JP2019517503A - Negative feedback regulation of HIV-1 by gene editing strategies - Google Patents

Abstract

A CRISPR-endonuclease gene editing composition comprising gRNA for targeting a specific viral sequence for cleavage by an endonuclease that introduces breaks into double stranded DNA recognized by guide RNA (gRNA). For example, placing the gene encoding Cas9 under the control of a minimal promoter spanning the 5'-LTR of HIV results in activation by the HIV-1 transactivator protein Tat. For example, coexpression of both HIV specific multiple gRNAs and endonucleases (eg Cas9) in cells results in modification and / or excision of viral DNA segments, resulting in eradication of the virus in vitro and in vivo Led. [Selected figure] Figure 1A

Description

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant Nos. P30 MH092177 (Khalili), P01DA037830 (Khalili), R01 MH 092371 (Khalili), and R01 NS 087971 (Khalili and Hu) awarded by National Institutes of Health. It was done. The government has certain rights in the invention.

  Embodiments of the present invention relate to gene editing complexes in the prevention, treatment and eradication of retroviral infection in a subject. In particular, the gene editing complex is clustered and regularly arranged short palindromic repeats under the control of a minimal viral promoter conditionally activated by a viral transcription regulator. Contains the endonucleases associated with Repeats, CRISPR).

Immediately after HIV-1 infection, the viral genome integrates into the host chromosome and is rapidly expressed in CD4 ⁺ T cells. HIV-1 replication leads to a marked reduction of CD4 ⁺ T cells (Alimonti, JB, et al. J. Gen. Virol. 84, 1649-1661 (2003), Okoye, AA, Picker, LJ Immunol Rev 254, 54 -64 (2013)). Often, after the acute infection phase, the virus enters a new phase called latency, the integrated proviral DNA continues to be expressed, and viral replication proceeds at very low levels. Under these circumstances, persistent viral replication weakens the weakened immune system to the development of AIDS and a wide range of opportunistic infections, and in the absence of treatment results in death within 3 years (3). At the molecular level, expression of the viral genome in both the acute and asymptomatic phase and its replication is controlled by a viral promoter that spans 450 nucleotides of the 5'-end repeat (LTR) (Garcia, JA, et al. EMBO J. 8, 765-778 (1989), Reddy, EP, Dasgupta, P. Pathobiology 60, 219-224 (1992)). A series of cellular transcription factors that recognize DNA sequences within the U3 region of the 5'-LTR and HIV-1 immediate early transcription activator (Tat) that interacts with TAR RNA sequences located within the viral leader region There is a co-operation between These interactions are required for strong initiation and efficient elongation in transcription from integrated copies of viral DNA (Marcello, A., et al. IUBMB Life 51, 175-181 (2001) Roebuck, KA et al, Gene Expr 8, 67-84 (1999)). Although current antiretroviral drugs are effective in suppressing the viral infection cycle, they do not yet contain any component that inhibits the expression of the viral genome at the transcriptional level, a fact that It supports the notion that the integrated copy of the virus may continue to express the viral genome, but at a very low level, in HIV-1 ⁺ patients who are living (Hatano, H., et al. AIDS 24, 2535-2539 (2010), Pasternak, AO, et al. J. Clin. Microbiol. 46, 2206-2211 (2008)). In fact, viral gene expression rises dramatically with the cessation of ART, allowing the production of early regulatory proteins of the virus such as Tat, organizing productive replication of the viral genome.

  In recent years, more attention has been paid to the development of effective and safe strategies for HIV-1 / AIDS treatment. In this regard, there are several approaches known as shock and kill strategies, including the activation of dormant virus and the elimination of latently infected cells that serve as virus reservoirs by boosting immune cells. This strategy was initially expected but showed limited efficacy and contradictory results (Archin, NM, et al. J. Infect. Dis. 210, 728-735 (2014), Manson McManamy, et Chem. Chemother. 23, 145-149 (2014), Siliciano, JD et al. J. Allergy Clin. Immunol. 134, 12-19 (2014)). More recently, the discovery of novel gene editing techniques is directed to inactivating viral DNA by introducing mutations into various regions of the viral genome and / or cellular genes that support HIV-1 infection. Some institutes were urged to examine feasibility (Khalili, K., et al. J. Neurovirol. 21, 310-321 (2015); White, MK, et al. Discov. Med. 19, 255-262 (2015); Yin, C., et al. AIDS 30, 1163-1170 (2016)).

  Embodiments of the present invention relate to compositions for conditional activation of CRISPR / Cas in the early stages of reactivation. These compositions completely and permanently remove viral replication prior to productive viral replication by removing viral promoters and / or segments of the viral gene that span viral coding sequences. In embodiments, the composition is associated with a clustered, regularly arranged short palindromic sequence repeat operably linked to a functional minimal viral promoter under control of the immediate early transcriptional activator. Containing the nucleic acid sequence encoding the endonuclease (Crisps / Cas), thereby conditionally activating the CRISPR / Cas in the early stages of viral replication. The isolated nucleic acid further comprises at least one guide RNA which is complementary to the target nucleic acid sequence present in the virus. The CRISPR / Cas excises segments of the viral genome, eg, segments that span the viral promoter and / or viral coding sequences. In these embodiments, the composition is adjusted to excise any virus. In a particular embodiment, the virus is a retrovirus.

  Other aspects are described below.

FIGS. 1A-1E show that expression of Cas9 by the HIV-1 LTR promoter is stimulated by Tat, resulting in cleavage of the viral promoter in the presence of gRNA. FIG. 1A is a schematic representation of the full-length HIV-1 LTR and various regulatory motifs within the enhancer and core regions, and a partial Gag gene. The scope of LTR deletion mutants generated for expression of Cas9 is shown. The positions of the gRNA target sequences and their distance from one another are shown. FIG. 1B shows a plasmid expressing Tat (pCMV-Tat) and pX260-LTR containing full-length LTR (-454 / + 66) or various variants thereof (-120 / + 66 or -80 / + 66). -Shows that co-transfection of TZM-bl cells with Cas9 increased the production level of Tat as tested by Western blot (upper panel). Expression of housekeeping α-tubulin (middle panel) and Tat (lower panel) is shown. FIG. 1C shows infection of TZM-bl cells with adenovirus expressing GFP or GFP Tat, followed by lentiviral transduction of expressing gRNAs A / B by Cas9 and U6 promoters by LTR _{-80 / + 66} promoter Performing at three different MOIs of 2, 4 and 8 shows that cleavage of the integrated HIV-1 LTR promoter DNA sequence in TZM-bl cells resulted in the emergence of a 205 bp DNA fragment ( Tested by PCR and DNA gel analysis). FIG. 1D is an SDS-PAGE illustrating the levels of Cas9, β-tubulin and Tat protein expressed in TZM-bl cells as described in FIG. 1C. FIG. 1E is a graph showing the results of a luciferase assay illustrating the transcriptional activity of HIV-1 LTR incorporated into TZM-bl cells after various treatments as described in FIG. 1C. FIGS. 2A-2C show that HIV-1 infection induces Cas9 to stimulate cleavage of incorporated viral DNA. LTR _{-80 / + 66-} Cas9 transduced with three different MOI (2, 4 and 8) lentivirus (LV-gRNA A / B) or control (empty LV) expressing gRNA A / B The reporter TZM-bl cell line is infected with HIV-1 _JRFL or HIV-1 _SF162, and after 48 hours, cells are harvested and protein expression is measured by Western blot (FIG. 2A), by induction of Cas9 after virus infection The level at which the incorporated HIV-1 LTR was cleaved was detected by PCR / DNA gene analysis (FIG. 2B), and the promoter transcription activity of incorporated HIV-1 was evaluated by a luciferase reporter assay (FIG. 2C). FIGS. 3A-3C show that, at the latent stage, stimulation with Cas9 Tat cleaves incorporated HIV-1 DNA in T cells, including HIV-1 reporters. 2D10 cells with integrated copies of the LTR- _{80 / + 66-} Cas9 gene were transduced with control (empty LV) or LV-gRNA A / B and subsequently transfected with pCMV or pCMV-Tat plasmid. After 48 hours, levels of various proteins were measured by Western blot as indicated (FIG. 3A). Genomic DNA to assess status of integrated HIV-1 DNA is measured by LTR specific PCR, excision efficiency is measured as a percentage of the ratio between truncated and full length amplicon, The units (AU) are represented by 0 to 0.5 (FIG. 3B). The level of reactivation of the integrated viral promoter after cleavage was assessed by flow cytometry and a representative scatter plot is shown (FIG. 3C). Red positive propidium iodide labeled dead cells were excluded from analysis. Figures 4A-4C show that treatment of cells with agents that activate latent virus induces the expression of Cas9 and cleavage of incorporated viral DNA in Jurkat 2D10 cells. 2D10 cells expressing LTR _{-80 / + 66-} Cas9 are treated with control (empty) or lentivirus expressing gRNAs A / B and after 24 hours cells are shown as PMA (P), Treated with TSA (T) or both (P / T) for 16 hours. Protein studies on the expression of Cas9-Flag, α-tubulin and GFP (which is indicative of integrated HIV-1 genome) were determined by Western blot (FIG. 4A). Genomic DNA to detect the level of excision in integrated LTR DNA by Cas9 and gRNA A / B was evaluated by PCR and the excision efficiency was measured as described in the legend of FIG. 3B (FIG. 4B) ). A GFP reporter assay by flow cytometry and a representative scatter plot are shown (FIG. 4C). Figures 5A-5G show that expression of LTR-Cas9 / gRNA protects cells from fresh HIV-1 infection. Figures 5A and 5B: Jurkat cells were co-transduced with LV-gRNA A / B and Lenti-LTR ( _{-80 / + 66} ) -Cas9-Blast. The next day, cells were infected with HIV-1 _{NL4-3-GFP-P2A-Nef} at an MOI of 0.01. On days 3 and 5 of infection, cells were harvested and excision levels were assessed by LTR specific PCR using genomic DNA as a template (FIG. 5A) and quantified as in FIG. 3B, 3C (FIG. 5B). Figure 5B). FIG. 5C: Shows the location of the excised fragment compared to control NL4-3 in direct sequencing analysis of the 205 bp DNA fragment after selection of 10 clones cloned into the TA vector and designated as trunc LTR 1-10. The position of gRNA corresponding to LTR A and LTR B in addition to the PAM sequence, and the primers used for amplification of the DNA are highlighted. FIG. 5D: Agarose gel showing the results of PCR analysis of DNA segments corresponding to UTR, Env and control β-actin DNA in experimental cells 3 and 5 days after HIV-1 infection. FIG. 5E: Results from flow cytometry quantifying the percentage of positive cells (indicative of viral expression) at three different times after infection. (For DNA) beta-(for RNA) globin and beta-actin was used as a reference, the virus DNA corresponding to the Gag sequence by TAQMAN ^TM (FIG. 5F) and quantitative detection of viral RNA (Figure 5G). FIG. 6 is a schematic of the negative feedback regulation of HIV-1 by CRISPR / Cas9. In the early stages of viral replication, basal transcription of the viral genome results in the production of Tat protein. The association of Tat with the TAR stem-loop structure within the leader of viral transcription at the badge causes the mobilization of several cellular regulatory proteins leading to the induction of viral transcription (solid thick arrow). At an early stage, Tat also stimulates a minimal viral promoter (represented as ltr) to promote transcription of the Cas9 gene. When newly synthesized Cas9 binds to various HIV-1 specific gRNAs, it then cleaves the viral genome and permanently inactivates LTR activity by excising a large DNA segment of viral DNA And thereby stop the expression and replication of the HIV-1 gene. FIG. 7 highlights the gRNA A / B target (medium gray (green), PAM dark gray (red) within the LTR in the reference HIV-1 NL4-3 genome (SEQ ID NO: 26) And the positions and nucleotide sequences of LTR specific primers (highlighted with light gray (blue)) used in PCR on genomic DNA of TZM-bl cells and in vitro infected Jurkat cells. Sequence and size of LTR specific PCR products (full length (SEQ ID NO: 27) and truncated (SEQ ID NO: 30)), and the predicted compiled fragment (SEQ ID NO: 29). FIG. 8 is a representative agarose analyzing LTR-specific PCR reaction used to quantify Cas9 / gRNA-mediated LTR ablation efficiency in experiments with FIGS. 3A-3C and 4A-4C Jurkat 2D10 reporter cell lines. Show a gel. 9A-9C are highlighted with LTR gRNA A / B targets (medium gray (green), PAM (dark gray) in the reference HIV-1 NL4-3 genome (SEQ ID NO: 26) And the location and nucleotide composition of LTR specific primers (highlighted by light gray (blue)) used to analyze excision in Jurkat 2D10 cells by PCR. Nucleotide sequence and size of amplicon ( The full length (SEQ ID NO: 31) and truncated LTR DNA (SEQ ID NO: 34)) and the predicted excised DNA fragment (SEQ ID NO: 33) are shown. FIG. 10A shows representative fluorescence microscopy images of transduced / infected Jurkat cells on day 5 of infection. Expression of BFP is indicative of the presence of vectors that express gRNAs. HIV-1 infection was observed by the level of GFP. FIG. 10B is a graph showing a quantitative comparison of cell numbers at various time points between control and experimental samples treated with LTR-Cas9. 11A-11C show early human transduced with a lentiviral cocktail containing lenti-LTR _{(-80 / + 66)} -Cas 9 (MOI 10), lenti-KLV-BFP-LTR A, B (MOI 3.3 each) Figure 2 is a graph showing results from fetal astrocytes and microglia. On day 3 of transduction, cells were infected with HIV-1 _{NL4-3-GFP-P2A-Nef} / VSV-G at MOI 1. One week after HIV-1 infection, cells were harvested and virus expression levels were determined by expression of GFP in flow cytometry (FIG. 11A), virus by TAQMAN qPCR and qRT-PCR using primer set and probe specific for Gag gene It was quantified at DNA level (FIG. 11B) and viral RNA (FIG. 11C).

DETAILED DESCRIPTION Embodiments of the present invention relate to HIV-1 transcriptional activator, compositions for conditional activation of CRISPR / Cas9 in the early stages of virus reactivation by Tat, and its use in methods. These compositions permanently remove viral replication prior to productive viral replication by removing viral promoters and / or segments of the viral gene that span viral coding sequences. Furthermore, the use of these compositions in the methods embodied herein alleviates any concerns due to unanticipated complications that may be caused by the high level of unnecessary and sustained expression of Cas9 in cells. Do.

Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice the tests of the present invention, preferred materials and methods are described herein. The following terms are used in the specification and claims of the present invention.

  It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

  The article "one of" ("a" and "an") is used herein to refer to a grammatical object of the article of one or more than one (ie, at least one). By way of example, "an element" means one element or more than one element. Thus, the recitation of "a cell" includes, for example, a plurality of cells of the same type. Furthermore, "including", "includes", "includes", "having", "has", "with", or variations thereof are As used in the specification and / or claims, such terms are intended to be included in a manner analogous to the term "comprising."

  As used herein, the terms "comprise", "comprise", "comprise" or "comprised" and variations thereof refer to items, compositions, devices, Apparatus, method, process, system etc. which are generic in terms of methods, processes, systems or other defined or described elements, or those specified elements-or as appropriate, It is meant to include equivalents-and other elements may be included, and also to include ranges / definitions such as defined items, compositions, devices, methods, processes, systems, etc.

  The term "about" as used herein in referring to amount, duration, and other measurable values is +/− 20%, +/− 10% from the specified value It is intended to include variations of +/- 5%, +/- 1%, or +/- 0.1%, such variations being suitable for performing the disclosed method. Alternatively, particularly with respect to biological systems or biological processes, the term may mean within 5 times of a certain value and within 2 times of an index. Where a specific value is stated in the application and claims, the term "about" should be assumed within the allowable error for the specific value, unless stated otherwise. It is.

  As used herein, the term "antiviral agent" refers to any molecule used to treat the virus, and any agent that alleviates any symptoms associated with the virus, such as antipyretics, antiinflammatory agents, chemotherapeutic agents, and Points to others. Antiviral agents include, but are not limited to: antibodies, aptamers, adjuvants, antisense oligonucleotides, chemokines, cytokines, immunostimulants, immunomodulators, B cell modulators, T cell modulators, NK Cell modulators, antigen presenting cell modulators, enzymes, siRNA, ribavirin, protease inhibitors, helicase inhibitors, polymerase inhibitors, helicase inhibitors, neuraminidase inhibitors, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, purine Nucleosides, chemokine receptor antagonists, interleukins, or combinations thereof.

  As used herein, the term "replacing" a retrovirus, such as human immunodeficiency virus (HIV), means that the virus is not replicable, the genome is deleted, fragmented, degraded, genetically Other physical, biological, chemical, or other physical, biological, or chemical agents that inhibit inactivation or transmission or infection of the virus into any other cell or subject, resulting in clearance of the virus in vivo. Or imply structural expression. In some cases, fragments of the viral genome may be detectable, but the virus can not replicate or infect others.

  As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

  "Coding" refers to other polymers in biological processes that have either the defined nucleotide sequences (i.e. rRNA, tRNA and mRNA) or the defined amino acid sequences and the biological properties resulting therefrom And nucleotides in polynucleotides that serve as templates for the synthesis of macromolecules, such as genes, cDNAs, or unique characteristics in specific sequences of mRNA. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, ie the nucleotide sequence identical to the mRNA sequence, usually provided in the sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA, are proteins or other products of that gene Or may be shown as encoding a cDNA.

  The term "expression" as used herein is defined as transcription and / or translation of a particular nucleotide sequence driven by its promoter.

  "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to the nucleotide sequence to be expressed. The expression vector comprises a cis-acting element sufficient for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include recombinant polynucleotides, such as cosmids, plasmids (eg, contained in naked or in liposomes) and viruses such as lentivirus, retrovirus, adenovirus, and adeno-associated virus. , All known in the art.

  "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide that has been partially or completely separated from its natural co-presence material is "isolated" ' The isolated nucleic acid or protein can be present in substantially purified form, or can be present in a non-natural environment, such as a host cell.

  An "isolated nucleic acid" is a nucleic acid segment or fragment that has been separated from the flanking sequences in a naturally occurring manner, ie, a DNA fragment that has been removed from the sequences that normally flank the fragment, ie, Sequences flanking fragments in the naturally occurring genome are shown. The term also applies to other components naturally associated with nucleic acids, ie, RNA or DNA which is naturally associated with nucleic acids in cells, or nucleic acids which have been substantially purified from proteins. The term is thus intended to, for example, be a vector, an autonomously replicating plasmid or virus, or an isolated molecule integrated into prokaryotic or eukaryotic genomic DNA or isolated from other sequences (ie PCR or restriction It comprises recombinant DNA, which is present as cDNA, or a genomic or cDNA fragment, generated by digestion of an enzyme. Also included are: deoxyribonucleosides, ribonucleosides, their substituted and alpha anomerized forms, peptide nucleic acids (PNA), immobilized nucleic acids (LNA), phosphorothioates, methylphosphonates, and other additional polys. Recombinant DNA which is part of a hybrid gene encoding a peptide sequence, complementary DNA (cDNA), linear or cyclic oligomers or natural and / or modified monomers or linkages.

