Hepatitis B virus (HBV) knockout

Abstract

An RNA molecule comprising a guide sequence portion of 17 to 50 consecutive nucleotides within any one of SEQ ID NOs: 1 to 18936, and compositions, methods and uses thereof.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 224,581, filed July 22, 2021, the contents of which are incorporated herein by reference.

[0002] Throughout this application, various publications are referenced, including those referenced in parentheses. The disclosures of all publications mentioned in their entirety in this application are incorporated by reference into this application to provide further description of the art to which this invention pertains and the art to which this invention may be used.

[0003] Reference to sequence listing This application incorporates by reference the nucleotide sequence present in the file entitled "220721_91770-A-PCT_Sequence_Listing_AWG.xml", which is 23,850 kb in size, was created on July 19, 2022 in IBM-PC machine format with MS-Windows® system compatibility, and is included as part of this application in an XML file filed on July 21, 2022. [Background technology]

[0004] Hepatitis B Virus and Liver Infections Hepatitis B virus (HBV) is the causative agent of hepatitis B liver infection (also called "hepatitis B"). According to the CDC, hepatitis B is a short-term illness for many people, but can become a long-term chronic infection that can lead to serious health problems such as cirrhosis or liver cancer. The risk of chronic infection is related to age at the time of infection: approximately 90% of infants with hepatitis B develop chronic disease, whereas only 2%-6% of adults with hepatitis B develop chronic disease. Efficient cure of chronic HBV infection requires elimination of HBV covalently closed circular DNA (cccDNA), a long-lived viral genomic intermediate that is the template for HBV replication and persistence. Summary of the Invention

[0005] Disclosed is an approach for disrupting HBV genomic DNA molecules in cells. In some aspects of the invention, this approach results in knocking out expression of HBV genes. In some embodiments, the disclosure provides methods for disrupting conserved regions or portions thereof of HBV sequences. In some embodiments, the disclosure provides methods for disrupting regulatory elements or portions thereof of HBV sequences. In some embodiments, the disclosure provides methods for disrupting coding sequences or portions thereof of HBV sequences. In some embodiments, HBV sequences are targeted and modified within HBV covalently closed circular DNA (cccDNA) molecules and / or within mammalian host genomic DNA molecules. In some aspects, the disclosure provides methods for targeting and modifying sequences of HBV covalently closed circular DNA (cccDNA) molecules.

[0006] The present disclosure provides a method for modifying a Hepatitis B Virus (HBV) gene in a cell containing HBV, comprising: At least one CRISPR nuclease, or a nucleotide sequence encoding a CRISPR nuclease; and A first RNA molecule comprising a guide sequence portion of 17 to 50 nucleotides, or a nucleotide sequence encoding the first RNA molecule. introducing into a cell a composition comprising Also provided is a method, wherein the complex of the CRISPR nuclease and the first RNA molecule makes a double-stranded break in the HBV gene.

[0007] In some embodiments of the present invention, a first RNA molecule is provided that includes a guide sequence portion of 17 to 50 contiguous nucleotides within a sequence shown in any one of SEQ ID NOs: 1 to 18936.

[0008] In some embodiments of the present invention, a composition is provided comprising an RNA molecule comprising a guide sequence portion of 17 to 50 contiguous nucleotides within any one of SEQ ID NOs: 1 to 18936, and a CRISPR nuclease.

[0009] In some embodiments of the present invention, there is provided a method for treating hepatitis B, comprising delivering to cells of a subject having hepatitis B a composition comprising an RNA molecule comprising a guide sequence portion of 17 to 50 contiguous nucleotides within any one of SEQ ID NOs: 1 to 18936, and a CRISPR nuclease.

[0010] In some embodiments of the present invention, a method for inactivating Hepatitis B virus in a cell is provided, the method comprising delivering to a cell containing the Hepatitis B virus a composition comprising an RNA molecule comprising a guide sequence portion of 17 to 50 contiguous nucleotides within any one of SEQ ID NOs: 1 to 18936, and a CRISPR nuclease. [Brief description of the drawings]

[0011] [Figure 1A] FIG. 1A shows a schematic diagram of the HBV cccDNA (linearized) molecule. [Figure 1B] FIG. 1B shows how HBV segments were cloned into a lentiviral vector for use in subsequent infection of HeLa cells. [Diagram 2]Figure 2 shows the activity of guide molecules targeting HBV in HeLa cells. Specific guide molecules were co-transfected with wild-type OMNI-79 (WT) nuclease or OMNI-79 V5570 variant nuclease to determine the on-target activity of the guide molecules. 72 hours after DNA transfection, cells were collected, genomic DNA was extracted, and the region containing the nuclease cleavage site was amplified and then analyzed by next-generation sequencing (NGS). The graph shows the editing rate (%) ± STDV of three independent transfections in cells transfected with lentivirus at a multiplicity of infection (MOI) of 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description Unless otherwise defined, all technical and / or 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 methods and materials similar or equivalent to those described in this specification can be used in the practice or testing of embodiments of the present invention, representative methods and / or materials are described below. In case of conflict, the specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0013] The term "a" or "an" is understood to refer to "one or more" of the listed components. It will be apparent to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Thus, the terms "a" or "an" and "at least one" have the same meaning in this application.

[0014] For the purpose of improving the understanding of the present teachings, and without in any way limiting the scope of the teachings, unless otherwise indicated, all numbers and other numerical values ​​expressing quantities, percentages or proportions used in this specification and claims are understood to be modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should be construed in light of at least the number of reported significant digits and by applying ordinary rounding techniques.

[0015] Unless otherwise stated, adjectives such as "substantially" and "about" modifying a condition or relationship characterizing an embodiment of the invention are understood to mean that the condition or characteristic is defined to the extent that tolerance is acceptable in the operation of the embodiment for its intended application. Unless otherwise indicated, the term "or" in this specification and claims is considered to be an inclusive "or" rather than an exclusive "or" and indicates at least one or any combination of the items it connects.

[0016] In the description and claims of this application, the verbs "comprise," "contain," and "have," and their conjugations, are used to indicate that the object of the verb is not necessarily an exhaustive list of components, elements, or moieties of the subject of the verb. Other terms in this specification are intended to be defined by their known meanings in the art.

[0017] In some aspects of the present invention, DNA nucleases are utilized to cleave DNA at target sites and induce cellular repair mechanisms, such as, but not limited to, non-homologous end joining (NHEJ). In classical NHEJ, the two ends of a double-stranded break (DSB) site are ligated together in a rapid but imprecise manner (i.e., frequently resulting in DNA mutations at the break site in the form of small insertions or deletions), whereas HDR uses an intact homologous DNA donor to replace the DNA surrounding the break site in a precise manner. HDR can also mediate precise insertion of exogenous DNA at the break site. Thus, the term "homology-directed repair" or "HDR" refers to a mechanism that repairs DNA damage in cells, for example, during repair of double-stranded and single-stranded breaks in DNA. HDR requires nucleotide sequence homology and uses a "nucleic acid template" (as used herein, nucleic acid template and donor template are synonymous) to repair double-stranded or single-stranded breaks (e.g., DNA target sequence). This can result in the transfer of genetic information, for example, from a nucleic acid template to a DNA target sequence. HDR can result in a change in the sequence of the DNA target (e.g., an insertion, deletion, mutation) if the sequence of the nucleic acid template differs from that of the DNA target and some or all of the nucleic acid template polynucleotide or oligonucleotide is incorporated into the DNA target sequence. In some embodiments, the entire nucleic acid template polynucleotide, a portion of the nucleic acid template polynucleotide, or a copy of the nucleic acid template is incorporated into the DNA target sequence.

