime (Polyplus, # 101000046) was used according to the direction of the supplier.

(2) Cells and viruses

HEp-2, HEK293T, U2OS and Vero cells were cultured as indicated by ATCC. HEP-2, HEK293T and Vero cells were cultured in Dulbecco's modified Eagle's Medium (DMEM, CORNING, 10013074) supplemented with 10% fetal bovine serum (FBS, gibco,42G 4086K). U2OS cells were cultured in McCoy's 5A (Procell, PM 150710) supplemented with 10% Fetal Bovine Serum (FBS). The dCAS9-Emerin expression cell line using the prokaryotic regulatory element Tet-on was prepared by lentiviral transduction of HEp-2 cells, screening for stable cell lines with puromycin (1 ug/ml) (Gibco, #A 1113803), inducing expression with Doxycycline (DOX) at 3ug/ml, and screening for high GFP expressing cells at 488nm by MoFLO Astrios EQ (Beckman Coulter Life Sciences). dCAS9-Emerin expressing cells were treated with DOX for at least 3 days prior to HSV-1 infection experiments.

HSV-1 (F) and recombinant HSV-1 (R8515) virus carrying GFP gene were prepared in HEp-2 cells and titrated in Vero cells by the virus plaque assay. Recombinant HSV-1 deleted for ICP0 gene (ΔICP0) was amplified in U2OS cells and titrated by a virus plaque assay. Viruses were stored as aliquots for one use at-80 ℃.

Except for the virus plaque assay and virus amplification, all infections were cells infected with HSV-1 virus at the required MOI for 2 hours on ice (referred to as 0 hpi). Infected cells were cultured in DMEM supplemented with 1% fbs at 37 ℃. Infected cells were harvested at the indicated time points, frozen and thawed three times, sonicated at 20% amp for 7 seconds, and titrated in Vero cells to determine HSV-1 titer.

(3) Lentivirus production

To produce lentiviruses, HEK293T cells were added to 60mm plates and transfected with packaging plasmids pCMV-dR8.91 and VSV-G, and either the dCAS9-Emerin-flag-T2A-GFP construct or a control vector in appropriate proportions using Polyethylenimine (PEI) (Sigma, 408727) as transfection reagent. The medium was changed 6-8 hours after transfection. 48 hours after transfection, the supernatant was filtered through a 0.45 μm filter and centrifuged at 1000g for 10 minutes to remove cell debris and lentiviruses were collected. The supernatant was added directly to the cells or stored frozen at-80 ℃.

(4) Fluorescence In Situ Hybridization (FISH)

HSV-1DNAFISH probes were prepared from BAC-HSV-1 by nick translation (Exon biotechnology, # 21076) using Cy 3-labeled dNTPs. A5 kb plasmid DNA FISH probe was prepared from this plasmid in a similar manner. According to the manufacturer's method, 2. Mu.g of template DNA was mixed with Cy 3-labeled dNTPs, nick translation buffer, nuclease-free water and nick translation enzyme. The mixture was then incubated at 15℃for 1-5 hours. The labeled probe is precipitated and redissolved in water without the nuclease. Telomeric FISH probes were purchased from PNA (TelG-Alexa 488, #f1008). The method of hybridization briefly comprises: cells were fixed with 4% paraformaldehyde (Sigma, #P6148) at Room Temperature (RT) for 15 min, incubated with 0.1% Tris-HCl pH7.0 for 10 min, pre-permeabilized with 0.8% Triton (Sigma, #T8787) in PBS for 10 min, incubated with 20% glycerol (Sangon Biotech, # A600232-0500) in PBS for 20 min, permeabilized with 0.8% Triton PBS for 30 min, treated with RNase A (Omega, # L15 UM) for 50 min at 37℃and incubated with 50% formamide (VETEC, # V900064) in 2 XSSC buffer (0.3M NaCl,30mM sodium citrate) for 10 min, washed with PBS between the two steps. Cells were incubated with probes diluted to 20 nanograms/milliliter in hybridization solution (2 XSSC, 50% formamide, 10% dextran sulfate (Sigma, #D8906), 1% Triton) for 10 minutes at 85℃and 20 hours at 37 ℃. After hybridization, cells were washed sequentially with PBS containing 75%, 50% and 25% wash buffer (2 XSSC, 70% formamide, 10% dextran sulfate).

For FISH and immunofluorescent staining, the cells were further processed and stained as described in the immunofluorescent staining section.