  The nucleic acid sequences may be "chimeric", ie composed of different regions. In the context of the present invention, a "chimeric" compound is an oligonucleotide comprising two or more chemical regions, such as a DNA region, an RNA region, a PNA region, and the like. Each chemical domain is comprised of at least one monomer unit, ie, a nucleotide. These sequences typically include at least one region whose sequence has been modified to exhibit one or more desirable properties.

  The term "target nucleic acid" sequence refers to a nucleic acid (often derived from a biological sample) designed to specifically hybridize an oligonucleotide. The target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding oligonucleotide directed to the target. The term target nucleic acid can refer to a specific partial sequence of the large nucleic acid to which the oligonucleotide is directed, or the entire sequence (eg, a gene or mRNA). Differences in usage will be apparent from the context.

  In the context of the present invention, the following abbreviations are generally used for the occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine and "T" is Refers to thymidine, and "U" refers to uridine.

  Unless otherwise specified, the amino acid sequences "nucleotide sequences encoding" are degenerate versions of one another and include all nucleotide sequences encoding the same amino acid sequence. Phrase nucleotide sequences encoding proteins or RNA may also include introns, to the extent that the nucleotide sequence encoding the protein may include introns in some versions.

  "Parenteral" administration of the immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

  The terms "patient" or "individual" or "subject" are used interchangeably herein to refer to a mammalian subject to be treated, with human patients being preferred. In some cases, methods of the invention include laboratory animals, veterinary applications, and diseases including rodents or primates including, but not limited to, mice, rats, and hamsters Used in the development of animal models.

  The term "polynucleotide" is a strand of nucleotides also known as "nucleic acid". As used herein, a polynucleotide is obtained by any means available in the art, including, but not limited to, all including both naturally occurring and synthetic nucleic acids The nucleic acid sequence of

  The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound comprising amino acid residues covalently linked by peptide bonds. The protein or peptide must contain at least 2 amino acids, and there is no limit on the maximum number of amino acids that can constitute the sequence of the protein or peptide. A polypeptide includes any peptide or protein comprising two or more amino acids linked together by peptide bonds. As used herein, this term is commonly referred to in the art as a short chain, also commonly referred to as peptides, oligopeptides and oligomers, and generally as a protein in the art, and the like. Shows both types of long chains. The “polypeptide” is, for example, a biologically active fragment, a substantially homologous polypeptide, an oligopeptide, a homodimer, a heterodimer, a variant of the polypeptide, a modified polypeptide, a derivative, among others. , Analogs, fusion proteins. Polypeptides include naturally occurring peptides, recombinant peptides, synthetic peptides, or combinations thereof.

  The terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. . A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with an exogenous nucleic acid. Transfection / transformation / transduced cells include the main subject cell and its progeny.

  "Treatment" is an intervention performed with the intention of preventing the progression of the disease or revising the condition or symptoms. Thus, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. "Treatment" can also be designated as palliative care. Those in need of treatment include those already with the disorder and those in which the disorder is to be prevented. Thus, "treating" or "treatment" of a condition, disease or condition includes: (1) the condition, disease or condition is afflicted or prone to the condition, disease or condition Preventing or delaying the appearance of clinical symptoms of the condition, disease or condition progressing in humans or other mammals, who have not yet experienced or shown clinical symptoms or subclinical symptoms of the condition; (2) the condition, Inhibiting the disease or condition, ie preventing, reducing or delaying the disease or its recurrence (in the case of maintenance therapy), or at least one clinical or subclinical condition thereof; or (3) alleviating the disease To regress the condition, disease or condition, or at least one of its clinical or subclinical conditions. The benefit of the individual to be treated is statistically significant or at least perceptible to the patient or physician.

  A "vector" is a composition that includes an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Examples of vectors include, but are not limited to, linear polynucleotides, polynucleotides related to ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "vector" includes plasmids or viruses that replicate autonomously. The term is intended to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as polylysine compounds, liposomes and the like. Examples of viral vectors also include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

  The terms "percent sequence identity" or "sequence identity" refer to the degree of identity between a given query sequence and the subject sequence.

  The term "exogenous" indicates that the nucleic acid or polypeptide is part of a recombinant nucleic acid construct or is encoded by a recombinant nucleic acid construct or is not in its natural environment. For example, the exogenous nucleic acid can be a sequence incorporated from one species into another, ie, a heterologous nucleic acid. Typically, such exogenous nucleic acids are incorporated into other species via recombinant nucleic acid constructs. An exogenous nucleic acid is unique to an organism and can also be a sequence reincorporated into the cells of that organism. An exogenous nucleic acid comprising a naturally occurring sequence is a non-naturally occurring sequence linked to a foreign nucleic acid, eg, a naturally occurring genetic sequence due to the presence of a non-naturally occurring regulatory sequence adjacent to the naturally occurring sequence in a recombinant nucleic acid construct. And often can be distinguished. In addition, stably transformed exogenous nucleic acids are typically incorporated at positions other than those at which the native sequence is found.

  The term "pharmaceutically acceptable" (or "pharmacologically acceptable"), as appropriate, is disadvantageous, allergic, or otherwise harmful when administered to an animal or human. Refers to molecular entities and compositions that do not react. As used herein, the term "pharmaceutically acceptable carrier" is any and all solvents, dispersion media, coatings, antimicrobials, which can be provided as a medium for pharmaceutically acceptable substances. It includes isotonic and absorption delaying agents, buffers, excipients, binders, binders, lubricants, gels, surfactants and the like.

  If any amino acid sequence is specifically referred to by Swiss Prot. Or Genbank Accession Number, this sequence is incorporated herein by reference. Information related to accession numbers, such as identification of signal peptides, extracellular domains, transmembrane domains, promoter sequences and translation initiation are also incorporated herein by reference in their entirety.

  Genes: All genes, gene names and gene products disclosed herein are intended to correspond to homologues from any species to which the compositions and methods disclosed herein are applicable. Be done. Where a gene or gene product derived from a particular species is disclosed, it is understood that the present disclosure is illustrative only and is not to be construed as limiting unless the context in which it appears is explicitly stated. Thus, for example, with respect to the genes or gene products disclosed herein, it is intended to encompass homologous genes and / or orthologous genes and gene products from other species.

  Scope: Throughout this disclosure, various aspects of the present invention can be presented in the form of a scope. It should be understood that the description in the form of a range is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the present invention. Thus, the recitation of a range should be considered as specifically disclosing all possible subranges and individual numerical values within that range. For example, the description in the range of 1 to 6 should be considered to specifically disclose sub-ranges of 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6 etc. And includes individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6. This applies regardless of the width of the range.

Compositions Antiretroviral therapy does not suppress low levels of viral genome expression and does not suppress latently infected cells such as resting memory T cells, monocytes, macrophages, microglia, astrocytes and lymphoid cells associated with the intestine. It does not target efficiently. In contrast to the therapies available prior to the present invention, the methods and compositions disclosed herein provide HIV in a subject infected at any stage of infection or a non-infected subject at risk for HIV infection. Useful for treating and eradicating

  Thus, the disclosed methods and compositions are useful for HIV-infected subjects in the latent period of infection. In addition, the HIV genome is a host cell when the guide RNA is associated with the CRISPR associated endonuclease operably linked to the HIV LTR minimal promoter responsive to Tat disclosed herein. It can be excised and eradicated. When the composition is administered as a nucleic acid, or contained in an expression vector, the CRISPR endonuclease may be encoded by the same nucleic acid or vector as the guide RNA sequence. Alternatively or additionally, the CRISPR endonuclease may be encoded on a nucleic acid or vector that is physically separate from the guide RNA sequence.

  We used CRISPR / Cas9 technology for the first time to excise the entire HIV-1 genome between the 5'- and 3'-LTR from the host chromosome of latently infected cells, protecting the cells from reinfection, A gene editing molecule specific for HIV-1 was developed (Khalili, K., et al. J. Neurovirol. 21, 310-321 (2015); Hu, W., et al. Proc. Natl. Acad. Sci. USA 111, 11461-11466 (2014); Kaminski, R., et al. Sci. Rep. 6: 22555 (2016)). Methods of excision include multiple specific guide RNAs that recognize various regions of 5'- and 3'-LTR DNA sequences, and Cas9, which introduces a cut into double stranded DNA at a site complementary to the guide RNA. The use of endonucleases is included (Hu, W., et al. Proc. Natl. Acad. Sci. USA 111, 11461-11466 (2014); Kaminski, R., et al. Sci. Rep. 6: 22555 ( 2016)). After removal of viral DNA, the remaining cellular DNA recombines by cellular DNA repair (Khalili, K., et al. J. Neurovirol. 21, 310-321 (2015); White, MK, et al. Discov. Med. 19, 255-262 (2015); Hu, W., et al. Proc. Natl. Acad. Sci. USA 111, 11461-11466 (2014); Kaminski, R., et al. Sci. Rep. 6 22555 (2016)). The multiplex use of gRNA to compile the HIV-1 genome by CRISPR technology is particularly important to alleviate any concerns related to the emergence of viruses resistant to the first gRNA treatment. In addition to the CRISPR / Cas9 technology, more recently, recombination based procedures have been developed with the ability to edit HIV-1 DNA sequences from the host genome (Karpinski, J. et al. Nat. Biotechnol. 34, 401 -409 (2016)).

  Negative feedback regulation: viral genomes, eg, HIV integrated into the genome of infected host cells, are RNA-guided, endonucleases associated with clustered, regularly arranged short palindromic repeats (Crisps), eg, Cas9 can be used to eliminate such HIV-infected cells. Successful therapeutic gene editing using CRISPR / Cas9 enzyme and guide RNA requires efficient and specific delivery and expression of Cas9 enzyme and guide RNA in target cells. This is difficult when the frequency of recipient cells in a tissue or cell population is low, such as HIV-infected cells in patients receiving highly active antiretroviral therapy (HAART).

  According to the invention, a CRISPR-related endonuclease, such as Cas9, is placed under the control of a minimal promoter of the HIV LTR that is responsive to Tat. Endonuclease expression is thereby activated in cells containing Tat protein. When the endonuclease is placed under the control of the HIV LTR minimal promoter responsive to Tat, it was produced exogenously (eg by transfection), and (eg latent virus reactivation) ) Endogenously produced Tat can activate expression of a (Plates) related endonuclease (eg, Cas9) in a cell line. In the studies presented in more detail in the Examples section, the composition allows conditional activation of CRISPR / Cas9 in the early stages of reactivation of the virus by the HIV-1 transcriptional activator, Tat .

  This strategy completely and permanently removes viral replication prior to productive viral replication by removing the entire viral gene or viral gene segment that extends to the viral promoter and / or viral coding sequences.

  FIG. 1A is a schematic representation of the full-length HIV-1 LTR, and various regulatory motifs within the enhancer and core regions, and a partial Gag gene. The range of LTR deletion mutants generated for expression of Cas9 is indicated. The positions of the target sequences of gRNA and their distance from each other are indicated. The HIV-1 LTR is approximately 640 bp in length and is divided into U3, R and U5 regions. Transcription of the HIV-1 genome is controlled by a series of cis-acting regulatory motifs that extend to the terminal repeat region of the 5 'end viral genome. The U3 region of the viral promoter occupies nucleotides -1 to -454 relative to the transcription initiation site +1 and has 3 subregions: regulation, enhancer, and core. Enhancers contain the NF-κB binding site (-127 to -80). The core domain contains a GC-rich TATA box (-80 to +1). The R region (+1 to +98) of LTR contains TAR, a region where the expressed RNA forms a stem-loop structure and provides a binding site for the viral transcriptional activator (Krebs et al, Lentiviral LTR-directed expression, sequence variation, disease pathogenesis. Los Alamos National Laboratory HIV Sequence: Compendium, pp. 29-70.2002).

  The LTR contains all of the signals necessary for gene expression and is involved in the integration of the provirus into the genome of the host cell. For example, the core promoter, enhancer and regulatory regions are found in U3, while TAR is found in R as shown in FIG. 1A. The binding site for Tat proteins and cellular proteins, TAR, consists of approximately the first 45 nucleotides of viral mRNA in HIV-1 and forms a hairpin stem loop structure. In HIV-1, the U5 region contains several subregions, such as poly A involved in dimerization and genomic packaging, PBS or primer binding sites, Psi or packaging signals, and DIS or dimer initiation sites including.

  Negative feedback regulation of HIV-1 by CRISPR / Cas9 is shown in FIG. In the early stages of viral replication, basic transcription of the viral genome results in the production of Tat protein. The association of Tat with the TAR stem-loop structure within the viral transcription leader at the badge (budge) leads to the mobilization of several cellular regulatory proteins that direct viral transcription (solid bold arrow). In the early stages, Tat also stimulates the minimal viral promoter (shown as ltr) which drives the transcription of the Cas9 gene. The newly synthesized Cas9, in association with various HIV-1 specific gRNAs, in turn cleaves the viral genome and excises large segments of viral DNA to stop expression and replication of the HIV-1 gene. Permanently inactivate LTR activity.

  Minimal promoter of LTR: According to the present invention, the HIV LTR promoter is operably linked to the minimal promoter of HIV LTR including at least the core region and TAR (element responsive to transactivation) region. There is provided a composition comprising an isolated nucleic acid encoding an endonuclease associated with The HIV LTR minimal promoter refers to an operable functional promoter, including those shorter than the full-length HIV LTR promoter. In certain embodiments, the minimal promoter comprises a TAR region. In certain embodiments, the minimal promoter comprises a core region and a TAR region. In certain embodiments, the minimal promoter comprises core and TAR regions with all or substantially all of the regulatory and / or enhancer regions. In another embodiment, the minimal promoter of the HIV LTR comprises a core region, a TAR region, and all or substantially all enhancer regions but does not include any regulatory regions. In certain embodiments, the HIV LTR minimal promoter comprises a combination of one or more mutations, modified bases, variants, locked nucleic acids thereof. The minimal promoter of the HIV LTR is responsive to Tat proteins. Thus, Tat can activate expression of a CRISPR-related endonuclease, eg, Cas9, operably linked to the minimal promoter of the HIV LTR. The disclosed compositions eradicate HIV in host cells in vitro or in vivo, to inactivate HIV in mammalian cells, to treat subjects with HIV infection, subjects at risk of infection It can be used to reduce the risk of HIV infection in and / or to reduce the risk of HIV transmission from an HIV-infected mother to its offspring. The therapeutic methods disclosed herein may be practiced in conjunction with other anti-retroviral therapies, such as HAART. The composition may be included as part of a kit for diagnostic, research, and / or therapeutic applications.

  Several advantages may be realized by a composition comprising a CRISPR-related endonuclease encoding sequence operably linked to the HIV LTR minimal promoter, including the core region of the HIV LTR promoter and the TAR region. By limiting the expression of endonucleases associated with CRISPR to cells with expression and / or replication of the HIV gene, the potential risk of toxic effects caused by continuous expression can be mitigated and / or eliminated. For example, the potential for toxicity induction due to the immunogenicity of the endonuclease associated with CRISPR can be reduced due to low and / or intermittent expression of the endonuclease according to the present invention, simultaneously infection Can cause the elimination or self-destruction of the HIV genome in an individual. In addition, the present invention may provide a preventative strategy for at-risk individuals to minimize the sustained expression of CRISPR-associated endonucleases. Thus, Tat-responsive HIV LTR minimal promoter-driven CRISPR-linked endonucleases are not associated with HIV infection to provide a safe treatment for subjects infected with HIV and uninfected at risk of infection. It can be used to vaccinate an individual.

  The minimal promoter of the HIV-1 LTR may comprise a nucleic acid comprising nucleotides from position -80 to +66 of the HIV-1 LTR promoter. In certain embodiments, the minimal promoter of the HIV-1 LTR may comprise a nucleic acid comprising the -120 to +66 position of the HIV-1 LTR promoter. Preferably, the minimal promoter of the HIV-1 LTR does not include sequences derived from regulatory regions. In some embodiments, the promoter comprises one or more mutations, deletions, insertions, variants, derivatives or combinations thereof. The promoter may also be a chimera comprising one or more chimeric compounds.

  Placing the CRISPR-related endonuclease under the control of the minimal promoter of the HIV LTR described herein makes the smaller sized nucleic acid a suitable delivery mechanism for gene therapy (eg, retrovirus) It is also beneficial because it can be easily packaged. Promoter constructs containing regulatory regions may be less suitable for gene therapy, for example, due to their variable size and / or variable effects on transcription of the CRISPR associated endonuclease.

  As mentioned above, the HIV genome is integrated into the host genome of an individual infected with HIV. This integrated sequence is then replicated by the host. Even in the incubation period, Tat can be produced by cells. The compositions of the present invention eliminate and / or reduce the presence of proviral polynucleotides in the host. Since the endonuclease associated with CRISPR is driven by a promoter responsive to Tat according to the present invention, any time Tat is present (eg, produced by an infected cell), the endonuclease is produced and nascent poly Degrades nucleotides. If the virus is not active, no endonuclease is produced. Thus, the potential toxic effects that continuous expression of the endonuclease may have on the cell and / or the host are avoided.

  CRISPR Related Endonuclease: The compositions disclosed herein may comprise a CRISPR related endonuclease, eg, a nucleic acid encoding Cas9. In some embodiments, one or more guide RNAs complementary to HIV target sequences may also be encoded. In bacteria, the CRISPR / Cas locus encodes an RNA-guided adaptive immune system against mobile genetic elements (viruses, transposable elements and mating plasmids). Three types of CRISPR systems (I-III) have been identified. The cluster of CRISPR contains a spacer, ie, a sequence that is complementary to the preceding mobile element. Clusters of CRISPR are transcribed and processed into mature CRISPR RNA (crRNA). The CRISPR-related endonuclease Cas9 belongs to the type II CRISPR / Cas system and has potent endonuclease activity to cleave target DNA. Cas9 is a short, trans-activated, serving as a guide for a unique target sequence of approximately 20 base pairs (bp) (called a spacer) and ribonuclease III-assisted processing of pre-crRNA It is guided by mature crRNA, which contains RNA (tracrRNA). The duplex of crRNA versus tracrRNA directs Cas9 to the target DNA through complementary base pairing of the spacer on crRNA and the complementary sequence on the target DNA (called the protospacer). Cas9 recognizes a motif (PAM) adjacent to the trinucleotide (NGG) protospacer to identify the cleavage site (the third nucleotide derived from PAM). crRNA and tracrRNA may be separately expressed or engineered artificial small guide RNAs (sgRNAs) via synthetic stem loops (AGAAAU) to mimic duplexes by natural crRNA / tracrRNA Can. Such sgRNAs can be synthesized similarly to shRNAs, or can be transcribed in vitro for direct RNA transfection, or can be expressed from RNA expression vectors promoted to U6 or HI, but are artificial The cleavage efficiency of sgRNA is lower than that of systems with separately expressed crRNA and tracrRNA.