[0018] In this specification, the term "targeting sequence" or "targeting molecule" refers to a nucleotide sequence or a molecule that includes a nucleotide sequence that can hybridize with a specific target sequence, for example, a targeting sequence has a nucleotide sequence that is at least partially complementary to the sequence to be targeted. The targeting sequence or targeting molecule may be a part of an RNA molecule that can form a complex with a CRISPR nuclease, alone or in combination with other RNA molecules, and the targeting sequence serves as the targeting portion of the CRISPR complex. When a molecule with a targeting sequence is present simultaneously with a CRISPR molecule, the RNA molecule, alone or in combination with one or more other RNA molecules (e.g., tracrRNA molecules), is capable of targeting the CRISPR nuclease to a specific target sequence. As a non-limiting example, the guide sequence portion of a CRISPR RNA molecule or a single guide RNA molecule may serve as a targeting molecule. Each possibility is a separate embodiment. The targeting sequence can be custom designed to target a desired sequence.

[0019] In this specification, the term "targeting" refers to the preferential hybridization of the targeting sequence of targeting molecule to the nucleic acid having the nucleotide sequence being targeted.The term "targeting" encompasses variable hybridization efficiency, and thus, although there is preferential targeting of the nucleic acid having the nucleotide sequence being targeted, it is understood that in addition to on-target hybridization, unintended off-target hybridization may also occur.It is understood that when an RNA molecule targets a sequence, the complex of the RNA molecule and the CRISPR nuclease molecule targets that sequence for nuclease activity.

[0020] A "guide sequence portion" of an RNA molecule refers to a nucleotide sequence that can hybridize to a specific target DNA sequence, e.g., the guide sequence portion has a nucleotide sequence that is partially or completely complementary to the DNA sequence targeted along the guide sequence portion. In some embodiments, the length of the guide sequence portion is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides, or about 17-50, 17-49, 17-48, 17-47, 17-46, 17-45, 17-44, 17-43, 17-42, 17-41, 17-40 nucleotides. , 17-39, 17-38, 17-37, 17-36, 17-35, 17-34, 17-33, 17-31, 17-30, 17-29, 17-28, 17-27, 17-26, 17-25, 17-24, 17-22, 17-21, 18-25, 18-24, 18-23, 18-22, 18-21, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-22, 18-20, 20-21, 21-22, or 17-20. Preferably, the entire length of the guide sequence portion is completely complementary to the DNA sequence to be targeted along the guide sequence portion. The guide sequence portion may be a portion of an RNA molecule capable of forming a complex with a CRISPR nuclease, with the guide sequence portion serving as the DNA targeting portion of the CRISPR complex. When a DNA molecule having a guide sequence portion is present simultaneously with a CRISPR molecule, the RNA molecule is capable of targeting the CRISPR nuclease to a specific target DNA sequence. Each possibility is a separate embodiment. An RNA molecule can be custom designed to target a desired sequence. Thus, a molecule that includes a "guide sequence portion" is a type of targeting molecule. In some embodiments, the guide sequence portion includes a guide sequence portion described herein, e.g., a guide sequence shown in any of SEQ ID NOs: 1-18936, or a different sequence by 1, 2, 3, 4, or 5 nucleotides or less. Each possibility is a separate embodiment.In some of these embodiments, the guide sequence portion is fully complementary to the target sequence and comprises a sequence that is the same as any of SEQ ID NOs: 1-18936. Throughout this application, the terms "guide molecule," "RNA guide molecule," "guide RNA molecule," and "gRNA molecule" are synonymous with a molecule that comprises a guide sequence portion, and the term "spacer" is synonymous with "guide sequence portion."

[0021] In some embodiments of the present invention, the RNA molecule comprises a guide sequence portion of 17 to 50 contiguous nucleotides within the sequence set forth in any one of SEQ ID NOs: 1 to 18936.

[0022] The RNA molecule and / or the guide sequence portion of the RNA molecule may have modified nucleotides. Exemplary modifications to nucleotides / polynucleotides may be synthetic and include polynucleotides with nucleotides containing bases other than naturally occurring adenine, cytosine, thymine, uracil, or guanine bases. Modifications to polynucleotides include polynucleotides with synthetic, non-naturally occurring nucleosides, e.g., locked nucleic acids. Polynucleotides may be modified to increase or decrease the stability of the RNA. An example of a modified polynucleotide is an mRNA with 1-methylpseudouridine. For example, see U.S. Pat. No. 8,278,036, WO 2015 / 006747, and Weissman and Kariko (2015), which are incorporated herein by reference, for examples of modified polynucleotides and their uses.

[0023] As used herein, "consecutive nucleotides" as referred to in a SEQ ID NO: refers to the nucleotides of the sequence in the order set forth in the SEQ ID NO:, without any intervening nucleotides.

[0024] In some embodiments of the invention, the guide sequence portion may be 25 nucleotides in length and may contain 20-22 contiguous nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-18936. In some embodiments of the invention, the guide sequence portion may be less than 22 nucleotides in length. For example, in some embodiments of the invention, the guide sequence portion may be 17, 18, 19, 20 or 21 nucleotides in length. In such embodiments, the guide sequence portion may consist of 17, 18, 19, 20 or 21 nucleotides in the sequence of 17-22 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-18936, respectively. For example, the guide sequence portion of the sequence of 17 contiguous nucleotides set forth in SEQ ID NO: 18937 may consist of any one of the following nucleotide sequences (nucleotides excluded from the contiguous sequence are crossed out):

[0025] [ka]

[0026] In some embodiments of the invention, the guide sequence portion may be greater than 20 nucleotides in length. For example, in some embodiments of the invention, the guide sequence portion may be 21, 22, 23, 24, or 25 nucleotides in length. In such embodiments, the guide sequence portion comprises 17-50 nucleotides that contain a sequence of 20, 21, or 22 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-18936, and nucleotides that are fully complementary to (a sequence of) nucleotides adjacent to the 3' end, 5' end, or both, of the target sequence.