(5) Quantitative real-time PCR

Total RNA was extracted using E.Z.N.A Total RNA Kit I (OMEGA, # R6834-02) and cDNA was synthesized using Evo M-MLV RT Kit (exact biology, # AG 11603). Total DNA was isolated using a viral DNA kit (OMEGA, #D3892-01). qPCR was performed on the StepOneTM system (ThermoFisher) using the Sybrgreen detection system (exact biology, # AG 11701). GAPDH was used as a loading control.

The nucleotide sequences of the primers used are shown in SEQ ID No.10 to SEQ ID No.31, and the specific steps are as follows:

ICP0 forward, 5'-ACTGCCTGCCCATCCTGGACA-3';

ICP0 reversal, 5'-CCATGTTTCCCGTCTGGTCC-3';

ICP27 forward, 5'-CGGGCCTGATCGAAATCCTA-3';

ICP27 reversal, 5'-GACACGACTCGAACACTCCT-3';

TK forward, 5'-CCAAAGAGGTGCGGGAGTTT-3';

TK reverse, 5'-CTTAACAGCGTCAACAGCGTGCCG-3';

VP16 forward, 5'-CCATTCCACCACATCGCT-3';

VP16 reverse, 5'-GAGGATTTGTTTTCGGCGTT-3';

us1 forward direction, 5'-ATCAGCTGTTTCGGGTCCTG-3';

us1 in reverse, 5'-TCGGCAGTATCCCATCAGGT-3';

us12 forward direction, 5'-AACGCACCAAACAGATGCAG-3';

us12 in reverse, 5'-CGTCCAAACCCACCGACATA-3';

Human GAPDH is positive, 5'-GAAGGTGAAGGTCGGAGTC-3';

human GAPDH reverse, 5'-GAAGATGGTGATGGGATTTC-3';

the ICP27 promoter is forward, 5'-CCGCCGGCCTGGATGTGACG-3';

the ICP27 promoter was reversed, 5'-CGTGGTGGCCGGGGTGCTC-3';

ICP8 promoter forward, 5'-CCACGCCCACCGGCTGATGAC-3';

the ICP8 promoter is reversed, 5' -TGCTTACGGTCAGGTGCTCCG-3;

the VP16 promoter is forward, 5'-GCCGCCGTACCTCGTGAC-3';

the VP16 promoter is reversed, 5'-CAGCCCGCTCCGCTTCTCG-3';

GAPDH promoter forward, 5'-TTCGACAGTCAGCCGCATCTTCTT-3';

the GAPDH promoter was inverted, 5'-CAGGCGCCCAATACGACCAAATC-3'.

(6) Cell viability assay

Cell viability assays were performed using Cell Counting Kit-8 (CCK-8, bimake, #B 34302). Fluorescence intensity was measured on a Synergy H1 microplate reader (Biotek) at a wavelength of 450 nm. Wells containing medium alone served as controls.

(7) Subcellular fractionation

Subcellular fractionation was performed according to the method described in Xu, P.and B.Roizman, proc Natl Acad Sci USA,2017.114 (19): p.E 3823-E3829. Briefly, 1X 10 was collected ⁶ Individual cells were centrifuged at 1000g for 5 min and resuspended in 0.3ml ice-cold buffer 1 (150mM NaCl,50mM HEPES[pH 7.4)]25. Mu.g/ml digoxin, 10. Mu.l/ml protease inhibitor), incubated at 4℃for 30 minutes, and then centrifuged at 4600rpm for 5 minutes. Collecting supernatant, washing and re-suspending the particles at 0.3m Ice-cold buffer 2 (150mM NaCl,50mM HEPES[pH 7.4)],1％[vol/vol]NP-40, 10. Mu.l/ml protease inhibitor) was incubated on ice for 30 min. The samples were centrifuged at 8,700rpm for 5 minutes to pellet the nuclei, and the supernatant was collected and combined with the previous collection to represent the cell fluid. The particles were washed and resuspended in 0.2 ml ice-cold buffer 3 (150mM NaCl,50mM HEPES[pH7.4)]，0.5％[wt/vol]Sodium deoxycholate, 0.5% [ wt/vol ]]SDS,1mM DTT, 10. Mu.l/ml protease inhibitor) was kept on ice for 30 min, followed by sonication (10 s,20% magnification). The final solution contains nuclear extract.