  The endonuclease associated with CRISPR may be Cas9 nuclease. Cas9 nuclease can have a nucleotide sequence identical to the wild-type Streptococcus pyogenes sequence. The endonuclease associated with CRISPR may be other species, eg, sequences from other Streptococcus, eg, thermophiles. The Cas9 nuclease sequence may be derived from, but not limited to, the following other species: Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides imici The snails, Lactobacillus, Lacto bacterium, Lactobacillus, Bacterosio, Aespaticus, Arachnida, Anemones, Anemone, Anemone, Anemone, Anemone, Anemone, Anemone, Anemone, Anemone, Anemone, Anemone, Anemone, Anemone, Anemone, Anemone, Anemone, Anemone, Anemone, Anemone. Clostridium difficle, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidiobacillus caldus, Acidiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp. (Garner), Irositosis, vetrosiococco watsoni, Pseudosormons watsoni, Pseudosormons watsoni, Pseudosormons latus, Angiore eta, Nio, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth, moth; , Or Acaryochloris marina. Psuedomona aeruginosa, Escherichia coli, or other sequenced bacterial genomes and archaebacteria, or other prokaryotic microorganisms are also sources of Cas9 sequences utilized in the embodiments disclosed herein. possible.

  The Cas9 sequence of wild-type Streptococcus pyogenes can be modified. The nucleic acid sequences can be codon-optimized or "humanized" for efficient expression in mammalian cells. The sequence may be, for example, a Cas9 nuclease sequence encoded by any of the expression vectors listed in Genbank Accession No. KM 099231.1 GI: 669193735; KM 099232.1 GI: 669193761; or KM 099233.1 GI: 669193765. Alternatively, the Cas9 nuclease sequence may be, for example, a sequence contained within a commercially available vector such as PX330 or PX260 of Addgene (Cambridge, Mass.). In some embodiments, the Cas9 endonuclease is the Cas9 endonuclease sequence of Genbank Accession No. KM 099231.1 GI: 669193757; KM 099232.1 GI: 669193761; or KM 099233.1 GI: 669193 765, or PX330 or PX260 (Addgene, Cambridge) , MA) can have amino acid sequences that are variants or fragments of any of the Cas 9 amino acid sequences. The Cas9 nucleotide sequence can be modified to encode biologically active variants of Cas9; these variants may, for example, be one or more mutations (eg, additions, deletions or substitutions) or By including such a combination of mutations, it may have or contain an amino acid sequence different from that of wild type Cas9. One or more substitution mutations may be substitution (eg, conservative amino acid substitution). For example, a biologically active variant of a Cas9 polypeptide has at least or about 50% sequence identity (eg, at least or about 50%, 55%, 60%) to wild-type Cas9 polypeptide. It may have an amino acid sequence of 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity). Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; Phenylalanine and tyrosine. The amino acid residue in the Cas9 amino acid sequence may be a non-naturally occurring amino acid residue. Naturally occurring amino acid residues include those that are naturally encoded by the genetic code as well as by non-standard amino acids (eg, amino acids having D-form instead of L-form). The peptides of the invention may also comprise amino acid residues which are modified forms of standard residues (eg pyrrolidine may be used instead of lysine and selenocysteine may be used instead of cysteine). Non-naturally occurring amino acid residues are those which are not found in nature, but which conform to the basic formula of amino acids and which can be incorporated into peptides. These include D-alloisoleucine (2R, 3S) -2-amino-3-methylpentanoic acid and L-cyclopentylglycine (S) -2-amino-2-cyclopentylacetic acid. As another example, reference can be made to the text or the World Wide Web (sites currently managed by the California Institute of Technology display the structure of unnatural amino acids well incorporated into functional proteins).

  Guide RNA Sequences: The compositions and methods of the present invention may comprise a sequence encoding a guide RNA that is complementary to a target sequence in HIV. The genetic diversity of HIV is reflected in the complex groups and subtypes that have been described. The collection of HIV sequences is organized in the Los Alamos HIV database and list (ie, the website of the sequences database is hitp: //www.hiv.lani.gov). The methods and compositions of the present invention can be applied to HIV from any of these various groups, subtypes, and circulating recombinant forms. These include, for example, the major groups of HIV-1 (often referred to as group M) as well as minority groups, ie, group N, group O and group P, and subtypes A, including, but not limited to: B, C, D, F, G, H, J and K. Or a group of HIV (for example, but not limited to, any of the following groups, N, O and P).

  The guide RNA can be a sequence that is complementary to a coding or non-coding sequence (ie, a target sequence). For example, the guide RNA is complementary to the terminal repeat (LTR) region of HIV other than that used to inform the minimal promoter responsive to Tat operably linked to the Cas9 gene. Can be Guide RNA can not target the sequence corresponding to the minimal promoter of HIV-1 LTR that is responsive to Tat as disclosed herein, but this results in the degradation of the construct itself Therefore, because the HIV LTR minimal promoter is driven by the minimal promoter driven by the CRISPR-related endonuclease, it eliminates potential advantages. Thus, the guide RNA is found within the reference or consensus sequence of the HIV-1 U3, R and / or U5 region without selecting a sequence that is part of HIV's minimal promoter responsive to Tat. Array can be included.

  In some embodiments, the guide RNA can be a sequence complementary to the coding sequence, eg, a sequence encoding one or more viral structural proteins (eg, gag, pol, env and tat). Thus, this sequence is a gag polyprotein, such as MA (matrix protein, p17); CA (capsid protein, p24); NC (nucleocapsid protein, p7); and P6 protein; pol, such as reverse transcriptase (RT) and RNase H, integrase (IN), and HIV protease (PR); cleavage products of env, eg gp160, or gp160, eg gp120 or SU, and gp41 or TM; or tat, eg 72-amino acid 1-exon Tat or 86 -101 amino acid may be complementary to a sequence within 2-exon Tat. In some embodiments, the guide RNA can be a sequence complementary to a sequence encoding an accessory protein, including, for example, vif, n willef (negative factor) vpu (virus protein U) and tev.

  In some embodiments, a guide RNA sequence is complementary to a structural or regulatory element (ie, a target sequence), such as RRE, PE, SLIP, CRS (a cis-acting repressor sequence) and / or INS. It can be. RRE, an element responsive to Rev, is an element of RNA encoded within the env region of HIV and approximately 77 nucleotides from the start of transcription in HIV-1 which spans the border of gp120 and gp41, approximately 200 nucleotides. Position to 8061). PE (element of Psi) corresponds to a set of four stem loop structures overlapping and overlapping the Gag start codon. SLIP is the "slippery site" of the TTTTTT, followed by a stem-loop structure. CRS (cis-acting repressing sequence), INS (inhibitory / labile RNA sequence) can be found, for example, at nucleotides 414 to 631 of the gag region in HIV-1.

  The guide RNA sequence may be a sense or antisense sequence. Guide RNA sequences generally include PAM. The sequence of PAM can vary depending on the specificity requirements of the CRISPR endonuclease used. In the CRISPR-Cas system from S. pyogenes, the target DNA is typically located immediately before the 5'-NGG proto spacer flanking motif (PAM). Thus, for Cas9 of S. pyogenes, the PAM sequence may be AGG, TGG, CGG or GGG. Other Cas9 orthologues may have different PAM specificities. For example, Cas9 from S. thermophilus requires 5'-NNAGAA for CRISPR 1, 5'-NGGNG for CRISPR 3), and 5'-NNNNGATT for Neisseria meningitidis. Do. Although specific sequences in the guide RNA may vary, regardless of the sequence, useful guide RNA sequences minimize off-target effects while achieving high efficiency and complete excision of the HIV integrated virus integrated into the genome It is an array to be The length of the guide RNA sequence is about 20 to about 60 or more nucleotides, such as about 20, about 21, about 22, about 23, about 23, about 24, about 25, about 26, about 27, about 28, about 29, About 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 45, about 50, about 55, about 60 or more nucleotides It can change. A useful selection method is to identify regions with very low homology between the foreign viral genome and the host cell genome, including the endogenous retroviral DNA, and off target human transcriptome or Bioinformatic screening using 12-bp + NGG target selection criteria to eliminate untranslated genomic sites (although); transcription within the HIV-1 LTR promoter (potentially conserved in the host genome) Avoiding factor binding sites; to increase specificity / efficiency to LTR-A- and -B-derived, 30 bp guide RNAs and also 20 bp guide RNAs-, chimeric crRNA-trac RNA based systems and WGS The pre-crRNA system reflects the original bacterial immune mechanism, Sanger sequencing and SURVEYOR asses to identify and eliminate potential off-target effects. Including the selection.

  The guide RNA sequences can be configured as a single sequence or as a combination of one or more different sequences, eg, as a multiplexed structure. The multiplex structure can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more without selection of sequences that are part of the HTV minimal promoter responsive to Tat. Combinations of different guide RNAs may be included, for example a combination of sequences within U3, R or U5. When the composition is administered to an expression vector, the guide RNA can be encoded by a single vector. Alternatively, multiple vectors can be engineered to each contain two or more different guide RNAs. Useful structures result in the excision of viral sequences between cleavage sites that result in the elimination of HIV genome or HIV protein expression. Thus, the use of two or more different guide RNAs facilitates the excision of viral sequences between the cleavage sites recognized by CRISPR endonucleases. The excised region can vary in size, from a single nucleotide to several thousand nucleotides.

Modified or mutated nucleic acid sequences: In some embodiments, any of the nucleic acid sequences are modified or derived from naturally occurring nucleic acid sequences, for example by mutation, deletion, substitution, modification of nucleobases, backbone, etc. obtain. Nucleic acid sequences include vectors, gene editors, gRNA and the like. Examples of some modified nucleic acid sequences contemplated for the present invention include modified backbones, such as phosphorothioates, phosphotriesters, methylphosphonates, short alkyl or cycloalkyl sugar linkages, or short heteroatom linkages or Included are those containing heterocyclic carbohydrate linkages. In some embodiments, modified oligonucleotides are those having a phosphorothioate backbone and a heteroatom backbone, CH ₂ —NH—O—CH ₂ , CH, —N (CH ₃ ) —O—CH ₂ [known as a methylene (methylimino) or MMI _{backbone], CH 2 --O - N (} CH 3) - CH 2, CH 2 --N (CH 3) - N (CH 3) - CH ₂ and O--N (CH ₃ )-CH ₂ --CH ₂ backbones are included, the natural phosphodiester backbone being represented as O--P--O--CH,) The amide scaffold disclosed by De Mesmaeker et al. Acc. Chem. Res. 1995, 28: 366-374 is also embodied herein. In some embodiments, a morpholino backbone structure (Summerton and Weller, US Pat. No. 5,034, 506), a nucleic acid sequence having a peptide nucleic acid (PNA) backbone wherein the phosphodiester backbone of the oligonucleotide is substituted with a polyamide backbone, the polyamide backbone Nucleic acid bases directly or indirectly attached to the aza nitrogen atom (Nielsen et al. Science 1991, 254, 1497). The nucleic acid sequence may also include one or more substituted sugar moieties. The nucleic acid sequence may also have a glycomimetic such as cyclobutyl instead of a pentofuranosyl group.

  The nucleic acid sequence may additionally or alternatively include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U) . Modified nucleobases are nucleobases which are found only rarely or transiently in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidine, in particular (also with 5-methyl-2'deoxycytosine). 5-methylcytosine, 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, also referred to and often referred to in the art as 5-Me-C, as well as synthetic nucleobases such as 2-aminoadenine 2- (Methylamino) adenine, 2- (imidazolylalkyl) adenine, 2- (aminoalkylamino) adenine or other heterosubstituted alkyladenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5- Hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexi ) Including adenine and 2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, 1980, pp 75-77; Gebeyehu, G., et al. Nucl. Acids Res. 1987, 15: 4513). "Universal" bases known in the art, such as inosine, may be included. A 5-Me-C substitution has been shown to increase the stability of nucleic acid duplexes by 0.6-1.2 ° C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

  Another modification of the nucleic acid sequences of the invention involves chemically linking one or more moieties or complexes to the nucleic acid sequences that enhance the activity of the oligonucleotide or cellular uptake. Such moieties include, but are not limited to, lipid moieties such as: cholesterol moieties, cholesteryl moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553), cholic acid (Manoharan et al. Bioorg. Med. Chem. Let. 1994, 4, 1053), thioethers such as hexyl-S-trityl thiol (Manoharan et al. Ann. NY Acad. Sci. 1992, 660). Manoharan et al. Bioorg. Med. Chem. Let. 1993, 3, 2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533), aliphatic chains such as dodecanediol or Undecyl residues (Saison-Behmoaras et al. EMBO J. 1991, 10, 11 1; Kabanov et al. FEBS Lett. 1990, 259, 327; Svinarchuk et al. Biochimie 1993, 75, 49), phospholipids such as diazo -Hexadecyl-rac-glycerol or triethylammonium 1,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al. Shea et al. Nucl. Acids Res. 1990, 18, 3777), polyamine or polyethylene glycol chains (Manoharan et al. Nucleosides & Nucleotides 1995, 14, 969), or adamantane acetate. (Manoharan et al. Tetrahedron Lett. 1995, 36, 3651). It is not necessary that all positions in any nucleic acid sequence be uniformly modified, in fact one or more of the above modifications may be incorporated into a single nucleic acid sequence, or within a nucleic acid sequence. It may even be incorporated within a single nucleoside of

In some embodiments, RNA molecules, such as crRNA, tracrRNA, gRNA, are designed to include one or more modified nucleobases. For example, known modifications of RNA molecules are described, for example, in Genes VI, Chapter 9 ("Interpreting the Genetic Code"), Lewis, ed. (1997, Oxford University Press, New York), and Modification and Editing of RNA, Grosjean and Benne, eds. (1998, ASM Press, Washington DC). The components of the modified RNA include: 2′-O-methyl cytidine; N ⁴ -methyl cytidine; N ⁴ -2′-O-dimethyl cytidine; N ⁴ -acetyl cytidine; 5-methyl cytidine; 5-hydroxymethylcytidine; 5-formylcytidine; 2'-O-methyl-5-formylcytidine;3-methylcytidine;2-thiocytidine;lysidine;2'-O-methyluridine2-thiouridine;2-thio-2'-O-methyluridine;3,2'-O-dimethyluridine; 3- (3-amino-3-carboxypropyl) uridine; 4-thiouridine; ribosylthymine; '-O-dimethyluridine;5-methyl-2-thiouridine;5-hydroxyuridine;5-methoxyuridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 5-carboxymethyluridine; 5-methoxycarbonylmethyl Uridine; 5-Methoxycarbonylmeth 5-methoxycarbonylmethyl-2'-thiouridine;5-carbamoylmethyluridine; 5-carbamoylmethyl 1-2'-O-methyluridine; 5- (carboxyhydroxymethyl) uridine; 5- (Carboxyhydroxymethyl) uridine methyl ester; 5-aminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyl-2-selenopyridine; 5- Carboxymethylaminomethyluridine; 5-carboxymethylaminomethyl-2'-O-methyl-uridine;5-carboxymethylaminomethyl-2-thiouridine;dihydrouridine;dihydroribosylthymine;2'-methyladenosine; 2-methyladenosine N ⁶ N methyl adenosine N ⁶ , N ⁶ -dimethyl adenosine N ⁶ 2'-O-trimethyl adenosine 2 methyl thio N ⁶ isopentenyl adenosine; N ^6- (cis-hydroxy isopentenyl)-adenosine; 2 methylthio-N ^6- (cis--hydroxy isopentenyl)-adenosine; N ^6- glycinyl carbamoyl) adenosine; N ⁶ threonyl carbamoyl adenosine N ⁶ -methyl-N ⁶ -threonylcarbamoyladenosine; 2-methylthio-N ⁶ -methyl-N ⁶ -threonylcarbamoyladenosine; N ⁶ -hydroxynorvalylcarbamoyladenosine; 2-methylthio-N ⁶ -hydroxynorvalyl 2'-O-ribosyl adenosine (phosphate); inosine; 2'O-methylinosine;1-methylinosine;1,2'-O-dimethylinosine;2'-O-methylguanosine; 1-methyl guanosine; N ² - methyl guanosine; N ^2, N ² - dimethyl-guanosine; N ^2, 2'-O- dimethyl ^{guanosine; N 2, N 2, 2'} -O- Torimechirugua Shin; 2'-O-ribosyl guanosine (phosphoric acid); 7-methyl guanosine; N ^2, 7-dimethyl-guanosine; N ^2, N ^2; 7-trimethyl guanosine; Waioshin; Mechiruwaioshin; unmodified hydroxy Wai but-thin Hydroxybutytocin; peroxivibutosin; cuocin; epoxycuiocin; galactosyl-cuocin; 7-cyano-7-deazaguanosine; also called arachiocin [7-formamide-7-deazaguanosine ; And 7-aminomethyl 1-7-deazaguanosine.

  Isolated nucleic acid sequences are produced by standard techniques. For example, PCR techniques can be used to obtain an isolated nucleic acid sequence comprising a nucleotide sequence as described herein, including a nucleotide sequence encoding a polypeptide as described herein. PCR can be used to amplify specific sequences, including sequences from RNA as well as DNA, genomic DNA or complete cellular RNA. Various PCR methods are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995. In general, sequence information from the end of the region of interest or beyond is used to design oligonucleotide primers that are identical or similar to the single strand opposite to the template to be amplified. Various PCR strategies are also available as site-specific modifications of the nucleotide sequence can be incorporated into the template nucleic acid.

  The isolated nucleic acid is also synthesized chemically and solely as a single nucleic acid molecule (eg, using automated DNA synthesis in the 3 'to 5' direction using phosphoramidite technology) or as a series of oligonucleotides It can be done. For example, one or more sets of long oligonucleotides (e.g.,> 50-100 nucleotides) contain short segments (e.g., about 15 nucleotides) with each set being complementary, and the set of oligonucleotides can be It may be synthesized to contain the desired sequence, such that upon annealing, a duplex is formed. DNA polymerase is used to extend the oligonucleotides, and each set of oligonucleotides results in a single double stranded nucleic acid molecule, which can then be ligated into the vector. The isolated nucleic acids of the invention can also be obtained by mutagenesis, for example by the naturally occurring part of the DNA encoding Cas9.