[0027] In some embodiments of the present invention, the CRISPR nuclease and the RNA molecule comprising the guide sequence portion bind to the target DNA sequence to form a CRISPR complex that cleaves the target DNA sequence. The CRISPR nuclease, for example, Cpf1, may form a CRISPR complex comprising the CRISPR nuclease and the RNA molecule without an additional tracrRNA molecule. Alternatively, the CRISPR nuclease, for example, Cas9, may form a CRISPR complex between the CRISPR nuclease, the RNA molecule, and the tracrRNA molecule. The guide sequence portion comprising a nucleotide sequence that can hybridize with a specific target DNA sequence and the sequence portion involved in CRISPR nuclease binding, for example, the tracrRNA sequence portion, may be present in the same RNA molecule. Alternatively, the guide sequence portion may be present in one RNA molecule, and the sequence portion involved in CRISPR nuclease binding, for example, the tracrRNA portion, may be present in another RNA molecule. A single RNA molecule comprising a guide sequence portion (e.g., a DNA-targeting RNA sequence) and at least one CRISPR protein-binding RNA sequence portion (e.g., a tracrRNA sequence portion) can form a complex with a CRISPR nuclease and act as a DNA-targeting molecule. In some embodiments, a first RNA molecule comprising a DNA-targeting RNA portion including a guide sequence portion and a second RNA molecule comprising a CRISPR protein-binding RNA sequence interact by base pairing to form an RNA complex that targets the CRISPR nuclease to the DNA target site, or fuse to form an RNA molecule that complexes with the CRISPR nuclease and targets the CRISPR nuclease to the DNA target site.

[0028] In some embodiments of the invention, the RNA molecule comprising the guide sequence portion may further comprise the sequence of a tracrRNA molecule. Such embodiments may be designed as a synthetic fusion of the guide portion of the RNA molecule with a transactivating crRNA (tracrRNA) (see Jinek et al., 2012). In such embodiments, the RNA molecule is a single guide RNA (sgRNA) molecule. Some embodiments of the invention may also form a CRISPR complex utilizing individual tracrRNA molecules and individual RNA molecules comprising the guide sequence portion. In such embodiments, the tracrRNA may hybridize to the RNA molecule via base pairing, which may be advantageous in certain applications of the invention described herein.

[0029] The term "tracr mate sequence" refers to a sequence that is sufficiently complementary to the tracrRNA molecule to hybridize with the tracrRNA through base pairing and promote the formation of a CRISPR complex (see U.S. Patent No. 8,906,616). In some embodiments of the present invention, the RNA molecule may further comprise a portion having a tracr mate sequence.

[0030] In the present invention, a "gene" includes a DNA region that codes for a gene product and all DNA regions that control the production of the gene product, whether or not the control sequence is contiguous with the coding and / or transcribed sequence. Thus, genes include, but are not necessarily limited to, promoter sequences, terminators, translation control sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0031] "Eukaryotic" cells include, but are not limited to, fungal cells (eg, yeast), plant cells, animal cells, mammalian cells, and human cells.

[0032] In this specification, the term "nuclease" refers to an enzyme capable of cleaving phosphodiester bonds between nucleotide subunits of nucleic acids. Nucleases may be isolated from natural sources or derived from natural sources. Natural sources may be living organisms. Alternatively, nucleases may be modified or synthetic proteins that retain phosphodiester bond cleavage activity. Genetic modification can be achieved using nucleases, such as CRISPR nucleases.

[0033] As used herein, the term "conserved region" refers to a region of a nucleotide or amino acid molecule that has sequence identity across several different species or strains. Similarly, as used herein, the term "conserved sequence" refers to a sequence that has sequence identity across several different species or strains. For example, a conserved sequence of HBV DNA is an HBV DNA sequence that has sequence identity across several different strains, variants, or serotypes of HBV. The sequence identity may be, for example, at least 70% sequence identity, 80% sequence identity, 81% sequence identity, 82% sequence identity, 83% sequence identity, 84% sequence identity, 85% sequence identity, 86% sequence identity, 87% sequence identity, 88% sequence identity, 89% sequence identity, or 90% sequence identity across several HBV strains, variants, or serotypes.

[0034] In some embodiments of the invention, there is provided a method for modifying a Hepatitis B virus (HBV) sequence in a cell, comprising the steps of: At least one CRISPR nuclease, or a nucleotide sequence encoding a CRISPR nuclease; and A first RNA molecule comprising a guide sequence portion of 17 to 50 nucleotides, or a nucleotide sequence encoding the first RNA molecule. introducing into a cell a composition comprising A method is provided in which a complex of a CRISPR nuclease and a first RNA molecule makes a double-stranded break in an HBV sequence.

[0035] In some embodiments, the guide sequence portion of the first RNA molecule comprises 17 to 50 contiguous nucleotides within the sequence set forth in any one of SEQ ID NOs: 1 to 18936.

[0036] In some embodiments, the double strand break is created in a Hepatitis B virus DNA sequence. In some embodiments, the double strand break is created in an HBV gene or a portion thereof, an HBV coding sequence or a portion thereof, an HBV regulatory sequence or a portion thereof, and / or a conserved HBV sequence.

[0037] In some embodiments, the double-stranded break is made up to 500 nucleotides upstream or downstream of the HBV coding sequence, HBV regulatory sequence, and / or conserved HBV sequence. Each possibility is a separate embodiment. In some embodiments, HBV conserved regions are targeted.

[0038] In some embodiments, the method further comprises introducing into the cell a second RNA molecule comprising a 17-50 nucleotide guide sequence portion, or a nucleotide sequence encoding the second RNA molecule, wherein the complex of the second RNA molecule and the CRISPR nuclease makes a further double-stranded break in the Hepatitis B virus sequence.

[0039] In some embodiments, the guide sequence portion of the second RNA molecule comprises 17 to 50 contiguous nucleotides within a sequence set forth in any one of SEQ ID NOs: 1 to 18936 other than the sequence of the first RNA molecule.

[0040] In some embodiments, a sequence of nucleotides is excised from a molecule that includes an HBV sequence.

[0041] In some embodiments, the sequence of nucleotides excised from the HBV sequence comprises an HBV gene or a portion thereof, an HBV coding sequence or a portion thereof, an HBV regulatory sequence or a portion thereof, and / or a conserved HBV sequence.

[0042] In some embodiments, the HBV gene or a portion thereof is ablated. In some embodiments, the HBV regulatory sequence or a portion thereof is ablated. In some embodiments, the HBV non-coding sequence or a portion thereof is ablated.

[0043] In some embodiments, the first or second RNA molecule comprises a guide sequence portion that targets a sequence located up to 500 base pairs from the HBV gene, HBV coding sequence, HBV regulatory sequence, and / or conserved HBV sequence to be excised by the first and second RNA molecules.

[0044] In some embodiments, the HBV sequences are excised from the HBV cccDNA molecule.

[0045] In some embodiments, the HBV sequences are excised from the genomic DNA molecule into which they are integrated.

[0046] In some embodiments, the cell is a liver cell or a hepatocyte.

[0047] In some embodiments, the cell is in a human subject.

[0048] In some embodiments, the human subject is suffering from chronic or acute hepatitis B.