(8) immunoWestern blotting

Lysates were separated on polyacrylamide gels, transferred to PVDF membranes and immunoblotted with the indicated antibodies. Antibodies used include: a mouse monoclonal anti-Flag antibody (Abways, #ab 0008), a mouse monoclonal anti-ICP 0 antibody (Santa, #sc-53070), a mouse monoclonal anti-ICP 8 antibody (Abcam, #ab 20194), a mouse monoclonal anti-VP 16 antibody (Santa, #sc-7545), a mouse monoclonal anti- β -actin antibody (Sino Biological, # 1000166), an anti-TK antibody, a mouse monoclonal anti-gapdh antibody (Abways, #ab 0037), a mouse monoclonal anti-Histone antibody (Sino Biological, # 100005). Rabbit polyclonal anti-dCas 9 antibody (abclon, # a 14997), goat anti-mouse IgG-HRP (Invitrogen, # 31430), goat anti-rabbit IgG (h+l) -HRP (Invitrogen, # 32460).

(9) Immunofluorescent staining

Cells were fixed overnight with methanol at-80℃and then permeabilized and reacted with PBS-TBH (0.1% Triton X-100in1XPBS,10%FBS,and 1%BSA (Sigma, #WXBD 5147V)), with primary antibodies overnight at 4℃or within 2 hours of RT, with secondary antibodies Alexa-Fluor-594-conjugated goat anti-rabit (Invitrogen, #A11012) or Alexa-Fluor-488-conjugated goat anti-mouse (Invitrogen, #A 32723) in the dark at RT for 30 minutes. Imaging was performed with a Zeiss confocal microscope (ZEISS imager.Z20).

(10) sgRNA design

HSV-1 (F) strain genome information was downloaded from GenBank (accession number: GU 734771.1). The sgRNAs were designed using Cistrome (http:// Cistrome. Org/SSC /). The control sgRNA (5'-GGGGTAGGCGGAGCCTCAGG-3', SEQ ID No. 32) had no known target sequence in the human genome and HSV-1 genome.

(11) In vitro transcription

In vitro sgRNA transcription was performed using the Ribomax T7 large-scale RNA production kit (Promega, #P1300). Briefly, 2 to 4. Mu.g of template DNA containing the T7 promoter was used for each reaction. The in vitro transcription mixture was incubated at 37℃for 3.5 hours and RNase-free DNase I was added to remove the DNA template. Transcribed RNA was treated with alkaline phosphatase (Thermo, # 01137175) for 1 hour and cleaned with E.Z.N.A.miRNA kit (Omega, # R6842-01). The final RNA was quantified using a Nanodrop spectrophotometer and sub-packaged and stored at-80 ℃.

(12) Confocal microscope and image processing

Cell samples were imaged with a Nikon microscope (N-STORM) and 100X lens. Images were processed using NIS Elements software, three-dimensional visualization and three-dimensional reorganization by time-lapse microscopy with Z-stack and Imaris software (Bitplane). DNA FISH Spots were established using Spots function, the surface of the nucleus was established as a membrane pattern using cell function, and then switched to a surface pattern. The number of plasmids or HSV-BACs on the nuclear envelope was quantified by using the reconstituted plasmids or HSV-BAC spots and the nuclear peripheral surface.

(13) Chromatin co-immunoprecipitation (ChIP)