  The two nucleic acids or the polypeptides they encode may be described as having a certain degree of identity with one another. For example, Cas9 protein and its biologically active variants can be described as showing a certain degree of identity. A short Cas9 sequence was placed at the Protein Information Research (PIR) site (pir.georgetown.edu), followed by "short identical identical in the NCBI website (ncbi.nlm.nih.gov/blast) The alignment may be organized by analysis of the "short locally identical sequences" using the Basic Local Alignment Search Tool (BLAST) algorithm.

  Percent sequence identity to Cas9 can be determined, the identified variants can be utilized as a CRISPR-related endonuclease and / or the utility as a pharmaceutical composition can be measured. . Naturally occurring Cas9 may be a query sequence, and a fragment of the Cas9 protein may be a sequence of interest. Similarly, a fragment of the Cas9 protein may be a query sequence, and the biologically active variant may be a sequence of interest. In order to determine sequence identity, the query nucleic acid or amino acid sequence allows the alignment of the nucleic acid or protein sequences provided over the full length (wide alignment), respectively, the computer program ClustalW (version 1.83, initially defined parameters) Can be used to align to one or more nucleic acid or amino acid sequences of interest. See Chenna et al., Nucleic Acids Res. 31: 3497-3500, 2003.

  Recombinant constructs and delivery vehicles: Recombinant constructs are also provided herein and for expressing Cas9 under the control of a minimal promoter of HIV LTR responsive to Tat, It can be used to transform cells. Recombinant constructs can also be utilized to express guide RNAs that are complementary to target sequences in HIV. The recombinant nucleic acid construct is operably linked to Cas9 and / or a regulatory region suitable for expressing a guide RNA complementary to a target sequence in HIV in the cell, Cas9 and / or herein It comprises a nucleic acid encoding a guide RNA which is complementary to the target sequence in the described HIV. It will be appreciated that large quantities of nucleic acid may encode a polypeptide having a particular amino acid sequence. Degeneration of the genetic code is known in the art. Many amino acids have triplets of more than one nucleotide that serve as codons for the amino acids. For example, the codons in the Cas9 coding sequence can be altered to the optimal expression possessed by a particular organism, using a table of codon biases appropriate for that organism.

The nucleic acids described herein can be contained in a vector. The vector may, for example, include an origin of replication, scaffold attachment regions (SARs) and / or markers. The marker gene can confer a selectable phenotype on the host cell. For example, the marker can confer resistance to biocides, such as resistance to antibiotics (eg, kanamycin, G418, bleomycin, or hygromycin). An expression vector can include a tag sequence designed to facilitate manipulation or detection (eg, purification or localization) of the expressed polypeptide. Tag sequences, such as green fluorescent protein (GFP), glutathione-S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG ^TM tag (Kodak, New Haven, CT) sequences are typically encoded Is expressed as a fusion with the expressed polypeptide. Such tags can be inserted anywhere within the polypeptide, including both the carboxyl or amino termini.

  Additional expression vectors can also include, for example, chromosomal, non-chromosomal segments and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids such as, for example, E. coli plasmids colEl, pCR1, pBR322, pMal-C2, pET, pGEX, pMB9 and variants thereof, plasmids such as RP4. Phage DNA, eg, a large number of derivatives of phage 1, eg, NM 989, and other phage DNAs, eg, M13 and filamentous single-stranded phage DNA; yeast plasmids, eg, 2μ plasmid or derivatives thereof, eukaryotic cells Useful vectors, such as those useful for insects or mammals; vectors derived from combinations of plasmids and phage DNA, such as plasmids modified for use with phage DNA or other expression control sequences.

  Several delivery methods are available for in vitro (cell culture) and in vivo (animal and patient) systems in conjunction with the minimal HIV LTR promoter responsive to Tat operably linked to the Cas9 gene. It can be done. In certain embodiments, a lentiviral gene delivery system may be utilized. Such systems provide a stable, long-term presence of genes in dividing or non-dividing cells with broad tropism and capacity for large DNA insertions. (Dull et al, J Virol, 72: 8463-8471 1998). In certain embodiments, adeno-associated virus (AAV) can be utilized as a delivery method. AAV is a nonpathogenic, single-stranded DNA virus, and has recently been used actively to deliver therapeutic genes to in vitro and in vivo systems (Choi et al, Curr Gene Ther, 5: 299- 310, 2005). An example of a non-viral delivery method may utilize nanoparticle technology. This platform has shown utility as pharmaceutical in vivo. Nanotechnology has improved transcytosis of drugs across tight epithelial and endothelial barriers. It provides targeted delivery of its payload to cells and tissues in a specific way (Allen and Cullis, Science, 303: 1818-1822, 1998).

  The vector can include regulatory regions. The term "regulatory region" refers to a nucleotide sequence that affects the initiation and rate of transcription or translation, the stability and / or mobility of the transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, responsive elements, protein recognition sites, inducible elements, protein binding sequences, 5 'and 3' untranslated regions (UTRs), transcription Includes start sites, terminal sequences, polyadenylation sequences, nuclear localization signals, and introns.

  The term "operably linked" refers to the arrangement of the regulatory regions and sequences to be transcribed in the nucleic acid to affect transcription and translation of such sequences. For example, to deliver the coding sequence under the control of a promoter, the translation initiation site of the translational reading frame in the polypeptide is typically between 1 and about 50 nucleotides downstream of the promoter Be placed. The promoter, however, can be placed as many as 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the translation initiation site. The promoter typically comprises at least one core (basal) promoter. The promoter may also comprise at least one regulatory element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). The choice of promoter to be included depends on several factors, which include, but are not limited to: efficiency, selectivity, inducibility, desirable expression levels, and preferential expression of cells or tissues I will not. It is routine for one skilled in the art to control the expression of the coding sequence by appropriately selecting and placing the promoter and other regulatory regions associated with the coding sequence.

  The vector delivers, for example, viral vectors (eg, adenovirus Ad, AAV, lentivirus, and vesicular stomatitis virus (VSV) and retrovirus), complexes including liposomes and other lipids, and polynucleotides to host cells. And other macromolecular complexes that can mediate that. The vector may also include other components or functionalities that further modulate gene delivery and / or gene expression or otherwise provide beneficial properties to the targeted cells. As described in more detail below and described below, such other components include, for example, components that affect cell binding or targeting (components that mediate specific binding to cell types or tissues) A component that affects the cellular uptake of the vector nucleic acid; a component that affects the localization of the polynucleotide in cells after inoculation (eg, an agent that mediates nuclear localization); including. Such components can be used to detect or select markers, eg, harvested cells, and can also include detectable and / or selectable markers expressing nucleic acid delivered to the vector. Such components can be provided as a natural property of the vector (eg, components that mediate binding and uptake, or use of a particular viral vector with functionality), or the vector It may be modified to provide. Other vectors include those described in Chen et al; BioTechniques, 34: 167-171 (2003). The wide variety of such vectors is known in the art and is generally available. "Recombinant viral vector" refers to a viral vector that comprises one or more heterologous gene products or sequences. As many viral vectors exhibit size limitations associated with packaging, heterologous gene products or sequences are typically introduced by replacing portions of one or more viral genomes. Such viruses may be replication defective and may be defective during delivery between replication and encapsidation of the virus (eg helper virus or packaging cells carrying gene products necessary for replication and / or encapsidation) By using the strain) requires the deletion function to be provided in trans. It has also been described that the modified viral vector to be delivered by the polynucleotide is carried outside the viral particle (see, eg, Curiel, DT, et al. PNAS 88: 8850-8854, 1991).

  Additional vectors include viral vectors, fusion proteins and scientific complexes. Retroviral vectors include Moloney murine leukemia virus and viruses based on HIV. One HIV-based viral vector contains at least two vectors, the gag and pol genes are derived from the HIV genome, and the env gene is derived from another virus. DNA virus vectors may be varicella vectors, such as herpespox virus vectors such as orthopox or avipox vectors, herpes simplex virus I virus (HSV) vectors [Geller, AI et al., J. Neurochem, 64: 487 (1995); , F., et al., In DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci .: USA Geller, AI, et al., Proc Natl. Acad. Sci USA: 87: 1149 (1990)], adenoviral vectors [LeGal LaSalle et al., Science, 259: 988 (1993); Davidson, et al., Nat. Genet. 3: 219 (1993); Yang, et al., J. Virol. 69: 2004 (1995)], and adeno-associated virus vectors [Kaplitt, MG, et al., Nat. Genet. 8: 148 (1994)].

  The polynucleotides described herein can be used with microdelivery vehicles such as cationic liposomes and adenoviral vectors. See Mannino and Gould-Fogerite, BioTechniques, 6: 682 (1988) for a discussion of procedures for liposome formation, targeting and delivery of contents. See also Felgner and Holm, Bethesda Res. Lab. Focus, 11 (2): 21 (1989) and Maurer, RA, Bethesda Res. Lab. Focus, 11 (2): 25 (1989).

  Replication-defective recombinant adenoviral vectors can be produced according to known techniques. Quantin, et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584 (1992); See Rosenfeld, et al., Cell, 68: 143-155 (1992).

  Another delivery method is to use single stranded DNA producing a vector capable of producing a product expressed in cells. See, for example, Chen et al, BioTechniques, 34: 167-171 (2003), which is incorporated herein by reference in its entirety.

  Another delivery method is to use single stranded DNA producing a vector capable of producing a product expressed in cells. See, for example, Chen et al, BioTechniques, 34: 167-171 (2003), which is incorporated herein by reference in its entirety. The polynucleotides described herein can be used with microdelivery vehicles such as cationic liposomes and adenoviral vectors. See Mannino and Gould-Fogerite, BioTechniques, 6: 682 (1988) for a discussion of procedures for liposome formation, targeting and delivery of contents. See also Felgner and Holm, Bethesda Res. Lab. Focus, 11 (2): 21 (1989) and Maurer, RA, Bethesda Res. Lab. Focus, 11 (2): 25 (1989).

  In certain embodiments of the invention, non-viral vectors can be used to generate transfections. Methods for non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or polycations or complexes of nucleic acids, naked DNA, Includes intake of artificial virions and drug-enriched DNA. Lipofections are described, for example, in US Pat. Nos. 5,049,386, 4,946,787: and 4,897,355), and lipofection reagents are commercially available (eg, Transfectam and Lipofectin). Cationic and neutral lipids suitable for lipofection that recognize sufficient receptors of the polynucleotide include those described in Jessee US Patent Publication No. 7,166, 298 or Jesse US Patent Publication No. 6,890, 554, each of which is incorporated herein by reference. The contents are incorporated by reference. Delivery can be to cells (eg, in vitro or ex vivo administration) or target tissues (eg, in vivo administration).

  Synthetic vectors are typically cationic lipids or polymers that can be complexed with negatively charged nucleic acids to form particles within an indicator diameter of 100 nm. The complex protects the nucleic acid from degradation by nucleases. In addition, cellular and localized delivery strategies must address the need for internalization, release, and distribution in appropriate subcellular compartments. Synthetic delivery strategies encounter additional difficulties, such as interaction of cationic delivery vehicles with blood components, uptake by the reticuloendothelial system, filtration of the liver, toxicity and the ability of the carrier to target the cells of interest . Modifying the surface of the cationic non-virus can minimize interactions with blood components, reduce uptake of the reticuloendothelial system, reduce toxicity, and increase sexual compatibility with target cells. Plasma protein binding (also called opsonization) is the main mechanism in the RES for recognizing circulating nanoparticles. For example, Kupffer cells in macrophages, eg liver, recognize opsonized nanoparticles via scavenger receptors.

  The nucleic acid sequences of the invention can be delivered to appropriate cells of interest. This may be achieved, for example, by the use of a biodegradable, microparticulate or microcapsule delivery vehicle of a size, which is sized to optimize phagocytosis by phagocytes such as macrophages. For example, PLGA (poly-lacto-co-glycolide) microparticles of about 1 to 10 μm in diameter may be used. The polynucleotide is encapsulated in such microparticles, taken up by macrophages and gradually biodegraded intracellularly, thereby releasing the polynucleotide. Once released, the DNA is expressed intracellularly. The second type of microparticles is not intended to be directly taken up by cells, but as a sustained release host of sustained release nucleic acid that is taken up by cells through biodegradation when released from the microparticles It is intended to work primarily. These polymer particles must therefore be large enough to eliminate phagocytosis (ie, greater than 5 μm, preferably greater than 20 μm). Another way of achieving uptake of nucleic acids is to use liposomes generated in a standard way. The nucleic acids may be incorporated alone into these delivery vehicles, or may be interdigitated into tissue specific antibodies, such as target cell type antibodies, which are generally latent infection bearing hosts of HIV infection. Alternatively, one can generate a molecular complex composed of a plasmid or other vector that is attached to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine binds to a ligand that can bind to the receptor of the target cell. Delivery of "bare DNA" (ie, without the delivery vehicle) to the intramuscular, intradermal or subcutaneous site is another way to achieve in vivo expression. A nucleic acid encoding an isolated nucleic acid sequence comprising a sequence encoding a guide RNA complementary to the CRISPR / Cas and / or HIV target sequences described above in related polynucleotides (eg, expression vectors) Array.

  In some embodiments, the complexes of the invention are high molecular weight complexes surrounded by nanoparticles, for example, a low molecular weight LPEI shell complexed with DNA and modified with polyethylene glycol (PEGylated) It can be formulated as nanoparticles comprising a core of linear polyethylenimine (LPEI). In some embodiments, the complex can be formulated as nanoparticles encapsulating the complex embodied herein. L-PEI has been used to efficiently deliver genes in vivo to a wide range of organs such as lung, brain, pancreas, retina, bladder and tumors. L-PEI can efficiently condense, immobilize and deliver nucleic acids in vitro and in vivo.

  In some embodiments, delivery of the vector can also be mediated by exosomes. Exosomes are lipid nanovesicles released by many cell types. They mediate intercellular communication by transporting nucleic acids and proteins between cells. Exosomes comprise RNAs, miRNAs and proteins derived from endocytic pathways. These can be taken up by target cells by endocytosis, fusion, or both. Exosomes can suppress delivery of nucleic acids to specific target cells.

  The expression construct of the present invention can be delivered using nanoclew. The nanocrew is a scaly DNA nanocomposite (Sun, et al., J. Am. Chem. Soc. 2014, 136: 14772-14725). They can be taken up by target cells and loaded with nucleic acid for release within the target cytoplasm. A method for constructing nanocrew, loading them and designing molecules to be released can be found in Sun, et al. (Sun W, et al., J. Am. Chem. Soc. 2014, 136: 14772-14725; Sun W, et al., Angew. Chem. Int. Ed. 2015: 12029-12033).

  The nucleic acids and vectors may also be applied to the surface of the device (eg, a catheter) or may be included in a pump, patch, or any other drug delivery device. The nucleic acids and vectors disclosed herein can be administered alone or in a mixture in the presence of a pharmaceutically acceptable excipient or carrier (eg, saline). The excipient or carrier is selected based on the mode and route of administration. Suitable pharmaceutical carriers, and pharmaceutical necessities for use in pharmaceutical formulations, can be found in the relevant references in the art, Remington's Pharmaceutical Sciences, EW Martin, and USP / NF (United States Pharmacopeia and the National Formulary). It is described in.

  In some embodiments of the invention, liposomes are used to effect transfection in cells or tissues. The pharmacodynamics of liposomal formulations of nucleic acids are largely determined by which nucleic acids are entrapped within the bilayer of the liposome. The encapsulated nucleic acid is protected from nuclease degradation, but not just that associated with the surface of the liposome. The encapsulated nucleic acids share the extended circulating life and biodistribution of the intact liposomes, while those associated at the surface introduce the pharmacology of the naked nucleic acid when separated from the liposomes. Nucleic acids can be encapsulated in liposomes by conventional passive loading techniques, such as ethanol droplet method (such as SALP), reverse phase evaporation method, ethanol dilution method (such as SNALP).

  Liposomal delivery systems provide stable formulations and provide improved pharmacokinetics and some "passive" or "physiological" targeting to tissues. The encapsulation of hydrophilic and hydrophobic substances, such as potential chemotherapeutic agents, is known. Schneider, U.S. Pat. No. 5,466,468, discloses parenteral liposome formulations containing synthetic lipids; Nucleoside analogues coupled with phospholipids, U.S. Pat. No. 5,580,571, pharmaceutically; Nyqvist disclosing a pharmaceutical composition in which the active compound is heparin or a fragment thereof comprising a defined lipid system comprising at least one amphiphilic and polar lipid component and at least one non-polar lipid component See U.S. Pat. No. 5,626,869.

  Liposomes and polymerosomes can contain multiple solutions and compounds. In certain embodiments, the complexes of the invention are linked or encapsulated to polymersomes. As a class of artificial vesicles, polymersomes are small hollow beads containing solutions, made using amphiphilic synthetic block copolymers to form vesicle membranes. Common polymersomes contain an aqueous solution in the nucleus and are useful for encapsulating and protecting reactive molecules such as drugs, enzymes, other proteins and peptides, and DNA and RNA fragments. is there. Polymersomes membranes provide a physical barrier that allows the inclusion material to be isolated from extraneous material, such as those found in biological systems. Since Polymerosomes can be produced from double emulsions by known techniques, Lorenceau et al., 2005, Generatoin of Polymerosomes from Double-Emulsions, Langmuir 21 (20): 9183-6 Please refer to.

  In some embodiments of the invention, the non-viral vector is modified to result in targeted delivery and transfection. PEGylation (ie, surface modification with polyethylene glycol) is the predominant method used to reduce opsonization and aggregation of non-viral vectors and minimize clearance by the reticuloendothelial system, Provides an extended circulatory life after intravenous (iv) administration. PEGylated nanomolecules are therefore often referred to as "stealth" nanomolecules. Nanomolecules that are not rapidly cleared from the circulation are likely to encounter infected cells.

  In some embodiments of the present invention, a targeted controlled release system is utilized that responds to the tissue's specific environment and external stimuli. Gold nanorods have strong absorption bands in the near infrared region, and the absorbed light energy is then converted into heat by the gold nanorods, which is the so-called "photothermal effect". The surface of the gold nanorods can also be modified with nucleic acids for controlled release, as near infrared light can penetrate deep into tissues. When the modified gold nanorods are irradiated with near-infrared light, nucleic acids are released due to the thermal degradation facilitated by the photothermal effect. The amount of nucleic acid released depends on the power of light irradiation and the exposure time.

  Regardless of whether the compositions are administered as nucleic acids or polypeptides, they are formulated in such a way as to facilitate their uptake by mammalian cells. Useful vector systems and formulations are described above. In some embodiments, the vector can deliver the composition to a particular cell type. Although not limiting the present invention, other methods of DNA delivery, such as chemical transfection such as calcium phosphate, DEAE dextran, liposomes, lipoplex, detergents, and perfluoro liquid drug, may be used. It is also contemplated for use as a delivery method, such as electroporation, microinjection, ballistic particles, and a "gene gun" system.