[0049] In some embodiments, the HBV sequences are located in an HBV covalently closed circular DNA (cccDNA) molecule.

[0050] In some embodiments, the HBV sequences are located in a genomic DNA molecule into which the HBV sequences are integrated.

[0051] In some embodiments of the present invention, a composition is provided comprising a first RNA molecule, the first RNA molecule comprising a guide sequence portion of 17 to 50 contiguous nucleotides within a sequence set forth in any one of SEQ ID NOs: 1 to 18936.

[0052] In some embodiments, the composition further comprises at least one CRISPR nuclease.

[0053] In some embodiments, the composition further comprises a second RNA molecule comprising a guide sequence portion of 17 to 50 contiguous nucleotides, wherein the second RNA molecule targets an HBV gene, and the guide sequence portion of the second RNA molecule is a sequence different from the sequence of the guide sequence portion of the first RNA molecule.

[0054] In some embodiments, the guide sequence portion of the second RNA molecule comprises 17 to 50 contiguous nucleotides within a sequence set forth in any one of SEQ ID NOs: 1 to 18936 other than the sequence of the first RNA molecule.

[0055] In some embodiments of the invention, a method of inactivating Hepatitis B virus in a cell is provided, comprising delivering to the cell a composition of any one of the above embodiments.

[0056] In some embodiments of the invention, a method of treating hepatitis B is provided comprising delivering to cells of a subject having hepatitis B a composition of any one of the above embodiments.

[0057] In some embodiments of the present invention, there is provided a use of a composition according to any one of the above embodiments for inactivating Hepatitis B virus in a cell, comprising delivering the composition to the cell.

[0058] In some aspects of the invention, there is provided a medicament for use in inactivating Hepatitis B virus in a cell, comprising the composition of any one of the above aspects, wherein the medicament is administered by delivering the composition to the cell.

[0059] In some aspects of the invention, there is provided a use of a composition according to any one of the above aspects for the treatment, amelioration or prevention of hepatitis B, comprising delivering the composition to cells of a subject having or at risk of having hepatitis B.

[0060] In some aspects of the invention, there is provided a medicament comprising the composition of any one of the above aspects for use in the treatment, amelioration or prevention of hepatitis B, wherein the medicament is administered by delivering the composition to cells of a subject having or at risk of having hepatitis B.

[0061] Some embodiments of the invention provide a composition of any one of the above embodiments for use in inactivating Hepatitis B virus in a cell.

[0062] In some aspects of the invention, there is provided a composition according to any one of the above aspects for use in the treatment, amelioration or prevention of Hepatitis B.

[0063] In some embodiments of the present invention, a kit for inactivating Hepatitis B virus in a cell is provided, comprising an RNA molecule, a CRISPR nuclease, and / or a tracrRNA molecule of any one of the embodiments set forth herein; and instructions for delivering the RNA molecule, the CRISPR nuclease, and / or the tracrRNA to the cell.

[0064] In some embodiments of the present invention, a kit for treating hepatitis B in a subject is provided, comprising an RNA molecule, a CRISPR nuclease, and / or a tracrRNA molecule of any one of the embodiments set forth herein; and instructions for delivering the RNA molecule, the CRISPR nuclease, and / or the tracrRNA to cells of a subject having or at risk of having hepatitis B.

[0065] In some embodiments of the present invention, an HBV gene editing composition is provided that includes an RNA molecule that includes a guide sequence portion of 17-50 contiguous nucleotides within any one of SEQ ID NOs: 1-18936. In some embodiments, the RNA molecule further includes a portion having a sequence that binds to a CRISPR nuclease. In some embodiments, the sequence that binds to a CRISPR nuclease is a tracrRNA sequence.

[0066] In some embodiments, the RNA molecule further comprises a portion having a tracr mate sequence.

[0067] In some embodiments, the RNA molecule may further comprise one or more linker moieties.

[0068] In some embodiments of the present invention, the nucleotide length of the RNA molecule may be up to 1000, 900, 800, 700, 600, 500, 450, 400, 350, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100. Each possibility is a separate embodiment. In some embodiments of the present invention, the nucleotide length of the RNA molecule may be 17 to 300, 100 to 300, 150 to 300, 100 to 500, 100 to 400, 200 to 300, 100 to 200, or 150 to 250. Each possibility is a separate embodiment.

[0069] In some embodiments of the invention, the composition further comprises a tracrRNA molecule.

[0070] In some embodiments of the present invention, there is provided a method of inactivating Hepatitis B virus in a cell, the method comprising delivering to the cell a composition comprising an RNA molecule comprising a guide sequence portion of 17 to 50 contiguous nucleotides within any one of SEQ ID NOs: 1 to 18936, and a CRISPR nuclease.

[0071] In some embodiments of the present invention, there is provided a method of treating hepatitis B, comprising delivering to cells of a subject having hepatitis B a composition comprising an RNA molecule comprising a guide sequence portion of 17 to 50 contiguous nucleotides within any one of SEQ ID NOs: 1 to 18936, and a CRISPR nuclease.

[0072] In some embodiments of the invention, at least one CRISPR nuclease and an RNA molecule are delivered to a subject and / or cell at substantially the same time or at different times.

[0073] In some embodiments, the tracrRNA molecule is delivered to a subject and / or cell substantially simultaneously or at different times as the CRISPR nuclease and RNA molecule.

[0074] In some aspects of the invention, methods are provided that include removing a sequence from an HBV genomic DNA molecule (e.g., HBV cccDNA), or a host genomic molecule into which an HBV sequence has been integrated, wherein a first RNA molecule, or first and second RNA molecules, are targeted to a region adjacent to the sequence to be removed.

[0075] In some embodiments of the present invention, a method is provided that includes removing a sequence from an HBV genomic DNA molecule or a host genomic molecule into which an HBV sequence has been integrated. In some embodiments, the sequence is a gene or a portion thereof. In some embodiments, the sequence is a protein-coding sequence or a portion thereof. In some embodiments, the method is aimed at removing an entire open reading frame of an HBV genomic DNA molecule or an entire gene of an HBV genomic DNA molecule. In some embodiments, the sequence is a non-coding sequence of HBV or a portion thereof. In some embodiments, the sequence is a regulatory element or a portion thereof. In some embodiments, the size of the excised sequence is between 10 base pairs (bp) and 100 bp, between 10 bp and 3000 bp, between 100 bp and 300 bp, between 100 bp and 600 bp, between 100 bp and 800 bp, between 100 bp and 1000 bp, between 250 bp and 300 bp, between 250 bp and 600 bp, between 250 bp and 800 bp, between 250 bp and 1000 bp, or between 250 bp and 3000 bp. Each possibility is a separate embodiment.

[0076] The compositions and methods of the present disclosure may be utilized to treat, prevent, ameliorate, or slow the progression of Hepatitis B.

[0077] In some embodiments, the method of inactivating Hepatitis B virus includes delivering two RNA guide molecules to a cell to target and inactivate HBV genes.

[0078] The present invention may use any one or a combination of the previously described strategies for inactivating Hepatitis B virus.