The method of chromatin co-immunoprecipitation detection is briefly as follows: at the indicated time points after infection, cells were treated with 1% paraformaldehyde (Sigma, #p6148) for 10 min, washed twice with PBS, resuspended in Sodium Dodecyl Sulfate (SDS) lysis buffer (1% SDS 10mM EDTA 50mM Tris,pH 8.1) containing protease inhibitors (Thermo Scientific, #eo 0492) and incubated on ice for 20 min. The cell lysate was sonicated in a 20 second pulse manner for 4 minutes to give DNA fragments of 200 to 500bp in length and further centrifuged at 13,000g for 10 minutes at 4 ℃. The supernatant was collected and assayed by phosphate buffered radioimmunochromatography assay buffer (0.1% SDS,1% sodium deoxycholate, 150mM NaCl,10mM Na) ₂ PO ₄ 2mM EDTA,1% NP-40) was diluted 10-fold,pre-cleaned at 4 ℃. At this time, 1% of the total volume was withdrawn as input. The following antibodies were used for overnight co-immunoprecipitation at 4 ℃): mouse immunoglobulin G (Cell Signaling Technology, 5415S) or an anti-histone antibody (anti-H3K 4me3 antibody (Abcam, # ab 8580), anti-H3K 9me3 antibody (Abcam, # ab 8898), anti-H3K 27me3 antibody (Abcam, # ab 6002), anti-H4K 20me3 antibody (Abcam, # ab 9053), anti-H3K 4AC antibody (Abcam, # ab 4441)). The immunocomplexes were collected by spin incubation with protein A/G beads (Santa, #sc-2003) at 4℃for 1 hour, washed sequentially with low salt wash buffer (150mM NaCl,20mM Tris HCl,pH 8.1,2mM EDTA,1% Triton X-100 and 0.1% SDS), high salt wash buffer (500mM NaCl,20mM Tris HCl,pH 8.1,2mM EDTA,1%Triton X-100 and 0.1% SDS), lithium chloride wash buffer (0.25M LiCl,1% NP-40,1% sodium deoxycholate, 1mM EDTA and 10mM Tris-HCl, pH 8.1) and Tris-EDTA buffer (10 mM Tris-HCl, pH 8,1mM EDTA), and with elution buffer (1% SDS,0.1M NaHCO) ₃ ) Incubation was performed for 10 min at RT, 10 min at 65℃and finally 10 min at RT. All samples were then treated with RNase A (Omega, #D10SG) and proteinase K (QIAGEN, # 160016374) and total DNA was purified using E.Z.N.A. gel extraction kit (Omega, #D2500-02) as qPCR template.

The amount of input and the percentage of immunoprecipitated DNA were measured by quantitative PCR using specific primers for viral promoters and cellular GAPDH pseudogenes. Prior to further comparison, the fraction of virus or host DNA that was immunoprecipitated with the relevant antibody was normalized to its input value.

(14) Statistical analysis

In this study, each experiment was repeated at least three times unless otherwise indicated. Data are expressed as mean ± sd calculated by GraphPad prism6.0 software. Two-tailed unpaired student t-test, a common one-factor or two-factor anova was used to calculate the P-values indicated in each plot. N.S it is not significant that P >0.05, "represents P.ltoreq.0.05," "represents P.ltoreq.0.01," "represents P.ltoreq.0.001, and" "represents P.ltoreq.0.0001.

Example 2: establishment of a two-component CRISPR-nuPin system mediating immediate nuclear marginalization of extrachromosomal DNA

The life cycle of many DNA viruses is in hours. In the exemplary inventive protocol presented in this example, to rapidly relocate the viral genome to the periphery of the host cell nucleus, the inventors designed a two-component system (hereinafter referred to as CRISPR-nuPin system): dcas9, which anchors the peripheral region of the nucleus within the nucleus, and sgrnas that specifically recognize and bind to the virus of interest. The platform is capable of mediating the inducible migration of viral DNA genome within the nucleus, particularly to the periphery of the nucleus (fig. 1A).

To achieve peripheral migration and ligation, flag-emerin-TEV (TEV protease recognition sequence) -GFP was fused to the C-terminus of Streptococcus pyogenes (Streptococcus pyogenes) dCAS9 (D10A & H840A) to form a 230kDa fusion protein (dCAS 9-emerin) (FIG. 3A). A nuclear localization signal (nuclear localization signal, NLS) was inserted at the N-and C-termini of dCS 9 to allow dCS 9-emiin to enter the nucleus. Additionally constructed Flag-tagged Emerin, which does not have NLS and dCas9 fragments. The experimental results show that dCas9-Emerin with NLS signal is localized in the nucleus, whereas Emerin without NLS is localized in the nucleus and cytoplasm (fig. 1B and 3A).

HEp-2 cell lines (dCS 9-emerin cells) expressing dCS 9-emerin were then induced by Doxycycline (DOX) using a lentiviral transduction system. A HEp-2 cell line expressing only the dCas9 protein was also established as a control cell line (dCAs 9 cells). It was confirmed by immunoblotting that dCas9-emerin fusion protein in dCas9-emerin cells was successfully induced by addition of DOX: co-localization of anti-Flag and anti-dCAS 9 antibodies along the DAPI-stained nuclear periphery under immunofluorescence microscopy indicated that the dCas9-emerin fusion protein was correctly localized on the nuclear envelope (endosomes) (FIGS. 1C, D, 3B, C). DOX treatment produced no detectable cytotoxicity on HEp-2 cells (FIG. 3D).