  In another embodiment, the composition comprises cells transformed or transfected with one or more CRISPR / Cas vectors and gRNA. In some embodiments, the methods of the present invention may be applied ex vivo. That is, cells of a subject can be removed from the body and treated with the composition in culture, for example, to excise HIV sequences, and the treated cells can be returned to the body of the subject. The cells may be cells of interest or may be matched haplotypes or cell lines. The cells can be irradiated to inhibit replication. In some embodiments, the cells are autologous, matched, or a combination thereof matched to human leukocyte antigen (HLA). In another embodiment, the cells may be stem cells. For example, embryonic stem cells or induced pluripotent stem cells (induced pluripotent stem cells (iPS cells)). Embryonic stem cells (ES cells) and induced pluripotent stem cells (induced pluripotent stem cells (iPS cells) have been established from many animal species including human. These cells remain pluripotent These types of pluripotent stem cells are the most useful source of cells for regenerative medicine, since they possess the ability to actively divide and can differentiate into almost any organ by induction that is appropriate for their differentiation. IPS cells can be constructed, inter alia, from autologous somatic cells and are therefore less likely to cause ethical or social problems when compared to ES cells produced by the destruction of the embryo. Rejection, which is an autologous cell and is the greatest obstacle to regenerative medicine or transplantation, can be avoided.

Transduced cells are generated for reinfusion by established methods. After a period of about 2 to 4 weeks in culture, cells can reach a count of between 1 × 10 ⁶ and 1 × 10 ¹⁰ . In this regard, the growth characteristics of cells vary from patient to patient and from cell type to cell type. About 72 hours prior to reinfusion of transduced cells, one aliquot is taken for analysis of phenotype and percentage of cells expressing the therapeutic agent. For administration, the cells of the invention can be administered at a rate determined by the side effects of the cell type at various concentrations, as well as the LD _{50 of} the cell type and the majority and overall health of the patient. Administration can be accomplished via single or divided doses. Adult stem cells can also be recruited using extraneously administered elements that stimulate their production and exit tissue or space, which can include, but is not limited to, bone marrow or adipose tissue.

Pharmaceutical Compositions As noted above, the compositions of the present invention may be produced in various ways known to those skilled in the art. Regardless of their primary source or the way in which they are obtained, the compositions disclosed herein are formulated for their use. For example, the nucleic acids and vectors described above can be formulated into compositions for application to cells in tissue culture, or for administration to patients and subjects. Any of the pharmaceutical compositions of the present invention may be formulated for use in formulating a drug, the particular use being therapeutic, eg having HIV infection or suffering from HIV infection and risk of HIV infection It may be shown below in connection with the treatment of a subject. When used as pharmaceuticals, any nucleic acid and vector may be administered in the form of a pharmaceutical composition. These compositions can be produced in a manner known in the pharmaceutical art and can be administered by different routes depending on whether local or systemic treatment is desired and on the area to be treated Can. Administration may be topical (including ophthalmic, as well as intranasal, intravaginal, intrarectal administration to mucous membranes), lung (eg, inhalation or insufflation of powder or aerosol including nebulizers; intratracheal, nasal cavity (Internal, epidermal, transdermal), ocular, oral, parenteral. Methods of delivery to the eye may include topical administration (eye drops), subconjunctival, periocular, intravitreal injection, or introduction with a balloon catheter, or an ophthalmic insert surgically placed in the conjunctival sac. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, eg, intrathecal or intracerebroventricular administration. Parenteral administration may be in the form of a single bolus dose, or may be, for example, by a continuous infusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders and the like. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

  The pharmaceutical composition may comprise, as an active ingredient, the nucleic acids and vectors described herein in combination with one or more pharmaceutically acceptable carriers. To make the compositions of the present invention, the active ingredient is typically mixed with excipients, diluted by excipients, or, for example, capsules, tablets, sachets, papers Or enclosed in a carrier in the form of another container. If the excipient acts as a diluent, it can be a solid, semi-solid, liquid substance (eg, saline), which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be tablets, pills, powders, troches, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or liquid media), lotions, creams, ointments, gels. It may be in the form of soft or hard gelatine capsules, suppositories, sterile injectable solutions, and sterile packaged powders. As known in the art, the type of diluent may vary depending on the intended route of administration. The resulting composition may include additional agents, such as preservatives. In some embodiments, the carrier can be or include lipid based or polymer based colloids. In some embodiments, the carrier material can be a liposome, hydrogel, microparticles, nanoparticles, or colloid formulated as block copolymer micelles. As mentioned above, the carrier material can form a capsule, which can be a polymer based colloid.

  The nucleic acid sequences of the invention can be delivered to appropriate cells of interest. This can be achieved, for example, by the use of biodegradable, microparticulate or microcapsule delivery vehicles of a size sized to optimize phagocytosis by phagocytes such as macrophages. For example, PLGA (poly-lacto-co-glycolide) microparticles of about 1 to 10 μm in diameter may be used. The polynucleotide is encapsulated in these microparticles, taken up by macrophages and gradually biodegraded intracellularly, thereby releasing the polynucleotide. Once released, the DNA is expressed intracellularly. The second type of microparticle is not intended to be directly taken up by cells, but as a sustained release host of nucleic acid which is taken up by cells only when released from the microparticle through biodegradation It is intended to work primarily. These polymer particles must therefore be large enough to prevent phagocytosis (ie, greater than 5 μm, preferably greater than 20 μm). Another way of achieving uptake of nucleic acids is to use liposomes generated in a standard manner. The nucleic acid can be incorporated alone into these delivery vehicles, or a tissue specific antibody, such as a target cell type that is generally a latent infection bearing host of HIV infection, eg, brain macrophages, microglia, astrocytes Cells and gastrointestinal tract-associated lymphoid cells can be co-incorporated. Alternatively, one can generate a molecular complex comprising a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine binds to a ligand that can bind to the receptor in the target cell. Delivery of "bare DNA" (ie without a delivery vehicle) to intramuscular, intradermal or subcutaneous sites is another way to achieve in vivo expression. A nucleic acid sequence encoding an isolated nucleic acid sequence comprising a sequence encoding an CRISPR-related endonuclease, in a related polynucleotide (eg, an expression vector), and optionally a guide RNA, as described above It is operably linked to the minimal HIV LTR promoter responsive to Tat.

  In some embodiments, the composition of the present invention comprises nanoparticles, eg, a core of high molecular weight linear polyethylenimine (LPEI) complexed with DNA, and is polyethylene glycol modified (PEGylated) low molecular weight LPEI It can be formulated as nanoparticles, surrounded by shells.

  The nucleic acids and vectors may also be applied to the surface of the device (eg, a catheter) or may be included in a pump, patch, or other drug delivery device. The nucleic acids and vectors disclosed herein can be administered alone or in mixtures, in the presence of a pharmaceutically acceptable excipient or carrier (eg, saline). The excipient or carrier is selected based on the mode and route of administration. Suitable pharmaceutical carriers, and pharmaceutical supplies for use in pharmaceutical formulations, are known references in the art, Remington's Pharmaceutical Sciences (EW Martin), and USP / NF (United States Pharmacopeia and the National Formulary). It is described in.

  In some embodiments, the composition can be formulated as a topical gel to prevent sexual transmission of HIV. The topical gel can be applied directly to the skin or mucous membrane of the male or female genital area prior to sexual activity. Alternatively or additionally, the topical gel may be applied to the surface or contained within a male or female condom or pessary.

  In some embodiments, the composition can be formulated as a nanoparticle that encapsulates a nucleic acid encoding a Cas9 or Cas9 variant operably linked to an HIV LTR promoter. The nucleic acid may additionally encode a guide RNA sequence that is complementary to the target HIV.

  The formulations of the present invention may comprise a vector encoding Cas9 and a guide RNA complementary to the target HIV. The guide RNA sequences may comprise sequences complementary to a single target region, or may comprise any combination of sequences complementary to multiple target regions as described above. Alternatively, the HIV LTR minimal promoter driven sequence encoding Cas9 and the sequence encoding the guide RNA sequence may be separate vectors.

  The compositions disclosed herein are generally and variously useful for the treatment of subjects having HIV infection. The method is useful for targeting any HIV, such as HIV-1 and HIV-2, and SIV, and any circulating recombinant forms thereof. The subject is treated efficiently regardless of when the clinically beneficial outcome occurs. This can mean, for example, complete resolution of the symptoms of the disease, reducing the severity of the symptoms of the disease, or delaying the progression of the disease. These methods include: a) identifying subjects with HIV infection (e.g. patients, more strictly speaking human patients); and b) Tat response under the control of the minimal promoter of HIV to the CRISPR. It may further comprise the step of providing to the subject a composition comprising a nucleic acid encoding a related nuclease, for example Cas9. These methods may further include providing the target with a target sequence of HIV, eg, a sequence encoding a guide RNA complementary to the HIV LTR.

  Subjects can be identified using standard clinical tests, eg, immunoassays that find the presence of HIV antibody or HIV polypeptide p24 in the serum of the subject, or through amplification analysis of HIV nucleic acid. The amount of such composition provided to the subject that results in the complete resolution of the infection symptoms, a decrease in the severity of the infection symptoms, or a delay in the progression of the infection is considered to be a therapeutically effective amount. . The methods of the invention may also include measuring steps to help optimize dosing and scheduling, and predict outcome. In some methods of the invention, it is first determined whether the patient has latent HIV infection, and then the subject is treated with one or more of the compositions described herein. Can be determined. Measuring can also be used to find the development of drug resistance and to rapidly differentiate responding patients from non-responding patients. In some embodiments, the method further determines the nucleic acid sequence of particular HIV harbored by the patient and further designing the guide RNA to be complementary to the particular sequence. May be included. For example, the nucleic acid sequence of the LTR U3, R, or U5 region of interest is determined, and from that it is exactly complementary to the patient's sequence without further selection of a sequence to be part of the minimal promoter of Tat response HIV. One or more guide RNAs can be designed.

  The compositions are also useful for treatment, eg, prophylactic treatment of subjects at risk for having a retroviral infection, eg, HIV infection. These methods a) identify subjects at risk for having HIV infection; b) Tat response Under the control of a minimal promoter of HIV-1 LTR, a nucleic acid encoding a nuclease associated with the CRISPR, such as Cas9, The method may further comprise the step of providing the composition comprising the subject. The sequences may additionally encode guide RNAs that are complementary to HIV target sequences, such as the HIV LTR. Subjects at risk for having HIV infection include, for example, sexually active individuals who participate in unprotected sexual activity, ie sexual activity without the use of condoms; sexually active individuals with another sexually transmitted disease; It may be a user of an intravenous drug; or a male who has not been circumcised. The subject at risk for having HIV infection may be, for example, an individual who may be contacted by people with HIV infection by occupation, such as a healthcare professional or a first responder. Subjects at risk for having HIV infection may be, for example, detainees in correctional settings, or sex workers, ie, occupational income or non-monetary matters, eg, for food, drugs, or for sexual retention. It may be an individual who uses an action.

  The composition may be administered to pregnant, lactating women with HIV infection to reduce the chance of mother-to-child HIV infection. Pregnant women who are infected with HIV can infect the offspring with the virus in utero via the placenta, through the birth canal at delivery, or through breast milk after delivery. The compositions disclosed herein can be administered to a mother infected with HIV prenatal, perinatal, postnatal, during lactation, or any combination prenatal, perinatal, postnatal. The compositions can be administered to the mother together with standard antiviral therapies as described below. The compositions of the invention may also be administered to the infant immediately after delivery in some embodiments, and periodically thereafter in some embodiments. Infants can also receive standard antiviral therapy.

  The composition can be administered to an individual who is not infected with HIV to prevent HIV infection. The composition can include delivering a therapeutically effective amount of the pharmaceutical composition. The pharmaceutical composition may comprise the CRISPR-related endonuclease described above, and a sequence encoding at least the HIV LTR promoter and the TAR region of the minimal promoter of the Tat-responsive HIV LTR.

  The methods disclosed herein include a wide range of species such as humans, non-human primates (eg monkeys), horses or other domestic animals, dogs, cats, ferrets or other mammals kept as pets, rats , Mice, or other experimental animals.

  The methods of the invention can be expressed in terms of drug formulation. Thus, the present invention includes the use of the agents and compositions described herein in the preparation of a medicament. The compounds described herein are useful for the manufacture of a medicament for use in therapeutic compositions and regimens, or in the treatment of the diseases or disorders described herein.

  Any of the compositions described herein can be administered to any part of the host's body for subsequent delivery to target cells. The composition can be delivered to, without limitation, mammalian brain, cerebrospinal fluid, joints, nasal mucosa, blood, lung, intestine, muscle tissue, skin, or abdominal cavity. From the delivery route point of view, the composition is orally, by intravenous, intracranial, intraperitoneal, intramuscular, subcutaneous, intramuscular, intrarectal, intravaginal, intrathecal, intratracheal, intradermal or intradermal injection. It may be administered by administration or intranasal administration, or by prolonged stepwise perfusion. As a further example, an aerosol preparation of the composition can be given to the host by inhalation.

  The dosage required may be influenced by the route of administration, the nature of the formulation, the nature of the patient's illness, the size of the patient, body weight, surface area, age, sex, other agents being administered, and the judgment of the attending clinician. Wide variations in the required dose should be expected in view of the diversity of cellular targets and the different efficiencies in different administration routes. Variations in these dose levels can be adjusted using standard empirical routines for optimization, as is well understood in the art. Administration can be single or multiple times (eg, 2 or 3 times, 4 times, 6 times, 8 times, 10 times, 20 times, 50 times, 100 times, 150 times or more). Encapsulation of the compound within a suitable delivery vehicle (eg, polymeric microparticles or implantable devices) can increase the efficiency of delivery.

  The duration of treatment with any of the compositions provided herein can be as short as a day, to as long as the lifespan of the host (eg, many years), or any length of time It can also be done. For example, the compound may be once a week (eg, 4 weeks to several months or many years); once a month (eg, 3 to 12 months or many years); 5 years, 10 years or more It may be administered once a year for an interval. It should also be noted that the frequency of treatment may also be variable. For example, the compounds of the invention may be administered on a daily, weekly, monthly, monthly basis (or twice, three times, etc.).

  An effective amount of any of the compositions provided herein may be administered to an individual in need of treatment. An effective amount can be determined by assessing the patient's response after administering a known amount of the particular composition. In addition, the level of toxicity, if any, can be determined by assessing the patient's clinical condition before and after administering a known amount of the particular composition. It should be noted that the effective amount of a particular composition administered to a patient can be adjusted according to the desired result, and the level of response and toxicity of the patient. Significant toxicity can vary with each particular patient, and depends on complex factors including, without limitation, the patient's condition, age, and tolerance to side effects.

  Any method known to one of skill in the art can be used to determine if a particular reaction has been elicited. Clinical methods that can assess the extent of a particular medical condition can be used to determine if a response is elicited. The particular method used to assess the response may depend on the patient's disease, the patient's age and sex, the other medication being administered, and the nature of the judgment of the attending clinician.

  The composition may also be administered with another therapeutic agent, such as an antiretroviral agent used in HAART, a chemotherapeutic agent, a transcriptional activator of HIV such as PMA, TSA, etc. Antiretroviral agents include reverse transcriptase inhibitors (eg, nucleoside / nucleotide reverse transcriptase inhibitors, zidovudine, emtricitibine, lamivudine, tenofovir; and non-nucleoside reverse transcriptase inhibitors such as efavirenz, nevirapine) Protease inhibitors such as tipiravir, darunavir, indinavir; entry inhibitors such as marabiroc; fusion inhibitors such as enfuviritide; or integrase inhibitors such as raltegravir (Raltegrivir), dolutegravir (dolutegravir) may be included. Anti-retroviral agents are agents that combine multiple types (multi-class combination agents), for example, a combination of enticitabine, efavirenz, and tenofovir; a combination of enticitabine; rilpivirine, and tenofovir; , A combination of entericitabine and tenofovir may also be included.

  In addition, any other symptoms that may be associated with viral infections, such as fever, chills, headaches, secondary infections, and one or more additional agents, together or as part of a pharmaceutical composition, Or it can be administered at separate times. These agents include, without limitation, antipyretics, antiinflammatory agents, chemotherapeutic agents, or combinations thereof.

  In certain embodiments, the antiviral agent comprises the following therapeutically effective amounts; antibodies, aptamers, adjuvants, antisense oligonucleotides, chemokines, cytokines, immunostimulatory agents, immunomodulators, B cell modulators, T cells Modulator, NK cell modulator, antigen presenting cell modulator, enzyme, siRNA, interferon, ribavirin, protease inhibitor, helicase inhibitor, neuraminidase inhibitor, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, purine nucleoside, Chemokine receptor antagonists, interleukins, vaccines or combinations thereof.

  Immunomodulatory molecules include, but are not limited to, cytokines, lymphokines, T cell costimulatory ligands, and the like. The immunomodulatory molecules positively and / or negatively affect the humoral and / or cellular immune system, in particular its interactions with its cellular and / or non-cellular components, its functions and / or other physiological systems. The immunomodulatory molecule may be selected from the group comprising: cytokines, chemokines, macrophage migration inhibitory factor (MIF; in particular, Bernhagen (1998), Mol Med 76 (3-4); 151-61 or Mets (1997), Adv Immunol 66, 197-223), T cell receptors or soluble MHC molecules. Such immunomodulatory effector molecules are known in the art and are described, inter alia, in Paul, "Fundaental immunology", Raven Press, New York (1989). In particular, known cytokines and chemokines are described in Meager, “The Molecular Biology of Cytokines” (1998), John Wiley & Sons, Ltd., Chichester, West Sussex, England; (Bacon (1998). Cytokine Growth Factor Rev 9 (2) Clin Cancer Res 12, 2682-6; Taub, (1994) Ther. Immunol. 1 (4), 229-46 or Michiel, (1992). Semin Cancer Biol 3 (1). , 3-15).

  Immune cell activities that can be measured include, but are not limited to: (1) cell proliferation by measuring DNA replication; (2) cytokines such as IFN-γ, GM-CSF, or TNF -improved cytokine production, including specific measurement of alpha, (3) cells mediating killing or lysing targets; (4) cell differentiation; (5) immunoglobulin production; (6) (7) chemotaxis production, meaning the ability to respond to chemotaxis agents or chemotactin (chemotactin), (8) of several other immune cell types Immunosuppression by inhibiting the ability, (9) Apoptosis, which refers to the fragmentation of activated immune cells under certain circumstances at the onset of aberrant activation.

  The enzymes of the invention in a lytic package for delivering cytotoxic T lymphocytes, or LAK cells to a target, are also of interest. Perforin, pore-forming protein, and Fas ligand are the major cytolytic molecules in these cells (Brandau et al., Clin. Cancer Res. 6: 3729, 2000; Cruz et al., Br. J. Cancer 81: 881, 1999). CTLs also express a group of at least 11 serine proteases called granzymes, with primary substrate specificities of 4 (Kam et al., Biochim. Biophys. Acta 1477: 307, 2000) . Low concentrations of streptolysin O and pneumococcal hemolysin promote granzyme B-dependent apoptosis (Browne et al., Mol. Cell Biol. 19: 8604, 1999).