[0079] In an embodiment of the invention, an RNA guide molecule is used to direct a CRISPR nuclease to a site within an HBV sequence in a cccDNA molecule or a host genome to create a double strand break (DSB) resulting in the insertion or deletion of nucleotides by inducing an error-prone non-homologous end joining (NHEJ) mechanism and the formation of a frameshift mutation, which may inactivate or knock out the HBV gene, for example, by generating a premature stop codon, generate a truncated protein, or cause nonsense codon-mediated mRNA decay of the HBV transcript.

[0080] In some aspects, the disclosure provides RNA guide sequences (also referred to as "RNA molecules") that bind or associate with and / or direct an RNA-guided DNA nuclease, e.g., a CRISPR nuclease, to a target sequence within the HBV genome or a host genome.

[0081] In some embodiments, the method includes contacting a site in the HBV genomic DNA molecule with an RNA guide molecule and a CRISPR nuclease, e.g., a Cas9 protein, where the RNA guide molecule and the CRISPR nuclease associate with a nucleotide sequence at the site in the HBV genomic DNA, thereby modifying or knocking out expression of a product encoded by the HBV genome.

[0082] In some embodiments, the RNA molecule and the CRISPR nuclease are introduced into a cell that harbors Hepatitis B. In some embodiments, the cell is in a human subject.

[0083] In some embodiments, the methods are utilized to treat a subject having a disease phenotype resulting from HBV infection, hi such embodiments, the methods improve, ameliorate, or prevent the disease phenotype.

[0084] The embodiments of the compositions described herein include at least one CRISPR nuclease, an RNA molecule, and a tracrRNA molecule, where the tracrRNA molecule is effective in a subject or cell simultaneously. The at least one CRISPR nuclease, the RNA molecule, and the tracrRNA may be delivered substantially simultaneously, or may be delivered at different times, but with simultaneous effects. For example, this includes delivering the CRISPR nuclease to a subject or cell before the RNA molecule and / or the tracrRNA are substantially present in the subject or cell.

[0085] In some embodiments, the cell is a hepatocyte or liver cell.

[0086] HBV knockout strategies include, but are not limited to, (1) truncation, for example by targeting a sequence in the HBV genome molecule with one guide RNA molecule to induce frameshift or nonsense codon-mediated decay, and (2) excision of HBV sequences or a large portion of the HBV gene, for example using two guide RNA molecules.

[0087] Truncation may be achieved by several approaches. For example, truncation may be achieved by targeting the coding sequence of the HBV genomic molecule with a single guide RNA molecule (e.g., a single guide RNA molecule or "sgRNA"). Alternatively, excision may be achieved by targeting the HBV genomic molecule with two different RNA molecules.

[0088] In some aspects, any of the editing compositions described herein may be accompanied by small molecules that modify chromatin, such as, but not limited to, methylation inhibitors and deacetylation inhibitors, which can increase excision by increasing the accessibility of the DNA nucleases of the editing composition to the HBV minichromosome.

[0089] Alternatively, an editing composition comprising a nuclease (e.g., a catalytically inactive CRISPR nuclease) fused to a chromatin modifier, such as, but not limited to, a demethylase or histone acetyltransferase, may increase delivery of the nuclease to the HBV minichromosome.

[0090] The editing composition may include multiple guide RNA molecules targeting different sites in the HBV sequence, for example to mediate excision of a regulatory element from the HBV sequence or to knock out an HBV gene.

[0091] CRISPR nucleases and PAM recognition In some embodiments, the nuclease is selected from a CRISPR nuclease or a functional variant thereof. In some embodiments, the nuclease is an RNA-guided DNA nuclease. In such embodiments, an RNA sequence that guides the RNA-guided DNA nuclease (e.g., Cas9 or Cpf1) binds to and / or directs the RNA-guided DNA nuclease to a sequence in the HBV genome. In some embodiments, the CRISPR complex does not further comprise a tracrRNA. Those skilled in the art will appreciate that RNA molecules can be engineered to bind to a selected target in the genome by methods commonly known in the art.

[0092] In this specification, the term "PAM" refers to a nucleotide sequence of a target DNA that is located near the DNA sequence to be targeted and is recognized by a CRISPR nuclease complex. The PAM sequence may vary depending on the type of nuclease. In addition, there are CRISPR nucleases that can target almost any PAM. In some embodiments of the present invention, the CRISPR system utilizes one or more RNA molecules with a guide sequence portion that guides the CRISPR nuclease to the target DNA site by forming Watson-Crick base pairs between the guide sequence portion and the protospacer of the target DNA site next to the protospacer adjacent motif (PAM), which is an additional requirement for target recognition. The CRISPR nuclease then cleaves the target DNA site, generating a double-stranded break in the protospacer. In a non-limiting example, a Type II CRISPR system utilizes a mature crRNA:tracrRNA complex that guides a CRISPR nuclease (e.g., Cas9) to a target DNA by forming Watson-Crick base pairs between the guide sequence portion of the crRNA and a protospacer on the target DNA next to a protospacer adjacent motif (PAM). One of skill in the art will appreciate that the engineered RNA molecules of the present invention are further designed to associate with a target genomic DNA sequence of interest next to a protospacer adjacent motif (PAM), e.g., a PAM that corresponds to a sequence associated with the CRISPR nuclease of interest.The PAM may be, for example and without limitation, NGG or NAG (where N is any nucleobase) for Streptococcus pyogenes Cas9 WT (SpCAS9); NNGRRT for Staphylococcus aureus (SaCas9); NNNVRYM for Jejuni Cas9 WT; NGAN or NGNG for SpCas9-VQR variant; NGCG for SpCas9-VRER variant; NGAG for SpCas9-EQR variant; NRRH (where N is any nucleobase, R is A or G, and H is A, C, or T) for SpCas9-NRRH variant; NRTH (where N is any nucleobase, R is A or G, and H is A, C, or T) for SpCas9-NRCH variant; NRCH (where N is any nucleobase, R is A or G, and H is A, C, or T) for the SpG variant of SpCas9; NG (where N is any nucleobase); NG or NA (where N is any nucleobase) for the SpCas9-NG variant of SpCas9; NR, NRN, or NYN (where N is any nucleobase, R is A or G, and Y is C or T) for the SpRY variant of SpCas9; and Streptococcus canis. canis Cas9 variant (ScCas9), NNG (N is any nucleobase); NNNRRT (N is any nucleobase and R is A or G) for Staphylococcus aureus SaKKH-Cas9 variant (SaCas9); NNNNGATT (N is any nucleobase) for Neisseria meningitidis (NmCas9); TTN (N is any nucleobase) for Alicyclobacillus acidiphilus Cas12b (AacCas12b); or TTTV (V is A, C, or G) for Cpfl. The RNA molecules of the invention are each designed to form a complex with one or more different CRISPR nucleases and are designed to target a polynucleotide sequence of interest utilizing one or more different PAM sequences corresponding to said CRISPR nucleases.