To test the time efficiency of this platform, dCAS9-emerin cells were first electroporated with either a 5kb plasmid (which contains the targeting sequence for sgRNA-2 described below) (FIGS. 3G, H) or 170kb BAC DNA (FIGS. 1E, F), and again with either their targeting sgRNA (sgRNA-2:5 '-GCGGTGGTGTGTGACAGGAG-3', SEQ ID No. 34) or control sgRNA (5'-GGGGTAGGCGGAGCCTCAGG-3', SEQ ID No. 32) 24 hours after the initial electroporation. Fig. 1E shows a representative three-dimensional image of the nucleus at a given time point after sgRNA electroporation (upper panel) and its three-dimensional reconstructed image (lower panel). Each sample group was counted for more than 500 spots and their distribution pattern at the nuclear margin or in the nuclear space (inner nuclear space) was plotted. A FISH-stained spot is classified as a spot located at the edge of the nucleus when it is partially exposed outside the DAPI region or fully exposed but still adjacent to the DAPI region after three-dimensional reconstruction. When a FISH stained spot is fully contained in the DAPI region, it is counted as a spot located in the nuclear space. FISH spots that are not connected to the DAPI region are rejected during the counting process. As shown in FIG. 1F, 10% -20% of BAC DNA was relocated to the nuclear border starting from the nuclear space 10 seconds after the electroporation of sgRNA, and more than 80% of BAC DNA was relocated to the nuclear border after 3 minutes of the electroporation of sgRNA, while the introduction of ctrl sgRNA had little detectable effect.

To test the efficiency of CRISPR-nuPin repositioning of host genomic sites, dCas9-emerin cells were electroporated with sgrnas directed against the telomere region (fig. 1g, h). More than 38% of the cell telomeres were detected to bind to the periphery of the nucleus 3 hours after sgRNA induction, whereas at 0 hours about 25%; ctrl sgRNA was about 21%. The results indicate that the CRISPR-nuPin system is capable of transiently relocating extrachromosomal DNA up to 170kb in size to the periphery of the nucleus. Note that a point in the three-dimensional reconstructed image represents a single or multiple plasmids or BACs.

Example 3: replication of HSV-1 located at the periphery of the nucleus is inhibited

To initiate replication, DNA viruses require injection of their genetic material into the host cell nucleus and initiation of transcription of viral genes. A single viral genome encounters a highly heterogeneous microenvironment upon entry into the nucleus. In fact, from a spatial localization point of view, during natural infection, approximately 25% of the HSV-1 genome localizes at the edge of the endonucleomembrane, and the remaining viral DNA, although penetrating further into the interior of the nucleus, remains near the periphery of the nucleus (FIG. 4A). The inventors have designed 5 sgRNAs for different numbers of targeting sites within the HSV-1 genome in the CRIPSR-nuPin system to mediate nuclear peripherization of the HSV-1 genome in an effort to investigate whether viral DNA replicates preferentially to the intranuclear space rather than the nuclear border. The targeting site bypasses the protein coding region of the virus (see figure 2B). All sgRNAs produced a significant inhibition of HSV-1 in dCas9-emerin cells (fig. 4C). Since sgRNA-2 (5'-GCGGTGGTGTGTGACAGGAG-3', SEQ ID No. 34) binds to a site in the region encoding the 8.3kb Latency Associated Transcript (LAT) precursor of HSV-1 that does not play an important role in HSV-1 cell replication and the other binding site is in the terminal repeat short region (TRs), it was used to mediate the relocation of the HSV-1 genome in the following experiments of this study.

To exclude the effects of non-specific factors such as binding of dCas 9-sgrnas to viral genomes or an sgRNA-induced innate immune response, HEp-2 cells expressing GFP, dCas9 or dCas9-emerin fusion proteins were transfected with plasmids encoding non-HSV-1 targeted sgRNA (Ctr sgRNA) or HSV-1 targeted sgRNA2 (HSV-1 sgRNA). These cells were not substantially different in supporting HSV-1 replication except for cells expressing dCAS9-emerin fusion protein and HSV-1sgRNA (FIGS. 4D, E, F). The kinetics of single cycle growth of HSV-1 infected dCAS9-emerin cells in the presence of Ctr sgRNA or HSV-1sgRNA in an amount of MOI value 1 was measured. In dCAS9-emerin cells expressing HSV-1sgRNA, the viral genome was directed to the periphery of the nucleus compared to the Ctr sgRNA group (FIG. 2A); binding of the viral genome to the endonucleomembrane resulted in reduced viral yield at all time points (including plateau phase) (fig. 2A). The platform provided by the present application maintained viral inhibition even though the fold of infection was increased to 20 infectious viruses per cell (fig. 2B, fig. 4G).