  Other suitable effectors are not toxic to the cells themselves, but can be made non-toxic to the cells either by metabolically altering the cells, or by converting non-toxic prodrugs to lethal agents. It encodes a polypeptide having the ability to be reactive to toxic compounds. Exemplary are herpes simplex virus and thymidine kinase (tk) as derived from catalytically equivalent variants. HSV tk converts the anti-herpes agent ganciclovir (GCV) into a toxic product that interferes with DNA replication in proliferating cells.

  Concurrent administration of two or more therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as the agents exert overlapping therapeutic effects. Simultaneous or sequential administration is considered to be administered on different days or weeks. The therapeutic agent can be administered under metronomic regimes, such as under continuous low doses of the therapeutic agent.

The dose, toxicity and therapeutic efficacy of such compositions can be determined, for example, to determine the LD ₅₀ (the dose fatal to 50% of the population) and the ED ₅₀ (the dose therapeutically effective to 50% of the population) , Cell cultures or by standard preparation procedures in experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index and may be expressed as LD ₅₀ / ED _50.

The data obtained from cell culture assays and animal studies can be used to devise a range of dosages for use in humans. The dosage of such compositions is preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage may vary within the range depending upon the dosage form employed and the route of administration utilized. For any composition used in the methods of the invention, the therapeutically effective amount can be estimated initially from cell culture assays. Doses, as determined in cell culture, to achieve a circulating plasma concentration range that includes the IC ₅₀ (ie, the concentration of test compound that achieves half-maximal suppression of symptoms) , Can be devised in animal models. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

  As described, a therapeutically effective amount (ie, an effective amount) of a composition means an amount sufficient to provide a therapeutically (eg, clinically) desired result. The composition may be administered from one or more times daily, including once every other day, to one or more times weekly. Certain factors, including but not limited to the severity of the disease or disorder, past treatment, the overall health and / or age of the subject, and other current illnesses, effectively affect the subject. One of ordinary skill in the art would understand that it may affect the dose and timing needed to treat. Furthermore, treatment of a subject with a therapeutically effective amount of a composition of the invention may include a single treatment or a series of treatments.

  The compositions described herein are suitable for use in the variety of drug delivery systems described above. In addition, in order to enhance the in vivo serum half-life of the compound to be administered, the composition may be encapsulated, introduced into the lumen of liposomes, produced as a colloid, or prolonged of the composition Other conventional techniques for providing serum half life may be used. Various methods produce liposomes, for example, as described in Szoka, et al., US Pat. Nos. 4,235,871, 4,501,728 and 4,837,028, each of which is incorporated herein by reference. It can be used to Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with an antibody specific for a tissue. Liposomes are targeted and selectively taken up by organs.

Also provided is a method of inactivating a retrovirus, eg, lentivirus, eg, human immunodeficiency virus in a mammalian cell, simian immunodeficiency virus, feline immunodeficiency virus, or bovine immunodeficiency virus. The human immunodeficiency virus can be HIV-1 or HIV-2. Human immunodeficiency virus may be a chromosomally integrated provirus. Mammalian cells include, but are not limited to, CD4 ⁺ lymphocytes, macrophages, fibroblasts, monocytes, T lymphocytes, B lymphocytes, natural killer cells, dendritic cells such as Langerhans cells and vesicles. It may be any cell infected with HIV, including dendritic cells, hematopoietic stem cells, endothelial cells, brain microglia cells, astrocytes and gut epithelial cells. Such cell types are typically those infected during primary infection, eg, CD4 ⁺ lymphocytes, macrophages, monocytes or Langerhans cells, and latent HIV-bearing hosts, ie, cell types that constitute latently infected cells. including.

  The method comprises a composition comprising an isolated nucleic acid encoding a CRISPR-related endonuclease, operably linked to a minimal promoter of HIV LTR, comprising a core region and a TAR region of the HIV LTR promoter. It may comprise exposing and / or contacting cells. The isolated nucleic acid can further comprise one or more guide RNAs, wherein the guide RNAs are complementary to target nucleic acid sequences in the retrovirus. The contacting step can be performed in vivo, ie, the composition can be directly administered to a subject with HIV infection. However, as the method is not limited thereto, the approaching step can also be performed ex vivo. For example, one or more cells, or tissue explants, can be removed from a subject with HIV infection, placed in culture, and then operable to a minimal promoter of HIV LTR. The linked CRISPR-related endonuclease, and optionally the guide RNA may be contacted with a composition comprising a guide RNA complementary to a nucleic acid sequence in HIV. As described above, the pharmaceutical composition may comprise a nucleic acid encoding the endonuclease associated with CRISPR operably linked to the minimal promoter of HIV LTR responsive to Tat.

  A composition is devised in a method for promoting mammalian cell uptake. Useful vector systems and formulations are described above. In some embodiments, a vector can deliver the composition to a particular cell type. Although the invention is not so limited, other methods of DNA delivery, such as calcium phosphate, DEAE dextran, liposomes, surfactants, scientific transfer using perfluorinated liquid drugs are also conceivable, and physical delivery methods, For example, electroporation, microinjection, ballistic particles, and "gene gun" systems are also contemplated.

  Standard methods, eg, immunoassays to find endonucleases associated with CRISPR, or nucleic acid based assays, eg, PCR to find guide RNA, were taken up by cells and / or expressed proteins incorporated It can be used to confirm that. The designed cells can then be reuptaked into the subject from which they are derived, as described below.

  In another embodiment, the composition comprises cells transformed or transfected with a minimal promoter vector of HIV LTR responsive to one or more Cas9 / Tat. In some embodiments, the methods of the present invention may be applied ex vivo. That is, the cells of the subject may be removed from the body, treated with the composition in culture medium to ablate the HIV sequences, and the treated cells may be returned to the body of the subject. The cells can be cells of interest, or haplotypes or cell lines can be matched. Cells can be irradiated to prevent replication. In some embodiments, the cells are autologous, a cell line, or a combination thereof, consistent with human leukocyte antigen (HLA). In another embodiment, the cells may be stem cells. For example, embryonic stem cells or induced pluripotent stem cells (induced pluripotent stem cells (iPS cells)). Embryonic stem cells (ES cells) and induced pluripotent stem cells (induced pluripotent stem cells (iPS cells)) have been constructed from many animal species, including humans. These types of pluripotent stem cells are capable of differentiating into almost any organ by induction suitable for their differentiation while retaining their ability to actively divide while maintaining pluripotency. It can be the most useful source of cells for regenerative medicine. iPS cells can be constructed, inter alia, from autologous somatic cells and are therefore less likely to cause ethical or social problems as compared to ES cells generated by the destruction of the embryo. Furthermore, iPS cells are autologous cells and can avoid rejection, which is the greatest obstacle to regenerative medicine or transplantation therapy.

  The compositions described herein are classified, eg, for use as a therapy for treating a retrovirus infection, eg, a subject having HIV infection, or a subject having a retrovirus infection, eg HIV infection. , Can be packaged in a suitable container. The container may comprise a composition comprising an endonuclease associated with CRISPR, such as Cas9 endonuclease, described above, and a nucleic acid sequence encoding a minimal promoter of the HIV LTR responsive to Tat. The sequence may additionally be a guide RNA complementary to the target sequence in HIV, or a vector encoding a nucleic acid, or one or more suitable stabilizers, carrier molecules, flavors, and / or for intended use. Others may be appropriate to In addition, a packaged product (eg, packaged for storage, shipping, or sale at a concentrated or ready-to-use concentration) comprising at least one disclosed composition, as described herein Sterile containers) and kits comprising one or more of the compositions of The product may comprise a container (eg, a vial, a bottle, a bottle, a bag or other) containing one or more compositions of the present invention. In addition, the article of manufacture may further comprise packaging material, instructions for use, syringes, delivery devices, buffers, or other control reagents for the treatment or measurement of diseases which require prophylaxis or treatment. In some embodiments, the kit may include one or more additional antiretroviral agents, such as reverse transcription inhibitors, protease inhibitors or entry inhibitors. An additional agent is complementary to the nucleic acid sequence encoding the CRISPR-related endonuclease, eg, Cas9 endonuclease, and optionally to a target sequence in HIV operably linked to the minimal promoter of the HIV LTR. It can be co-encapsulated in the same container as the guide RNA, or the vector encoding the nucleic acid, or it can be encapsulated separately.

  The product may also include instructions (eg, printed labels or inserts or other media that describe the use of the product (eg, audio or video tapes)). The instructions may be associated with the container (eg, attached to the container) and may describe the manner in which the composition is to be administered (eg, frequency and route of administration), instructions therefor, and other uses. . The composition may be ready for administration (e.g., the dose is in a suitable unit), one or more additional pharmaceutically acceptable adjuvants, carriers or others. And / or additional therapeutic agents. Alternatively, the composition may be provided in a concentrated form with diluents and instructions for dilution.

  While various embodiments of the present invention are described above, it should be understood that they have been presented by way of illustration only and not limitation. Numerous modifications to the disclosed embodiments can be made without departing from the spirit or scope of the present invention in accordance with the disclosure herein. Thus, the breadth and scope of the present invention should not be limited to any of the above described embodiments.

  All documents mentioned herein are incorporated by reference. All publications and patent documents cited in the present application are incorporated by reference for the same purpose as if each individual published publication or patent document is separately indicated. By reference to the various citations in this document, applicants are not entitled to recognize any particular citation as "prior art" of the present invention.

EXAMPLES The following non-limiting examples are provided to illustrate selected embodiments of the present invention. Modifications and changes in proportions of elements of components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention.

Example 1: Negative Feedback Regulation of HIV-1 by Gene Editing In the work presented herein, the techniques of gene editing have been improved and viral reactivation by Tat, a transcriptional activator of HIV-1. A new strategy has been developed that allows for conditional activation of CRISPR / Cas9 at an early stage. This new strategy permanently removes viral replication prior to productive viral replication by removing the viral promoter and / or the segment of the viral gene that extends to the viral coding sequence. Furthermore, this strategy solves some of the concerns attributable to unforeseen complexities that may be caused by high levels of unwanted and sustained expression of Cas9 in cells.

Materials and Methods Preparation of plasmids. The full-length and truncated LTR promoter sequences were obtained by PCR using pNL4-3 HIV vector (NIH AIDS Reagent Program # 114) as a template and the primers described in Table 1 (SEQ ID NO: 1-4) . The PCR product is gel purified, subcloned directly into a TA vector (Invitrogen), excised with Kpn1 or Xba1 and Nco1 restriction enzymes, and Kpn1-Nco1 or Xba1-Nco1 digested pX260-U6-DR-BB-DR-Cbh- It was ligated to the NLS-hSpCas9-NLS-H1-shorttracr-PGK-puro plasmid (Addgene # 42229). Consequently, the original Cbh promoter (Xba1-Kpn1-Cbh-Nco1) of the pX260 plasmid was removed and replaced with one of the LTR promoters (Xba1- or Kpn1-LTR-Nco1). For the construction of lentiviral LTR-Cas9 construct, lentiple Cas9-Blast (Addgene # 52962) is treated with Nhe1 / Xba1 resulting in removal of the EFS promoter sequence and digestion of the PCR product Nhe1-LTR-80 The Lenti-LTR-80 / + 66-Cas9-Blast plasmid was generated which was substituted by / + 66-Xba1 (Table 1, SEQ ID NOs: 5, 6). The pKLV-U6-LTR A / B-PGK puro2 ABFP lentiviral plasmid was previously described (Kaminski, R., et al. Sci. Rep. 6: 22555 (2016)). The pcDNA3.1 control vector is purchased from Invitrogen and pCMV-Tat86 has been described previously (Gallia, GL, et al. Proc. Natl. Acad. Sci. USA 96, 11572-11577 (1999)). pKLV-U6-LTR A / B-PGKpuro2ABFP was prepared by cotransfecting HEK293T cells with pMDLg / pRRE (Addgene 12251), pRSV-Rev (Addgene 12253) and pCMV-VSV-G (Addgene 8454) Packaged in retroviral particles. The following vectors were used to package Cas9 in lentiviral particles: Lenti-LTR-80 / + 66-Cas9-Blast, psPAX2 (Addgene 12260) and pCMV-VSV-G (Addgene 8454).

Cell culture. The TZM-bl reporter cell line was obtained from the NIH AIDS Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH). Jurkat, the Clone E6-1 and U937, were purchased from ATCC (TIB-152 ^TM and CRL1593.2 ^TM). The Jurkat 2D10 reporter cell line has been previously described (Pearson, R., et al. J. Viral. 82: 12291-12303 (2008)). TZM-bl cells were cultured in high glucose DMEM supplemented with 10% FBS and gentamycin (10 μg / ml). Jurkat cells and Jurkat 2D10 cells were cultured in RPMI medium containing 10% FBS and gentamycin (10 μg / ml). Primary cultures of human astrocytes and microglia were obtained from the Tissue Culture Core facility at the Comprehensive NeuroAIDS Center (CNAC) of the Department of Neuroscience, Department of the Lewis Katz School of Medicine, Temple University, Philadelphia.

Stable cell lines and subcloning. TZM-bl cells were plated at 1 × 10 ⁵ cells / well in 6-well plates and transfected with 1 μg of pX260-LTR _{(-80 / + 66)} -Cas9 plasmid using Lipofectamine 2000 reagent (Invitrogen) . The following day, cells were transferred to 100-mm dishes and cultured in the presence of 1 μg / ml of puromycin (Sigma). After two weeks, surviving clones were isolated using a cloning cylinder (Corning). Two million Jurkat 2D10 cells were electroporated with 10 μg of pX260-LTR _{(-80 / + 66)} -Cas9 plasmid (Neon System, Invitrogen, 3 × 10 ms / 1350 V impulses). After 48 hours, the medium was replaced with medium containing 0.5 μg / ml puromycin. After one week, puromycin for selection was removed and cells were grown for another week. Next, cells were diluted to a concentration of 10 cells / ml, placed in a 96-well plate at 50 μl / well, and cultured for 2 weeks. Both single cell clones of TZM-bl pX260-LTR _{(-80 / + 66)} -Cas9 and Jurkat 2D10 pX260-LTR _{(-80 / + 66)} -Cas9 are the control empty pCMV plasmid (pcDNA3.1) Alternatively, Western blot was selected for Cas9-FLAG expression after transfection with pCMV-Tat plasmid. Single cell clones with undetectable / very low levels of Cas9 under control conditions and very high levels of Cas9 under Tat overexpression were expanded and used for further experiments.

Lentiviral packaging. HEK 293T cells were cotransfected with the packaging lentiviral vector mixture at a concentration of 30 μg total DNA / 2.5 × 10 ⁶ cells / 100 mm dish using CaPO ₄ precipitation in the presence of chloroquine (50 μM). The next day, the medium was replaced, the supernatant was collected after 24 and 48 hours, clarified at 3000 RPM for 10 minutes, filtered through a 0.45 μm filter, and (2 hours, 25000 RPMI, 20% sucrose cushion was used ) It was concentrated by ultracentrifugation. The lentiviral pellet was resuspended in HBSS by gentle agitation overnight, aliquoted and titered on HEK 293T cells. Lenti-LTR _{(-80 / + 66)} Cas9-Blast lentivirus is titrated by FLAG immunocytochemistry, pKLV-U6-LTR A / B-PGKpuro2 ABFP lentivirus is titrated by BFP fluorescence microscopy It was measured.

Stock of viruses. Fusion PCR (Heckman, KL, et al. Nat. Protoc. 2, 924-932 (2007)), EGFP gene, P2A auto-cleavage type to generate HIV-1 _{NL4-3-EFGP-p2A-Nef} Amplifies the N-terminus of the peptide (Kim, JH, et al. PLoS One 6, e 18556 (2011)) and the HIV splice acceptor and in frame HIV-1 Nef for expression of the original HIV-1 Nef Was used for The DNA was then cloned into the BamHI and XhoII restriction sites of the HIV-1 provirus clone pNL4-3 (Adachi, A., et al. J. Viral. 59, 284-291 (1986)). . A self-cleaving P2A peptide derived from swine fever virus-1 that is present between GFP and Nef enabled expression of HIV-1 Nef in full-length (Edmonds, TG, et al. PBMC. Virology 408, 1-13 (2010)). To generate pEcoHIV-NL4-3-EGFP, following previously published gene manipulation strategies (Potash, MJ, et al. Proc. Natl. Acad. Sci. USA 102, 3760-3765 (2005)) , the coding region of gp120 of HIV-1 _NL4-3 were PCR amplified from pHCMV-EcoEnv, substituted with gp80 derived ecotropic murine leukemia (Sena-Esteves, M., et al. J. Viral. Methods 122, 131-139 2004, Addgene plasmid 15802).
HIV-1 _{NL4-3-EGFP-P2A-Nef} Reporter Virus is prepared by transfecting HEK 293T cells with pNL4-3-EGFP-P2A-Nef plasmid (University of Pittsburgh Medical School), as a lentiviral stock Treated (see above) and titered by GFP-FACS on Jurkat cell lines. Crude stocks of HIV-1 JRFL and SF162 used were prepared from the supernatant of PBMCs infected with HIV-1 for 6 days, clarified at 3000 RPM for 10 minutes and filtered through a 0.45 μm filter. Virus was titered using a Gag p24 ELISA.

  HIV-1 infection in vitro. Jurkat cells and U937 cells are infected by spinoculation at 2700 RPM for 2 hours in a 500 μl inoculum containing 8 μg / ml polybrene, then resuspended and left for 4 hours, then 500 μl of growth medium Was added. The next day, cells were washed 3 times with PBS and resuspended in growth medium. For infection of astrocytes and microglia, primary human fetal brain cells are incubated for 4 hours with virus stock diluted in Opti-MEM medium in the presence of polybrene (8 μg / ml) and then 1 ml Transduction / infection was performed by adding overnight growth medium and incubating overnight. The next day, cells were washed 3 times with PBS and fresh growth medium was added.