[0093] In some embodiments, RNA-guided DNA nuclease, for example CRISPR nuclease, can be used to cause DNA cleavage at desired position in the genome of cells, either double-stranded or single-stranded in nature.The most commonly used RNA-guided DNA nuclease is from CRISPR system, but other RNA-guided DNA nucleases are also contemplated for use in genome editing compositions and genome editing methods described herein.See, for example, US Patent Application Publication No. 2015 / 0211023, which is incorporated herein by reference.

[0094] There are many different CRISPR systems that can be used to practice the present invention. The CRISPR system may be a type I, type II or type III system. Non-limiting examples of suitable CRISPR proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, Casl0, Casl These include Od, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csxl0, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966.

[0095] In some embodiments, the RNA-guided DNA nuclease is a CRISPR nuclease derived from a type II CRISPR system (e.g., Cas9). CRISPR nucleases have been shown to be effective in the detection and characterization of Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Neisseria meningitidis, Treponema denticola, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridomogenes, and Streptosporangium roseum. roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas species sp.), Crocosphaera watsonii, Cyanothece sp.), Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicellulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.), Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, or a species encoding a CRISPR nuclease with a known PAM sequence. CRISPR nucleases encoded by uncultured bacteria may also be used in the context of the present invention (see Burstein et al. Nature, 2017). Variants of CRISPR proteins with known PAM sequences, such as SpCas9 D1135E variant, SpCas9 VQR variant, SpCas9 EQR variant, or SpCas9 VRER variant, may also be used in the context of the present invention.

[0096] Thus, RNA-guided DNA nucleases of the CRISPR system, such as Cas9 protein or modified Cas9, or homologs or orthologues of Cas9, or other RNA-guided DNA nucleases belonging to other CRISPR systems, such as Cpf1 and its homologs and orthologues, may be used in the compositions of the invention.Other CRISPR nucleases may also be used, such as those described in WO2020 / 223514 and WO2020 / 223553, which are incorporated herein by reference.

[0097] In certain embodiments, the CRISPR nuclease may be a "functional derivative" of a naturally occurring Cas protein. A "functional derivative" of a native sequence polypeptide is a compound that has qualitative biological properties in common with the native sequence polypeptide. "Functional derivatives" include, but are not limited to, fragments of the native sequence, and derivatives of native sequence polypeptides and their fragments, provided that they have biological activity in common with the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability to hydrolyze DNA substrates into fragments. The term "derivative" encompasses both amino acid sequence variants of the polypeptide, and covalent modifications and fusions thereof. Suitable derivatives of Cas polypeptides or fragments thereof include, but are not limited to, mutants, fusions, and covalent modifications of Cas proteins or fragments thereof. Cas proteins, including Cas proteins or fragments thereof, as well as derivatives of Cas proteins or fragments thereof, may be obtainable from cells or may be chemically synthesized, or may be obtained by a combination of these methods. The cells may be cells that naturally produce a Cas protein, or cells that naturally produce a Cas protein and have been engineered to produce an endogenous Cas protein at a higher expression level, or to produce a Cas protein from an exogenously introduced nucleic acid encoding the same or a different Cas than the endogenous Cas. In some cases, the cells do not naturally produce a Cas protein and have been engineered to produce a Cas protein.

[0098] In some embodiments, the CRISPR nuclease is Cpf1. Cpf1 is a single RNA-guided endonuclease that utilizes T-rich protospacer adjacent motifs. Cpf1 cleaves DNA by staggered DNA double-strand breaks. Two Cpf1 enzymes from Acidaminococcus and Lachnospiraceae have been shown to efficiently edit genomes in human cells (see Zetsche et al., 2015).

[0099] Thus, RNA-guided DNA nucleases of Type II CRISPR systems such as Cas9 protein or modified Cas9 or homologs, orthologs or variants of Cas9, or other RNA-guided DNA nucleases belonging to other CRISPR systems such as Cpf1 and its homologs, orthologs or variants may be used in the present invention.

[0100] In some embodiments, the guide molecule comprises one or more chemical modifications that confer new or improved properties (e.g., stability against degradation, hybridization energy, or binding to RNA-guided DNA nucleases). Suitable chemical modifications include, but are not limited to, modified bases, modified sugars, or modified internucleoside linkages. Non-limiting examples of suitable chemical modifications include 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2'-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-O-methylpseudouridine, "β,D-galactosylqueosine", 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methyladenosine, 1-methylguano ... Chirpseudouridine, 1-methylguanosine, 1-methylinosine, "2,2-dimethylguanosine", 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, "β,D-mannosylqueuosine", 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine uridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N-((9-β-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine, N-((9-β-D-ribofuranosylpurin-6-yl)N-methylcarbamoyl)threonine, uridine-5-oxyacetic acid-methyl ester, uridine-5-oxyacetic acid, wybutoxocine, queuosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine , 4-thiouridine, 5-methyluridine, N-((9-β-D-ribofuranosylpurin-6-yl)-carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, wybutosine, "3-(3-amino-3-carboxy-propyl)uridine, (acp3)u", 2'-O-methyl (M), 3'-phosphorothioate (MS), 3'-thioPACE (MSP), pseudouridine or 1-methylpseudouridine.Each possibility represents a separate aspect of the present invention.

[0101] Cellular delivery The compositions described herein may be delivered to a target cell by any suitable means. The compositions of the present invention may be targeted to cells that contain HBV and / or express HBV products. For example, in one embodiment, the RNA molecule is specifically targeted to a site in the HBV genome and the target cell is a hepatocyte that harbors HBV. Delivery to the cell may be performed in vitro, ex vivo, or in vivo. Additionally, the nucleic acid compositions described herein may be delivered as one or more of a DNA molecule, an RNA molecule, a ribonucleoprotein (RNP), a nucleic acid vector, or a combination thereof.

[0102] In some embodiments, one of the compositions described herein is delivered to a cell in vivo. In some embodiments, the cell is a hepatocyte. In some embodiments, the composition is delivered to the liver of a subject. The composition may be delivered to a cell by known in vivo delivery methods, including, but not limited to, viral transduction using, for example, lentivirus or adeno-associated virus (AAV), nanoparticle delivery, and the like. Additional detailed delivery methods are described throughout this section. Non-limiting examples of suitable AAV serotypes include AAV8, liver-tropic AAV LK03.

[0103] In some embodiments, any one of the compositions described herein is delivered to a cell ex vivo. In some embodiments, the cell is a hepatocyte. The compositions may be delivered to cells by known ex vivo delivery methods, including, but not limited to, nucleofection, electroporation, viral transduction, e.g., using lentivirus or adeno-associated virus (AAV), nanoparticle delivery, liposomes, etc. Additional detailed delivery methods are described throughout this section.

[0104] In some embodiments, the RNA molecules in the composition include chemical modifications. Non-limiting examples of suitable chemical modifications include 2'-O-methyl (M), 2'-O-methyl, 3' phosphorothioate (MS), or 2'-O-methyl, 3' thioPACE (MSP), pseudouridine and 1-methylpseudouridine. Each possibility is a separate aspect of the present invention.