Example 4: HSV-1 genome located at the periphery of the nucleus is subject to transcriptional repression

HSV-1 transcribes three sets of viral genes in chronological order. In host cells in which the viral genome is located around the nucleus, the mRNA levels of these three groups of representative viral genes drop sharply to a significantly lower basal level before transcription of these viral genes is fully activated. Although the initiation of full-speed transcription (full-speed transcription) of the representative alpha gene ICP27 of HSV-1 was delayed by 30 minutes in HSV-1sgRNA transfected dCAS9-emerin cells compared to the control, the transcription rate was also at a comparable level after initiation. The total amount of ICP27 mRNA synthesized by HSV-1 located to the periphery of the nucleus was 10-fold lower than HSV-1 freely infecting the nucleus (FIG. 2C). Representative beta (TK) and gamma (VP 16) genes also have similar patterns, with slightly longer delay times for full-speed transcription initiation (FIG. 2C). Delayed transcription initiation resulted in delayed accumulation of viral proteins such as ICP8, IC P0, VP16, and TK (FIG. 2D), and inefficient formation of replication centers (FIGS. 5A-B).

Observations from this set of experiments indicate that deposition of the HSV-1 genome in the perinuclear region results in reduced initial basal transcription and delayed full-speed transcription initiation of the viral gene cascade, severely delaying viral protein accumulation, ultimately resulting in inefficient replication of the virus.

The periphery of the nucleus is known as a region with heterochromatin distribution. To investigate whether the viral genome around the nucleus is subject to histone-mediated transcriptional inhibition, the inventors performed ChIP-qPCR to quantify the activity of the HSV-1 a, β and γ gene promoter regions and the associated levels of the inhibitory modified histones. For viral genomes located around the nucleus, the association of promoters of representative viral genes with active histones (H3K 4me3 and H3K9 ac) was significantly reduced and the association with inhibitory histones (H4K 20me3, H3K9me3 and H3K27me 3) increased compared to those of non-manipulated viral DNA within the nucleus (fig. 2E). Furthermore, the inhibitory histone (represented by H3K9me 3) packaging of the viral genome around the nucleus gradually increased over time during the first hour of infection (fig. 2F). Treatment with sodium butyrate (SBt) whole cell inhibited Histone Deacetylase (HDAC) activity, alleviating to some extent the transcriptional inhibition of the perinuclear viral genome, but did not completely counteract the adverse effect of the perinuclear on HSV-1 transcription (fig. 2G).

Example 5: replication of adenovirus localized around the nucleus is inhibited

It is further verified that the CRISPR-nuPin system provided by the application, which mediates the migration of viral DNA genome in the nucleus to the periphery of the nucleus, can also inhibit the replication of adenovirus.

1. Adenovirus test sample preparation:

a recombinant adenovirus was constructed using Agilent company AdEasy XL Adenoviral System, pShuttle-CMV as the shuttle plasmid, and pAderasy-1 (deleted E1 and E3 genes) as the backbone plasmid.

Wherein a viral shuttle plasmid pShuttle-CMV-mCherry (comprising mCherry fluorescent protein coding sequence) was constructed by molecular cloning, and a PmeI linearized adenovirus shuttle plasmid was used to add the linearized adenovirus shuttle plasmid to an electrocompetent cell (AdEasier-1 cell). Resuscitates after the electric rotation. Colony PCR primers (Adeno F1: ACTGATTATGACACGCAT, SEQ ID No.38; adeno R1: CGTGCAATCCATCTTGTT, SEQ ID No. 39) were designed on backbone plasmids to identify the desired colonies. Extracting pAderasy-1 plasmid by alkaline lysis method, and then carrying out enzyme digestion with PacI for identification: the pAderasy-1 plasmid should be digested with a large fragment (approximately 30 kb) and a small fragment of 3kb or 4.5 kb.

The recombinant adenovirus plasmid obtained was reconverted into NEB stable 3 competent cells.

Recovery of linearized large fragment vector: pacI single enzyme digestion linearizes the recombinant adenovirus and then makes DNA purification.

Rescue of recombinant adenovirus: 293A cells were transfected with the recovered linearized large fragment vector. Rescue, amplification and identification of recombinant adenovirus are carried out.