Detection and quantification of HIV-1 DNA. Genomic DNA was isolated from the cells using the NUCLEOSPIN Tissue kit (Macherey-Nagel Inc. Bethlehem, PA) according to the manufacturer's protocol. 100 ng of extracted DNA for LTR-specific PCR (see Table 1, SEQ ID NO: 7-14, β-actin forward (F) and reverse (R) are SEQ ID NO: 15, 16 respectively) but, FAILSAFE ^TM PCR kit and buffer D (Epicentre Technologies Corp., Madison , WI) was used to be subjected to PCR under the following PCR conditions. Separated with 98 ° C. for 5 minutes, 30 cycles (98 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 30 seconds), 72 ° C. for 7 minutes, and 2% agarose gel. The PCR products were subjected to agarose gel electrophoresis, gel purified, cloned into a TA vector (Invitrogen Corp., Carlsbad, CA) and sent for sequencing by Sanger method (GENEWIZ Global, South Plainfield, NJ). HIV-1 DNA is a cell as a reference beta - was quantified using specific TAQMAN ^TM qPCR in 5'-UTR and Env genes of globin gene and HIV-1 (Table 1, SEQ ID NO: 17 25). Prior to qPCR, genomic DNA from infected cells was diluted to 10 ng / μl and then 5 μl (= 50 ng) were required for each well's reaction. Reaction mixtures were prepared according to a simplified procedure (Liszewski, MK, et al. Methods 47, 254-260 (2009)) using Platinum TAQ DNA Polymerase (Invitrogen Corp., Carlsbad, Calif.). Since U1 cell genomic DNA contains two single copies of HIV-1 provirus per diploid genome, equal to the copy number of the beta-globin gene, the standard substance is from a series of dilutions of U1 cell genomic DNA It was prepared. The conditions for qPCR are as follows. 45 cycles at 98 ° C. for 5 minutes (98 ° C. for 15 seconds, 62 ° C. for 30 seconds with collection, 72 ° C. for 1 minute). The reaction is carried out, data LIGHTCYCLER480 ^TM were analyzed (Roche Holding AG, Basel, Switzerland ) at.

Reverse transcription and PCR. Total RNA was extracted from cells using the RNEASY kit (Qiagen, Hilden, Germany) with on-column DNAse I digestion. Next, 1 μg of RNA was used for M-MLV reverse transcription reaction (Invitrogen Corp., Carlsbad, CA). cDNA was diluted cell beta as a reference - using actin gene and of HIV-1 Gag and Env genes in specific TAQMAN ^TM qPCR (Table 1, SEQ ID NO: 17-25) and analyzed by relative quantification It was quantified by the same protocol as genomic DNA except for the above.

Flow cytometry. Expression of GFP in Jurkat 2D10 cells was quantified in living cells using a GUAVA EASYCYTE Mini flow cytometer (Guava Technologies, Inc., Billerica, Mass.). Jurkat cells infected with HIV-1 _{NL4-3-GFP-P2A-Nef} were first fixed for 10 minutes with 2% paraformaldehyde and then washed 3 times with PBS. Cell viability was assessed using propidium iodide staining. To 200 μl of viable cell suspension, PI solution was added to a final concentration of 10 μg / ml. The samples were incubated for 5 minutes at room temperature in the dark. After incubation, samples were obtained using a GUAVA EASYCYTE Mini flow cytometer.

  Western blot. Whole cell lysates, Jurkat cells, on ice for 30 minutes, TNN buffer [50 mM Tris pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA pH 8, 1 Protease inhibitor to mammalian cells 1 × The mixture (Sigma)] was prepared by incubation and then precleared by spinning at maximum speed for 10 minutes at 4 ° C. 50 μg of lysate is denatured with 1 × Laemli buffer and separated by SDS-polyacrylamide gel electrophoresis in Tris-glycine buffer and onto nitrocellulose membrane (Bio-Rad Laboratories Inc., Hercules, Calif.) Transcribed. Membranes are blocked with 5% milk / PBST for 1 hour, followed by mouse anti-flag M2 monoclonal antibody (1: 1000, Sigma-Aldrich, St. Louis, MO) or mouse anti-alpha-tubulin monoclonal antibody (1: 2000) Incubated. After washing with PBST, the membrane was incubated with conjugated goat anti-mouse antibody (1: 10,000) for 1 hour at room temperature. Membranes were scanned and analyzed using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, Nebr.).

result
In order to identify the minimal DNA element of the viral promoter which remains responsive to Tat but lacks the sequences corresponding to gRNAs A and B initially used to edit HIV-1 DNA, 5'-LTR The DNA coding sequence corresponding to the Cas9 gene was placed in the pX260 expression vector containing three different segments of the HIV-1 promoter spanning the U3 to R regions of (Figure 1A). After validating this cloning strategy by sequencing the DNA of each construct, the level of expression of Cas9 by each vector and the responsiveness of the vector to Tat was determined with pX260 or pX260-LTR-Cas9 and CMV-Tat. Were tested in co-transfected TZM-bl cells. The results from the Western blot showed activation of Cas9 expression in all three constructs, including a plasmid containing a minimal DNA promoter sequence located between -80 and +66 (FIG. 1B). This result was particularly important for this study as the promoter sequences are outside the DNA sequences corresponding to gRNAs A and B (FIG. 1B). Second, the DNA fragment corresponding to LTR _{(-80 / + 66)} -Cas9 was wrenched to evaluate the effect of Tat protein on editing of the copy of the integrated HIV-1 DNA expressing the luciferase reporter gene. It was cloned into a viral vector (LV) and used to transduce TZM-bl cells. Results from PCR amplification of LTR revealed the detection of a 205 bp DNA fragment in cells expressing gRNAs A and B and Tat protein (FIG. 1C, comparison of lanes 1-5 with lanes 6-8). The positions of the primers used for PCR amplification and the expected amplicons are illustrated in FIG. 1A (see also FIG. 7). The expression of Cas9, Tat and α-tubulin (control for equal loading) is shown in FIG. 1D.

Next, a luciferase assay was tested for the effect of viral DNA excision on viral promoter activity. The results show that the luciferase activity gradually decreases in response to the activation of Cas9 by Tat, supporting the results from the DNA assay, suggesting that cleavage of the DNA causes the inhibition of the promoter activity of the virus in these cells (FIG. 1E). An additional study examined in detail the activation of Cas9 upon infection of TZM-bl cells with HIV-1. To achieve this goal, LTR _{(-84 / + 66)} -Cas9 reporter TZM-bl cells are transduced with LV-gRNAs A / B for 24 hours, after which the cells are HIV-1 at three different MOI. It was infected by _JRFL or HIV-1 _SF162 . After 48 hours, cells were harvested for evaluation of DNA excision by PCR, evaluation of expression of incorporated promoter sequences by luciferase assay, and evaluation of expression of Cas9 by Western blot. The results of these experiments revealed that the DNA fragment of 205 bp after cutting in cells infected with HIV-1 _JRFL or HIV-1 _SF162 is detected, HIV-1 _JRFL or these cells HIV- 1 _It was suggested that Tat production by _{SF162 transactivated the} LTR _{(-80 / + 66)} promoter and Cas9 production (FIG. 2A). In addition, the results from the luciferase assay reveal a marked decrease in luciferase activity in the cells, with Cas9 activated by Tat produced _upon infection with HIV-1 _JRFL or HIV-1 _SF162 , and HIV- integrated. The experiment was again demonstrated to be effective in stopping the activity of the 1 luciferase gene. Induction of Cas9 in infected cells is shown in FIG. 2B. Results from Western blots show that infection of cells with HIV-1 JRFL or HIV-1 SF162 activates the truncated LTR promoter, LTR _{(-80 / + 66)} , resulting in the production of Cas9 protein in cells It was clarified that it was done (FIG. 2C).

In additional experiments, the ability of Tat-mediated activation of LTR-Cas9, with gRNAs A / B, has been demonstrated in human T lymphocyte cell line 2D10 (Pearson, R., et al. J. Viral. 82: 12291 -12303 (2008)), tested for excluding the HIV-1 genome. 2D10 cells are single round HIV-1 _{NL4 with} a portion of the Gag and Pol genes deleted from its genome and the Nef gene replaced by the gene encoding the reporter green fluorescent protein (GFP) _Have an embedded copy of _{-3 in} the latent state. Enhanced levels of Tat protein and activation of Cas9 in 2D10 cells (shown in FIG. 3A) edit the viral LTR by activation of Cas9 in LV-gRNAs A / B transduced cells (See FIG. 3B, similarly, lanes 1-8 in FIG. 8). Thus, a marked decrease in the number of GFP positive cells is detected in the presence of Tat (FIG. 3C), which causes Tat activation to lose the ability of the truncated promoter for expression of viral DNA Suggested that it caused suppression of GFP in GFP-positive cells. DNA sequences corresponding to gRNAs, excision of DNA fragments and positions of PCR primers are shown in FIGS. 9A-9C, SEQ ID NO: 26-40. Basal levels of Cas9 expression and viral DNA excision can be attributed to constitutive but not minimal expression of Tat in the latent 2D10 cell line.

  Previous observations suggesting the ability of PMA and / or TSA to stimulate copies of proviral DNA incorporated into 2D10 cells (Pearson, R., et al. J. Viral. 82: 12291-12303 (2008)) Taking into account, the effects of PMA and TSA were evaluated for activation of Cas9 in latently infected T cell models. As apparent from FIG. 4A, treatment of 2D10 cells with PMA and TSA alone or in combination increased the expression level of Cas9. In parallel experiments, PCR analysis was performed to detect LTR DNA, revealing the level of excision of viral DNA, as evidenced by the appearance of the 205 bp DNA fragment (see lanes 9-14 in FIG. 8) It was shown to increase (FIG. 4B). Testing of viral activation by measuring GFP levels of cells using Western blot or quantifying with green fluorescent cells (FIG. 4C), which is an indicator of viral activation (FIG. 4C) Showed a significant decrease. Thus, production of Cas9 based on activation of the minimal viral promoter (-80 / + 66) by either Tat expressed upon reactivation of silent proviral DNA or Tat expressed by PMA and TSA Exerts a negative effect on the expression of the latent viral genome causing editing of the integrated viral DNA copy in cells containing gRNAs A and B.

In the next set of experiments, HIV-1 replication levels of Jurkat T cells containing LTR-Cas9 were tested. Cells were transduced with lentiviral vectors (LV) expressing gRNAs A and B and LTR-88 / + 60-Cas9. Twenty-four hours later, transformed cells are infected with HIV-1 _{NL4-3-EGFP-P2A-Nef} , and after 3 and 5 days, the cells are harvested and 190 bp spread of viral DNA into gRNA A and B target sequences. It was tested for excision of DNA fragments. As shown in FIGS. 5A and 5B, infection of cells with HIV-1 resulted in the appearance of a 205 bp amplicon three days after infection with an increase in intensity at five days (FIGS. 5A and B) . This observation, similar to the results shown in FIGS. 3A-3C, shows that an increase in the level of Tat in the course of HIV-1 infection stimulates the expression of LTR-Cas9, whereby LTR DNA is cleaved. Provide evidence. Direct sequencing of the 205 bp band seen on day 5 results in Cas9 / gRNA A and Cas9 / gRNA B, which cause various insertion deletion (InDel) mutations detected at the binding of the 5 'and 3' fusion sites. Cleavage sites within the LTR were revealed (Figure 5C). Although the 5-UTR (nt +97 to +235) and the envelope (env) genes (nt +5828 to +5977) are both located between the 5 'LTR and the 3' LTR, viral DNA corresponding to their respective regions The examination of the segment of B showed a substantial decrease in the strength of the 139 bp amplicon corresponding to the 5'-UTR and the 150 bp amplicon corresponding to the env gene at day 5 compared to day 3 ( Figure 5D). These observations show that cleavage of Cas9 / gRNA A (at the 5 'LTR) and cleavage by Cas9 / gRNA B (at the 3' LTR) from the HIV-1 genome that extends between the 5 'LTR and 3' LTR Although excision of large DNA fragments also provides evidence that treatment of cells with Cas9 and gRNAs A and B can occur, this phenomenon has been previously reported (Hu, W., et al. Proc. Natl. Acad. Sci. USA 111, 11461-11466 (2014), Kaminski, R., et al. Sci. Rep. 6: 22555 (2016)). Quantitative analysis of results from flow cytometry showing expression of reporter GFP, an indicator of viral gene activation, results in substantial inhibition (64%) of day 3 GFP-positive cells and day 5 (84 %), Reveal further inhibition on day 8 (88%). The presence of lentivirus carrying genes encoding gRNAs and marker BFP, and expression of GFP in the cells was monitored by fluorescence microscopy, and cell culture properties were examined by phase contrast microscopy (FIG. 10A). Quantitative analysis shows no change in the total number of cells, which is similar to the previous observation (Kaminski, R., et al. Sci. Rep. 6: 22555 (2016)) It suggested that there was no associated toxicity. Consistent with the results from PCR gel analysis (shown in FIG. 5D), the results from qPCR and qRT-PCT show a marked reduction in the copy number levels of the viral DNA corresponding to the Gag gene, ie day 3 after infection 55%, day 5 84%, and a marked reduction in Gag RNA levels, ie 91% 3 days post infection and 96% 5 days post infection (FIGS. 5F and 5G). A similar series of studies in primary cultures of human microglia and astrocytes was also performed. The results from these studies show that viral gene expression and the presence of viral DNA in HIV-1 infected cells transduced with LVs expressing LTR-Cas9 and gRNAs (as shown in FIGS. 11A-11C) It showed that it was suppressed significantly. In summary, these observations show that the use of a novel autoregulatory phenomenon by adopting viral proteins containing Tat triggers an editing strategy with CRISPR / Cas9 to ablate the viral genome and permanently suppress viral replication Provide evidence that

Consideration
Since being discovered in 1985 (Arya, SK, et al. Science 229: 69-73 (1985), Sodroski, J., et al. Science 227, 171-173 (1985)), Tat trans of HIV-1 Activator proteins have been shown to be very important regulatory proteins, due to their role in the expression of the viral genome at the transcriptional level and the virulence effect on non-infected cells. According to the mechanism, Tat binds to an RNA sequence (nucleotides +1 to +59) located downstream of the transcription initiation site, a so-called transcription responsive region or TAR. Binding of Tat to TAR triggers a series of molecular and biochemical events that lead to pre-transcriptional initiation and initiation complex formation close to the transcription initiation site (nucleotide +1). This complex contains a series of cellular proteins with the ability to phosphorylate or acetylate components of the complex, including pTEF and RNA polymerase II, to promote the transcriptional elongation of RNA (for review see Mbonye, U. , et al. Virology 454-455, 328-339 (2014), Taube. R., et al. Viruses 5, 902-927 (2013)). In addition, Tat and NF-κB (Taylor, JP, Khalili, K. Adv. Neuroimmunol. 4, 291-303 (1994)), p300 / CBP and GCN5 (Calm E., et al. J. Biol. Chem. 276, 28179-28184 (2001), Kiernan, RE, et al. EMBO J. 18, 6106-6118 (1999), Ott, M., et al. Curr. Biol. 9, 1489-1492 (1999)). Interaction with various transcription factors, including those described above, can affect the transcription of all other viral and cellular genes that contribute to the disease spectrum found in HIV-1 positive AIDS patients (Gibellini, D., et al. , et al. New Microbial. 28, 95-109 (2005)). Tat also plays an important role in the productive replication of latent virus in the virus reservoir, once transcription from the reactivated viral promoter results in the initial transcription of the virus and production of Tat. Tat's unique importance in HIV-1 replication and the pathogenesis of AIDS provides a strong rationale to become a potential target for drug discovery as well as vaccine development. In fact, some potent inhibitors, some have the ability to interfere with Tat-TAR interactions, and some prevent communication between Tat and its intracellular partners, but they do not It has shown varying degrees of efficacy when acting on replication (Tabarrini. O., et al. Future Med Chem 8, 42 1-442 (2016)). The strategy utilized in this study stimulates the expression of Cas9, promotes excision of segments of the viral genome, and allows transcription and replication of the HIV-1 gene in cells with productive or latent HIV-1. It is to adopt Tat to remove permanently. Here, the HIV-1 suicide pathway was induced by Tat and designed to include viral genome editing using CRISPR / Cas9 technology (illustrated in FIG. 6). According to this pathway, Tat production in cells cleaves to GC rich, TATA box and TAR (-80 to +66) regions, in addition to stimulating its own promoter with full-length 5'-LTR sequences The expression of Cas9 is enhanced through the same mechanism by a type of minimal promoter sequence. The production of Cas9, and the binding of Cas9 to gRNAs designed to target the LTR DNA sequence outside (-80 to +66), excises the gene segment and inserts a deletion in the full-length viral promoter ( InDel) mutations can be induced to permanently eradicate HIV-1 in cells. In addition to the expected 417 bp DNA fragment representing the full length LTR sequence, the results from the short range amplification of LTR DNA showed a second DNA fragment of size 227 bp found only in Tat expressing cells. This 227 bp DNA fragment was generated by binding the remaining 5'-LTR to the remaining 3'-LTR after cleavage with Cas9 / gRNA A in either the 5'-LTR or 3'-LTR . Ligating the remaining DNA fragment derived from 5'-LTR after cleavage with Cas9 / gRNA with the remaining DNA fragment derived from 3'-LTR creates a new template for gene amplification and has been reported previously It appears that an amplicon of similar size (227 bp) appears as it does (Kaminski, R., et al. Sci. Rep. 6: 22555 (2016)).

  The CRISPR / Cas9 gene editing strategy has attracted attention in recent biomedical research because of its unique ability to edit the genome accurately and efficiently, and its ease of implementation and applicability. However, there are several areas that require careful attention. For example, designing the most specific and effective gRNAs is important to avoid off-target effects. The strategies developed herein were employed to maximize specificity and avoid off-target editing, ultra-deep sequencing of the entire genome, and various as described. Other tests (Hu, W., et al. Proc. Natl. Acad. Sci. USA 111, 11461-11466 (2014), Kaminski, R., et al. Sci. Rep. 6: 22555 (2016)) Verified by Treatment of cells with a single RNA may cause the appearance of mutant HIV-1 as a result of incorrect NHEJ repair at the cleavage site, a mutant that is resistant to the single gRNA used initially. It may cause the development of the virus (Wang, G., et al. Mol. Ther. 24, 522-526 (2016a), Wang, Z., et al. Cell Rep. 15, 481-489 (2016b)) . The use of multiple gRNAs resolves some of the above concerns, as the introduction of multiple double-stranded breaks throughout the HIV-1 genome causes excision of larger viral DNA segments from the host genome. And any opportunity to develop a replicable virus is permanently excluded (Khalili, K., et al. J. Neurovirol. 21, 310-321 (2015), Hu, W., et al. Proc. Natl. Acad. Sci. USA 111, 11461-11466 (2014), Kaminski, R., et al. Sci. Rep. 6: 22555 (2016), Ebina, H., et al. Sci. Rep. 3, 2510 ( 2013), Liao, HK, et al. Nature Comm. 6, 6413 (2015)). The second problem is the controlled expression of Cas9 to avoid unnecessary persistence of Cas9 protein expression which may non-specifically damage the host genome over time and / or induce an immune response. About. The strategy herein for conditional expression of Cas using HIV-1 Tat is to stimulate silent gene editing molecules to express at the early stages of reactivation of the virus and excise HIV-1 DNA. Will provide a new approach.