[0105] A suitable viral vector system may be used to deliver the nucleic acid composition, for example the RNA molecule contained in the composition of the present invention. Conventional viral and non-viral based gene transfer can be used to introduce the nucleic acid and target the tissue. In certain embodiments, the nucleic acid is administered for in vivo or ex vivo gene therapy. Non-viral vector delivery systems include naked nucleic acid and nucleic acid complexed with a delivery vehicle (e.g. liposome or poloxamer). The delivered template molecule used during HDR may be, for example, an adeno-associated virus (AAV) based vector, a single stranded donor oligonucleotide (ssODN), or a PCR-generated double stranded DNA molecule. Such templates may be delivered, for example, by lipid nanoparticles (LNP). Alternatively, the RNA template molecule may be delivered, for example, by a lentivirus based delivery system.

[0106] For reviews of gene therapy procedures, see Anderson (1992); Nabel & Felgner (1993); Mitani & Caskey (1993); Dillon (1993); Miller (1992); Van Brunt (1988); Vigne (1995); Kremer & Perricaudet (1995); Haddada et al. (1995), and Yu et al. (1994).

[0107] Methods of non-viral delivery of nucleic acids and / or proteins include electroporation, lipofection, microinjection, biolistics, particle gun acceleration, virosomes, liposomes, immunoliposomes, lipid nanoparticles (LNPs), polycation or lipid:nucleic acid conjugates, artificial virions, and uptake of nucleic acids by facilitators, or the nucleic acids and / or proteins can be delivered by bacteria or viruses (e.g., Agrobacterium, Rhizobium sp. NGR234, Sinorhizobium meliloti, Mesorhizobium loti, tobacco mosaic virus, potato virus X, cauliflower mosaic virus, and cassava vein mosaic virus). Delivery to plant cells can be achieved by the use of recombinant endothelial cells (e.g., Chung et al., 2006). Sonoporation, e.g., using the Sonitron 2000 system (Rich-Mar), can also be used to deliver nucleic acids. Cationic lipid-mediated delivery of proteins and / or nucleic acids is also contemplated for in vivo, ex vivo or in vitro delivery. (See Zuris et al. (2015); Coelho et al. (2013); see also Judge et al. (2006) and Basha et al. (2011)).

[0108] Non-viral vectors, such as transposon-based systems (e.g., recombinant Sleeping Beauty transposon system or recombinant PiggyBac transposon system), may be delivered to target cells and used to transpose the polynucleotide sequences of, or encoding, the molecules of the composition in the target cells.

[0109] Other representative nucleic acid delivery systems include those offered by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) and Copernicus Therapeutics Inc. (see, e.g., U.S. Pat. No. 6,008,336). Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386, 4,946,787 and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam®, Lipofectin® and Lipofectamine® RNAiMAX). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those disclosed in WO 91 / 17424 and WO 91 / 16024. Delivery to cells (ex vivo administration) or target tissues (in vivo administration) is possible.

[0110] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those of skill in the art (see, e.g., Crystal, Science (1995); Blaese et al., (1995); Behr et al., (1994); Remy et al. (1994); Gao and Huang (1995); Ahmad and Allen (1992); U.S. Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028 and 4,946,787).

[0111] Another method of delivery involves packaging the nucleic acid to be delivered into an EnGeneIC delivery vehicle (EDV). The EDV is delivered specifically to the target tissue using a bispecific antibody, where one arm of the antibody has specificity for the target tissue and the other arm has specificity for the EDV. The antibody carries the EDV to the target cell surface, where it is carried into the cell by endocytosis. After entering the cell, the contents are released (see MacDiarmid et al., 2009).

[0112] The use of RNA or DNA viral systems for viral delivery of nucleic acids takes advantage of the highly evolved methods of targeting viruses to specific cells in the body and transporting the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or used to treat cells in vitro and the modified cells are administered to patients (ex vivo). Traditional viral systems for delivering nucleic acids include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated viral, vaccinia viral and herpes simplex viral vectors for gene transfer.

[0113] Retroviral tropism can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and usually have high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats and are capable of packaging up to 6-10 kb of foreign sequence. A minimal set of cis-acting LTRs is sufficient for vector replication and packaging, and is used to integrate therapeutic genes into target cells and permanently express the transgene. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchschacher et al. (1992); Johann et al. (1992); Sommerfelt et al. (1990); Wilson et al. (1989); Miller et al. (1991); WO 1994 / 026877).

[0114] At least six viral vectors are currently available for gene transfer in clinical trials, which utilize an approach involving complementation of a defective vector with a gene inserted into a helper cell line to generate the transducing agent.

[0115] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (see Dunbar et al., 1995; Kohn et al., 1995; Malech et al., 1997). PA317 / pLASN was the first therapeutic vector used in a gene therapy trial (Blaese et al., 1995). Transduction efficiency of the MFG-S packaging vector was greater than 50% (Ellem et al., (1997); Dranoff et al., 1997).

[0116] Packaging cells are used to form viral particles capable of infecting host cells. Such cells include 293 cells that package adenovirus, AAV, and Psi-2 or PA317 cells that package retrovirus. Viral vectors used in gene therapy are usually generated by producer cell lines that package nucleic acid vectors into viral particles. The vectors usually contain the minimum viral sequences required for packaging and subsequent integration into the host (if applicable), with other viral sequences being replaced with expression cassettes that code for the proteins to be expressed. Missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy usually only have inverted terminal repeats (ITRs) from the AAV genome that are required for packaging and integration into the host genome. The viral DNA is packaged in a cell line that lacks other AAV genes, i.e., ITR sequences, but contains a helper plasmid that codes for rep and cap. The cell line is also infected with adenovirus as a helper. The helper virus promotes the replication of AAV vectors and the expression of AAV genes from the helper plasmid. Due to the lack of ITR sequences, the helper plasmid is not packaged in large quantities. Adenovirus contamination can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV. In addition, AAV can be produced on a clinical scale using the baculovirus system (see U.S. Pat. No. 7,479,554).

[0117] In many gene therapies, it is desirable for the gene therapy vector to be delivered with a high degree of specificity to a particular tissue. Thus, viral vectors can be modified to have specificity for a given cell by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is selected to have affinity for a receptor known to be present on the cells of interest. For example, Han et al. (1995) reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and that the recombinant virus infects certain human breast cancer cells expressing the human epidermal growth factor receptor. This principle can be extended to other virus-target cell combinations, where the target cell expresses a receptor and the virus expresses a fusion protein containing a ligand for a cell surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) with specific binding affinity for virtually any selected cellular receptor. This description applies primarily to viral vectors, but the same principles can be applied to non-viral vectors. Such vectors can be engineered to contain specific uptake sequences that favor uptake by specific target cells.