Recombinant adenoviruses (carrying mCherry fluorescence) were titered in 293T cells by the following fluorescence method.

Adenovirus fluorescent assay for titer

Well-grown 293T cells were digested and counted and diluted to 1X 10 ⁵ Per ml, 96-well plates, 100 μl/well, were added and 10 wells were prepared for each virus. Placing at 37deg.C, 5% CO ₂ Culturing in an incubator.

The next day, 10-fold gradient dilutions were made in EP tubes of adenovirus stock to be tested, 10 dilutions in succession. The dilution method is as follows: 10 tubes of EP 1.5ml were prepared for each virus, 90. Mu.l of culture medium was added to each tube, 10. Mu.l of virus stock was added to the first tube, and after mixing, 10. Mu.l was aspirated and added to the second tube for mixing. In this wayBy analogy, ten dilutions (10 to 10 ^-8 ). Sucking the original culture medium in the 96-well plate, and adding diluted virus liquid. And marking.

On the third day, 100. Mu.l of complete culture solution was added to each well to facilitate cell growth.

The results were observed on the fifth day and titers were calculated: the results were observed under a fluorescence microscope and the number of fluorescent cell clones in 96 wells in the last two fluorescent dilution gradients were counted. Assuming X (number of fluorescent cells at last dilution) and Y (number of fluorescent cells at penultimate dilution), the titers (TU/ml) = (X+Y X10) ×1000/2/content of virus solution (μl) in X wells. TU/ml refers to the number of bioactive viral particles contained per milliliter.

2. CRISPR-nuPin system for adenovirus genome

CRISPR-nuPin platforms for adenovirus inhibition were prepared using the methods as disclosed in examples 2 and 3.

Wherein, the pcDNA3.1 plasmid encoding NLS-dCS 9-NLS-emerin-Flag-T2A-GFP is constructed by adopting the method disclosed in the example 2, and the plasmid encoding sgRNA are transfected in 293T cells, so that the positive expression efficiency can reach more than 80%. Adenovirus infection experiments in 3 were performed 24 hours after transfection.

In addition, sgrnas were designed for adenovirus genome sequences: 5'-CTGTTGTATGTATCCAGCGG-3' (SEQ ID No. 40); control sgrnas were designed: 5'-GGGGTAGGCGGAGCCTCAGG-3' (SEQ ID No. 32). The control sgrnas have no known target sequences in the human genome and ADV genome.

3. Intracellular adenovirus inhibition assay

The effect and effect of CRISPR-nuPin with sgRNA against ADV genome on ADV was observed and tested using the method as described in example 3.

293T cells expressing dCAS9-emerin under Dox induction were transfected with plasmids expressing ADV sgRNA or control sgRNA. ADV infection was performed 24 hours after transfection with moi=20, and viral DNA was collected from cells 0, 12, 24 and 48 hours after infection, respectively. It was observed by fluorescence that in dCas9-emerin cells expressing ADV sgrnas, the viral genome was directed to the periphery of the nucleus compared to the control sgrnas. In addition, the adenovirus genome content in the samples was quantified by real-time fluorescent quantitative PCR (qPCR primer information, for adenovirus E4 gene: forward primer: 5'-CTAACCAGCGTAGCCCCGA-3' (SEQ ID No. 41), reverse primer: 5'-TGAGCAGCACCTTGCATTTT-3' (SEQ ID No. 42), and adenovirus DNA content in the samples at 12, 24 and 48 hours was compared with the adenovirus original infection amount at 0 hour. The results are shown in FIG. 6, in which it was seen that replication of ADV virus was inhibited after ADV sgRNA was added to dCAS9-emerin cells.

The foregoing is illustrative of the present application and is not to be construed as limiting thereof. The practice of the application will employ, unless otherwise indicated, conventional techniques of organic chemistry, polymer chemistry, biotechnology, and the like, it being apparent that the application may be practiced otherwise than as specifically described in the foregoing description and examples. Other aspects and modifications within the scope of the application will be apparent to those skilled in the art to which the application pertains. Many modifications and variations are possible in light of the teachings of the application and, thus, are within the scope of the application.