FIGS. 1A-1E show that expression of Cas9 by the HIV-1 LTR promoter is stimulated by Tat, resulting in cleavage of the viral promoter in the presence of gRNA. FIG. 1A is a schematic representation of the full-length HIV-1 LTR and various regulatory motifs within the enhancer and core regions, and a partial Gag gene. The scope of LTR deletion mutants generated for expression of Cas9 is shown. The positions of the gRNA target sequences and their distance from one another are shown. FIG. 1B shows a plasmid expressing Tat (pCMV-Tat) and pX260-LTR containing full-length LTR (-454 / + 66) or various variants thereof (-120 / + 66 or -80 / + 66). -Shows that co-transfection of TZM-bl cells with Cas9 increased the production level of Tat as tested by Western blot (upper panel). Expression of housekeeping α-tubulin (middle panel) and Tat (lower panel) is shown. FIG. 1C shows infection of TZM-bl cells with adenovirus expressing GFP or GFP Tat, followed by lentiviral transduction of expressing gRNAs A / B by Cas9 and U6 promoters by LTR _{-80 / + 66} promoter Performing at three different MOIs of 2, 4 and 8 shows that cleavage of the integrated HIV-1 LTR promoter DNA sequence in TZM-bl cells resulted in the emergence of a 205 bp DNA fragment ( Tested by PCR and DNA gel analysis). FIG. 1D is an SDS-PAGE illustrating the levels of Cas9, β-tubulin and Tat protein expressed in TZM-bl cells as described in FIG. 1C. FIG. 1E is a graph showing the results of a luciferase assay illustrating the transcriptional activity of HIV-1 LTR incorporated into TZM-bl cells after various treatments as described in FIG. 1C. FIGS. 2A-2C show that HIV-1 infection induces Cas9 to stimulate cleavage of incorporated viral DNA. LTR _{-80 / + 66-} Cas9 transduced with three different MOI (2, 4 and 8) lentivirus (LV-gRNA A / B) or control (empty LV) expressing gRNA A / B The reporter TZM-bl cell line is infected with HIV-1 _JRFL or HIV-1 _SF162, and after 48 hours, cells are harvested and protein expression is measured by Western blot (FIG. 2A), by induction of Cas9 after virus infection The level at which the incorporated HIV-1 LTR was cleaved was detected by PCR / DNA gene analysis (FIG. 2B), and the promoter transcription activity of incorporated HIV-1 was evaluated by a luciferase reporter assay (FIG. 2C). FIGS. 3A-3C show that, at the latent stage, stimulation with Cas9 Tat cleaves incorporated HIV-1 DNA in T cells, including HIV-1 reporters. 2D10 cells with integrated copies of the LTR- _{80 / + 66-} Cas9 gene were transduced with control (empty LV) or LV-gRNA A / B and subsequently transfected with pCMV or pCMV-Tat plasmid. After 48 hours, levels of various proteins were measured by Western blot as indicated (FIG. 3A). Genomic DNA to assess status of integrated HIV-1 DNA is measured by LTR specific PCR, excision efficiency is measured as a percentage of the ratio between truncated and full length amplicon, The units (AU) are represented by 0 to 0.5 (FIG. 3B). The level of reactivation of the integrated viral promoter after cleavage was assessed by flow cytometry and a representative scatter plot is shown (FIG. 3C). Red positive propidium iodide labeled dead cells were excluded from analysis. Figures 4A-4C show that treatment of cells with agents that activate latent virus induces the expression of Cas9 and cleavage of incorporated viral DNA in Jurkat 2D10 cells. 2D10 cells expressing LTR _{-80 / + 66-} Cas9 are treated with control (empty) or lentivirus expressing gRNAs A / B and after 24 hours cells are shown as PMA (P), Treated with TSA (T) or both (P / T) for 16 hours. Protein studies on the expression of Cas9-Flag, α-tubulin and GFP (which is indicative of integrated HIV-1 genome) were determined by Western blot (FIG. 4A). Genomic DNA to detect the level of excision in integrated LTR DNA by Cas9 and gRNA A / B was evaluated by PCR and the excision efficiency was measured as described in the legend of FIG. 3B (FIG. 4B) ). A GFP reporter assay by flow cytometry and a representative scatter plot are shown (FIG. 4C). Figures 5A-5G show that expression of LTR-Cas9 / gRNA protects cells from fresh HIV-1 infection. Figures 5A and 5B: Jurkat cells were co-transduced with LV-gRNA A / B and Lenti-LTR ( _{-80 / + 66} ) -Cas9-Blast. The next day, cells were infected with HIV-1 _{NL4-3-GFP-P2A-Nef} at an MOI of 0.01. On days 3 and 5 of infection, cells were harvested and excision levels were assessed by LTR specific PCR using genomic DNA as a template (FIG. 5A) and quantified as in FIG. 3B, 3C (FIG. 5B). Figure 5B). FIG. 5C: Shows the location of the excised fragment compared to control NL4-3 in direct sequencing analysis of the 205 bp DNA fragment after selection of 10 clones cloned into the TA vector and designated as trunc LTR 1-10. The position of gRNA corresponding to LTR A and LTR B in addition to the PAM sequence, and the primers used for amplification of the DNA are highlighted. FIG. 5D: Agarose gel showing the results of PCR analysis of DNA segments corresponding to UTR, Env and control β-actin DNA in experimental cells 3 and 5 days after HIV-1 infection. FIG. 5E: Results from flow cytometry quantifying the percentage of positive cells (indicative of viral expression) at three different times after infection. (For DNA) beta-(for RNA) globin and beta-actin was used as a reference, the virus DNA corresponding to the Gag sequence by TAQMAN ^TM (FIG. 5F) and quantitative detection of viral RNA (Figure 5G). FIG. 6 is a schematic of the negative feedback regulation of HIV-1 by CRISPR / Cas9. In the early stages of viral replication, basal transcription of the viral genome results in the production of Tat protein. The association of Tat with the TAR stem-loop structure within the leader of viral transcription at the badge causes the mobilization of several cellular regulatory proteins leading to the induction of viral transcription (solid thick arrow). At an early stage, Tat also stimulates a minimal viral promoter (represented as ltr) to promote transcription of the Cas9 gene. When newly synthesized Cas9 binds to various HIV-1 specific gRNAs, it then cleaves the viral genome and permanently inactivates LTR activity by excising a large DNA segment of viral DNA And thereby stop the expression and replication of the HIV-1 gene. FIG. 7 highlights the gRNA A / B target (medium gray (green), PAM dark gray (red) within the LTR in the reference HIV-1 NL4-3 genome (SEQ ID NO: 26) And the positions and nucleotide sequences of LTR specific primers (highlighted with light gray (blue)) used in PCR on genomic DNA of TZM-bl cells and in vitro infected Jurkat cells. Sequence and size of LTR specific PCR products (full length (SEQ ID NO: 27) and truncated (SEQ ID NO: 30)), and the predicted compiled fragment (SEQ ID NO: 29). FIG. 8 is a representative agarose analyzing LTR-specific PCR reaction used to quantify Cas9 / gRNA-mediated LTR ablation efficiency in experiments with FIGS. 3A-3C and 4A-4C Jurkat 2D10 reporter cell lines. Show a gel. 9A-9C are highlighted with LTR gRNA A / B targets (medium gray (green), PAM (dark gray) in the reference HIV-1 NL4-3 genome (SEQ ID NO: 26) And the location and nucleotide composition of LTR specific primers (highlighted by light gray (blue)) used to analyze excision in Jurkat 2D10 cells by PCR. Nucleotide sequence and size of amplicon ( The full length (SEQ ID NO: 31) and truncated LTR DNA (SEQ ID NO: 34)) and the predicted excised DNA fragment (SEQ ID NO: 33) are shown. FIG. 10A shows representative fluorescence microscopy images of transduced / infected Jurkat cells on day 5 of infection. Expression of BFP is indicative of the presence of vectors that express gRNAs. HIV-1 infection was observed by the level of GFP. FIG. 10B is a graph showing a quantitative comparison of cell numbers at various time points between control and experimental samples treated with LTR-Cas9. 11A-11C show early human transduced with a lentiviral cocktail containing lenti-LTR _{(-80 / + 66)} -Cas 9 (MOI 10), lenti-KLV-BFP-LTR A, B (MOI 3.3 each) Figure 2 is a graph showing results from fetal astrocytes and microglia. On day 3 of transduction, cells were infected with HIV-1 _{NL4-3-GFP-P2A-Nef} / VSV-G at MOI 1. One week after HIV-1 infection, cells were harvested and virus expression levels were determined by expression of GFP in flow cytometry (FIG. 11A), virus by TAQMAN qPCR and qRT-PCR using primer set and probe specific for Gag gene It was quantified at DNA level (FIG. 11B) and viral RNA (FIG. 11C). FIG. 12 is a list showing each base sequence corresponding to SEQ ID NO: 1 to 25 in the sequence listing and the primer name.

Claims (45)

Operably linked to the HIV LTR minimal promoter, which is within the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter and contains elements responsive to the transcriptional transactivator (Tat), An isolated nucleic acid sequence encoding an endonuclease associated with a clustered, regularly arranged short palindromic sequence repeat (Pants), and / or at least one isolated nucleic acid encoding at least one guide RNA A nucleic acid which is complementary to a target nucleic acid sequence in HIV.
Pharmaceutical composition which comprises.   The pharmaceutical composition according to claim 1, wherein the minimal promoter of the human immunodeficiency virus (HIV) long terminal repeat (LTR) further comprises a core sequence.   The minimal promoter (LTR) of human immunodeficiency virus (HIV) long terminal repeat (LTR) contains a nucleic acid sequence having at least about 75% sequence identity to the nucleic acid sequence from about position -120 to about +66. The pharmaceutical composition according to claim 1.   4. The human immunodeficiency virus (HIV) terminal repeat (LTR) minimal promoter comprises a nucleic acid sequence from about position -120 to about position +66. Pharmaceutical composition as described in-.   5. The human immunodeficiency virus (HIV) terminal repeat (LTR) minimal promoter comprises a nucleic acid sequence from about position -80 to about position +66. Pharmaceutical composition as described in-.   The pharmaceutical composition according to claim 1, wherein the target nucleic acid sequence in HIV comprises a sequence within the coding or non-coding region of HIV.   7. The pharmaceutical composition according to claim 6, wherein the noncoding region comprises a terminal repeat of HIV or a sequence within a terminal repeat of HIV.   8. The pharmaceutical composition according to claim 7, wherein the sequence within the HIV long terminal repeat comprises the sequence within the U3, R or U5 region, excluding the sequence of any of the HIV LTR minimal promoter.   The pharmaceutical composition according to any one of claims 1 to 8, further comprising a plurality of guide RNA nucleic acid sequences that are complementary to a plurality of target nucleic acid sequences of HIV.   The pharmaceutical composition according to any one of claims 1 to 9, wherein the endonuclease associated with CRISPR is Cas9.   The pharmaceutical composition according to any one of claims 1 to 10, wherein the CRISPR-related endonuclease is optimized for expression in human cells.   The medicament according to any one of claims 1 to 11, further comprising a sequence encoding a short transactivation type RNA (tracrRNA), wherein the tracrRNA is fused to a sequence encoding a guide RNA. Composition.   The pharmaceutical composition according to any one of claims 1 to 12, wherein the isolated nucleic acid sequence is operably linked to an expression vector comprising a lentiviral vector, an adenoviral vector or an adeno-associated viral vector. object.   Operably linked to the HIV LTR minimal promoter, which is within the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter and contains elements responsive to the transcriptional transactivator (Tat), An isolated nucleic acid sequence comprising a sequence encoding an endonuclease associated with a clustered, regularly arranged short palindromic sequence repeat (Crisp).   15. The isolated nucleic acid sequence of claim 14, wherein the minimal promoter of the human immunodeficiency virus (HIV) long terminal repeat (LTR) further comprises a core sequence.   The minimal promoter (LTR) of human immunodeficiency virus (HIV) long terminal repeat (LTR) contains a nucleic acid sequence having at least about 75% sequence identity to the nucleic acid sequence from about position -120 to about +66. 16. An isolated nucleic acid sequence according to claim 15,   The isolated promoter according to claim 15, wherein the minimal promoter of the human immunodeficiency virus (HIV) long terminal repeat (LTR) comprises the nucleic acid sequence from the position of about -120 to the position of about +66. Nucleic acid sequence.   16. The isolated promoter according to claim 15, wherein the minimal promoter of the human immunodeficiency virus (HIV) long terminal repeat (LTR) comprises the nucleic acid sequence from the position of about -80 to the position of about +66. Nucleic acid sequence.   19. An isolated nucleic acid sequence according to any one of claims 14-18, wherein the CRISPR-related endonuclease is Cas9.   20. The isolated nucleic acid sequence of any one of claims 14-19, wherein the CRISPR-related endonuclease is optimized for expression in human cells.   21. Any one of claims 14 to 20, wherein the isolated nucleic acid sequence is operably linked to an expression vector, said expression vector comprising a lentiviral vector, an adenoviral vector or an adeno-associated viral vector. An isolated nucleic acid sequence according to   Operably linked to the HIV LTR minimal promoter, which is within the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter and contains elements responsive to the transcriptional transactivator (Tat), An isolated nucleic acid sequence encoding an endonuclease associated with a clustered, regularly arranged short palindromic sequence repeat (Pants) and / or at least one isolated encoding at least one guide RNA Comprising administering to a subject infected with HIV a composition comprising a nucleic acid, wherein the at least one guide RNA is complementary to a target nucleic acid sequence present in HIV. A method for treating a subject infected with HIV.   The method according to claim 22, wherein the HIV infection is an active infection or a latent infection.   23. The method of claim 22, wherein the pharmaceutical composition is administered topically or parenterally.   23. The method of claim 22, wherein the minimal promoter of the human immunodeficiency virus (HIV) long terminal repeat (LTR) further comprises a core sequence.   The minimal promoter (LTR) of human immunodeficiency virus (HIV) long terminal repeat (LTR) contains a nucleic acid sequence having at least about 75% sequence identity to the nucleic acid sequence from about position -120 to about +66. 23. The method of claim 22.   27. Any one of claims 22-26, wherein the minimal promoter of the human immunodeficiency virus (HIV) long terminal repeat (LTR) comprises a nucleic acid sequence from about position -120 to about +66. The method described in.   The minimal promoter (LTR) of human immunodeficiency virus (HIV) long terminal repeat (LTR) contains a nucleic acid sequence having at least about 75% sequence identity to the nucleic acid sequence from about position -80 to about +66. 28. The method of any of claims 22-27.   29. A human immunodeficiency virus (HIV) terminal repeat (LTR) minimal promoter comprising a nucleic acid sequence from about position -80 to about position +66. The method described in.   30. The method according to any one of claims 22-29, wherein the CRISPR-related endonuclease is Cas9.   31. The method of any of claims 22-30, wherein the CRISPR-related endonuclease is optimized for expression in human cells.   An expression vector is operably linked to the HIV LTR minimal promoter, which is within the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter and contains elements responsive to transcriptional transactivation factors (Tat). An isolated nucleic acid sequence encoding an endonuclease associated with CRISPR, and at least one isolated nucleic acid encoding at least one guide RNA, wherein the at least one guide RNA is HIV 32. A method according to any one of claims 22 to 31, comprising a nucleic acid which is complementary to a target nucleic acid sequence present in the genome.   The first expression vector is a minimal promoter of HIV LTR that is within the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter and contains elements responsive to the transcriptional transactivator (Tat). An operably linked, isolated nucleic acid sequence encoding a CRISPR-related endonuclease, wherein the second expression vector comprises at least one isolated encoding at least one guide RNA. 33. The method of any of claims 22-32, wherein the at least one guide RNA comprises a nucleic acid that is complementary to a target nucleic acid sequence in the HIV genome.   34. The method of claim 33, wherein the first expression vector and the second expression vector are coexpressed in a host cell in vitro or in vivo.   34. The method according to claim 32 or 33, wherein the expression vector comprises a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.   34. A method according to claim 32 or 33, wherein one expression vector encodes multiple guide RNAs and / or multiple expression vectors each encode one or more guide RNAs.   38. The method of any of claims 22-37, further comprising administering one or more Tat activators, an antiviral agent, or a combination thereof.   An expression vector is operably linked to the HIV LTR minimal promoter, which is within the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter and contains elements responsive to transcriptional transactivation factors (Tat). A target sequence that is associated with a clustered, regularly arranged short palindromic repeat (Pants) related endonuclease and / or at least one guide RNA (gRNA) in proviral DNA Containing at least one isolated nucleic acid sequence encoding a complementary gRNA, eradicating HIV integrated into the genome of a latently infected host cell, HIV latently infected in vitro or in vivo An expression vector for eradicating HIV nucleic acid sequences integrated into the host cell's genome.   a second guide of which the gRNA nucleic acid sequence is complementary to a first target sequence which is in proviral DNA and a second guide which is complementary to a second target sequence which is in proviral DNA 39. The expression vector of claim 38, comprising at least one gRNA.   The minimal promoter (LTR) of human immunodeficiency virus (HIV) long terminal repeat (LTR) contains a nucleic acid sequence having at least about 75% sequence identity to the nucleic acid sequence from about position -80 to about +66. 39. The expression vector of claim 38.   41. The human immunodeficiency virus (HIV) terminal repeat (LTR) minimal promoter comprises a nucleic acid sequence from about position -80 to about position +66. The expression vector as described in.   Clustered, regularly arranged short palindromic repeats (Crisps) operably linked to a minimal functional viral promoter, whereby the minimal viral promoter is under the control of the immediate early transcription activator. An isolated nucleic acid sequence encoding an endonuclease related to (Crisps / Cas). Clustered, regularly arranged short palindromic repeats (Crisps) operably linked to a minimal functional viral promoter, whereby the minimal viral promoter is under the control of the immediate early transcription activator. An isolated nucleic acid sequence encoding an endonuclease associated with the gene (Crisps / Cas), and / or at least one guide RNA which is complementary to the target nucleic acid sequence present in the virus Nucleic acid,
A composition comprising   Furthermore, an isolated nucleic acid sequence comprising a CRISPR-related endonuclease (Crisps / Cas) operably linked to a minimal viral promoter, and at least one encoding at least one guide RNA 42. The composition according to 42, comprising an isolated nucleic acid, the at least one guide RNA comprising an expression vector encoding a nucleic acid complementary to a target nucleic acid sequence in the viral genome.   The first expression vector comprises an isolated nucleic acid sequence comprising a CRISPR related endonuclease operably linked to a minimal promoter of the viral long terminal repeat (LTR) of HIV. A second expression vector is at least one isolated nucleic acid encoding at least one guide RNA, said at least one guide RNA being complementary to a target nucleic acid sequence in the viral genome 43. The composition of claim 42 comprising.

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