Claims (11)

1. A method of inhibiting a virus in a subject comprising administering to the subject a system that binds to a viral nucleic acid sequence and localizes the virus to a region of nuclear repressor gene expression, wherein the region of nuclear repressor gene expression is the periphery of the nucleus (nuclear periphery),

the system has:

a chimeric polypeptide comprising a localization domain (localization domain) and a nucleic acid binding domain (binding domain), wherein the localization domain is a polypeptide that localizes to a region within the nucleus that inhibits gene expression, and the nucleic acid binding domain is a binding moiety that specifically recognizes and binds to a viral nucleic acid sequence.

2. The method according to claim 1, wherein the viral nucleic acid is DNA, such as genomic nucleic acid of a virus, or DNA in a viral replication cycle.

3. The method according to claim 1, wherein the region of nuclear repressor gene expression is the nuclear envelope, e.g. the layer of the nuclear envelope facing the nuclear space.

4. The method of claim 1, wherein the nucleic acid binding domain comprises a Cas protein, a Zinc Finger Nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a Argonaute (Ago) protein.

5. The method of claim 4, wherein the nucleic acid binding domain comprises a Cas protein,

preferably, wherein the Cas protein substantially lacks DNA cleavage activity;

and optionally, wherein the system comprises a small guide RNA complexed with the Cas protein, the small guide RNA hybridizing to a viral nucleic acid sequence of interest.

6. The method of claim 1, wherein the localization domain comprises a perinuclear specific protein such as Emerin, lap2β, lamin a or lamin B, or a lamin-related protein such as lamin.

7. The method of claim 1, wherein the virus is a DNA virus or a retrovirus, such as an adenovirus, a poxvirus, a papovavirus, a herpes virus or human immunodeficiency virus 1 (HIV-1).

8. A system for specifically recognizing and binding viral nucleic acid sequences and localizing viral nucleic acids to a region of nuclear repression of gene expression, as defined in any of claims 1-7,

optionally, the system is for inhibiting a virus in a subject.

9. An isolated nucleic acid for encoding a system which specifically recognizes and binds a viral polypeptide or nucleic acid sequence and localizes the virus to a region of the nucleus in which gene expression is inhibited, said system being as defined in any one of claims 1 to 7,

the nucleic acid comprises a nucleotide sequence encoding a chimeric polypeptide comprising a localization domain, which is a polypeptide localized in a region of the nucleus that inhibits gene expression, and a nucleic acid binding domain, which is a binding moiety that specifically recognizes and binds to a viral nucleic acid sequence.

10. A pharmaceutical composition comprising a system as defined in any one of claims 1 to 8 which specifically recognizes and binds a viral polypeptide or nucleic acid sequence and localizes the virus to a region of the nucleus in which gene expression is inhibited, or a nucleic acid as defined in claim 9, together with a pharmaceutically acceptable carrier or excipient,

Optionally, the pharmaceutical composition is for inhibiting a virus in a subject.

11. A method for preventing or treating a virus-related disease, wherein a system is administered to the subject that specifically recognizes and binds to a viral polypeptide or nucleic acid sequence and localizes the virus to a region of nuclear repressor gene expression, said system being as defined in any of claims 1-8,

preferably, wherein the virus is a virus whose viral genome has DNA or DNA occurs during the replication cycle, more preferably wherein the virus is a DNA virus or retrovirus, including adenovirus, poxvirus, papovavirus, herpesvirus or human immunodeficiency virus 1 (HIV-1), such as human herpes simplex virus type 1 (HSV-1), human herpes simplex virus type 2 (HSV-2), cytomegalovirus (CMV), varicella Zoster (VZV), human herpesvirus-4 (Human herpesvirus, HHV-4) also known as Epstein-Barr virus (EBV) or EBV, human herpesvirus-6 (HHV 6), human herpesvirus-7 (HHV 7), human herpesvirus-8 (HHV 8), pseudorabies virus, hepatitis B Virus (HBV), human papilloma virus (Human Papillomavirus, HPV), adenovirus _， Human immunodeficiencyVirus 1 (HIV-1), etc.;

preferably, wherein the disease is selected from hepatitis b, oral herpes, herpetic keratitis, herpetic pannicus, traumatic herpes, herpetic eczema, neonatal herpes, genital herpes, atypical genital herpes, herpetic cervicitis, herpetic proctitis, herpetic encephalitis, herpetic meningitis, herpetic meningoeencephalitis, disseminated herpes simplex infection, alzheimer's disease and dementia, infectious mononucleosis, chronic active epstein barr virus infection, epstein barr virus-related nasopharyngeal carcinoma, epstein barr virus-related lymphoma, aids and HPV-related cervical cancer.