[0118] Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravitreal, intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial injection) or local application, as described below. Alternatively, vectors can be delivered ex vivo to cells, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirate, tissue biopsy) or universal donor hematopoietic stem cells, and then re-implanted into the patient after selection of cells that have incorporated the vector. Exemplary, but non-limiting, ex vivo approaches may include removing tissues (e.g., peripheral blood, bone marrow and spleen) from the patient for culture, transferring the nucleic acid to the cultured cells (e.g., hematopoietic stem cells), and then transplanting the cells into the patient's target tissue (e.g., bone marrow and spleen). In some embodiments, the stem cells or hematopoietic stem cells may be further treated with a survival enhancer.

[0119] Ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., by re-infusion of the transfected cells into a host) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from a subject, transfected with a nucleic acid composition, and re-infused into the subject (e.g., patient). A variety of cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney, "Culture of Animal Cells, A Manual of Basic Technique and Specialized Applications (6th edition, 2010) and references cited therein in the discussion of methods for isolating and culturing cells from patients).

[0120] Suitable cells include, but are not limited to, eukaryotic cells and / or cell lines. Non-limiting examples of such cells, or cell lines derived from such cells, include COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2 / 0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6 cells, plant cells (differentiated or undifferentiated), as well as insect cells, such as Spodoptera fugiperda (Sf), or fungal cells, such as Saccharomyces, Pichia, and Schizosaccharomyces. In certain embodiments, the cell line is a CHO-K1, MDCK, or HEK293 cell line. Additionally, primary cells may be isolated and used ex vivo to be reintroduced into the subject to be treated after treatment with an inducible nuclease system (e.g., CRISPR / Cas). Suitable primary cells include peripheral blood mononuclear cells (PBMCs) and other blood cell subsets, including but not limited to CD4+ T cells or CD8+ T cells. Suitable cells also include stem cells, such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neural stem cells, and mesenchymal stem cells.

[0121] In some embodiments, stem cells are treated ex vivo for cell transfection and gene therapy. The advantage of using stem cells is that they can be differentiated in vitro into other cells or introduced into a mammal (such as a cell donor) and transplanted into the bone marrow. Methods are known for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such as GM-CSF, IFN-γ, and TNF-α (see Inaba et al., 1992, for non-limiting examples).

[0122] Stem cells are isolated for transduction and differentiation by known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies that bind unwanted cells such as CD4+ and CD8+ (T cells), CD45+ (pan B cells), GR-1 (granulocytes), and Iad (differentiated antigen presenting cells) (see, for non-limiting examples, Inaba et al., 1992). Modified stem cells may also be used in some embodiments.

[0123] Vectors (e.g., retroviruses, liposomes) carrying therapeutic nucleic acid compositions can also be administered directly to an organism to transduce cells in vivo. Administration is by routes typically used to introduce molecules into ultimate contact with blood or tissue cells, including, but not limited to, injection, infusion, and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and although multiple routes of administration of a particular composition can be used, a particular route will often provide a more rapid and effective response than another route. In some embodiments, the composition is delivered by IV injection.

[0124] Suitable vectors for introducing transgenes into immune cells (eg, T cells) include non-integrating lentiviral vectors (see, eg, US Patent Application Publication No. 2009 / 0117617).

[0125] Pharmaceutically acceptable carriers are determined in part by the composition being administered, as well as by the method used to administer the composition. Thus, there are a wide variety of suitable formulations of pharmaceutical compositions available, e.g., as described in Remington's Pharmaceutical Sciences, 17th ed., 1989.

[0126] The compositions and methods of the invention may also be used in the manufacture of a medicament for treating Hepatitis B in a patient.

[0127] Mechanism of action of HBV knockout methods Without being bound by any theory or mechanism, the present invention may be utilized to apply CRISPR nuclease to cleave HBV genomic DNA molecules, block their replication, and block expression from HBV genomic DNA molecules to prevent or treat Hepatitis B. Specific guide subsequences may be selected from Table 1 based on the HBV sequence to be targeted and the type of CRISPR nuclease to be used (required PAM sequence). Alternatively, HBV sequences integrated into the host genome may be targeted so that the CRISPR nuclease makes DNA cuts in the host genome.

[0128] One strategy for knocking out the HBV gene is to target the HBV sequence with an RNA molecule to mediate truncation or nonsense codon-mediated degradation (NMD).As a non-limiting example, a frameshift in HBV may be introduced by using an RNA molecule to target CRISPR nuclease to the HBV coding sequence and mediate a double-stranded break, which leads to the generation of a frameshift mutation and the expression of a truncated protein or nonsense codon-mediated degradation (NMD) of the HBV transcript.

[0129] Alternatively, the HBV gene may be knocked out by an excision strategy utilizing two RNA molecules. The same strategy may be implemented to excise regulatory elements that would prevent replication of the HBV genome.

[0130] An editing composition comprising a nuclease (e.g., a catalytically inactive CRISPR nuclease) fused to a chromatin modifier, such as, but not limited to, a demethylase or histone acetyltransferase, may increase delivery of the nuclease to the HBV minichromosome.

[0131] Alternatively, any of the editing compositions described herein may be accompanied by small molecules that modify chromatin, such as, but not limited to, methylation inhibitors and deacetylation inhibitors, which can increase excision by increasing the accessibility of the DNA nucleases of the editing composition to the HBV minichromosome.

[0132] One or more editing compositions comprising multiple guide RNA molecules targeting multiple sites in the HBV sequence may be utilized, for example, to mediate excision of a portion of the HBV sequence (e.g., a regulatory element) or to knock out an HBV gene from the HBV sequence.

[0133] In some embodiments, one or more editing compositions are delivered to a cell by one or more delivery vehicles. As a non-limiting example, two guide RNA molecules with different guide sequence portions and at least one CRISPR nuclease may be delivered to a cell, such that a first guide RNA molecule targeting a first site is delivered by a first delivery vehicle (e.g., a first AAV particle) and a second guide RNA molecule targeting a second site is delivered by a second delivery vehicle (e.g., a second AAV particle). In another example, guide RNA molecules with different guide sequence portions are delivered to a cell by a first delivery vehicle, and at least one CRISPR nuclease or a nucleic acid encoding the at least one CRISPR nuclease is delivered to the cell by a second vehicle. Additionally, a first editing composition comprising a first CRISPR nuclease-RNA guide complex may be delivered to a cell by a first delivery vehicle, and a second editing composition comprising a second CRISPR nuclease-RNA guide complex may be delivered to a cell by a second delivery vehicle.

[0134] Examples of RNA guide sequences that specifically target HBV Table 1 shows guide sequence segments designed to specifically target the HBV genome. Each engineered guide molecule is further designed to bind to a target genomic DNA sequence of interest located adjacent to a protospacer adjacent motif (PAM) (e.g., a PAM corresponding to the sequence NGG or NAG, where N is any nucleobase). The guide sequence is designed to work with one or more different CRISPR nucleases, including, but not limited to, SpCas9WT (PAM sequence: NGG), SpCas9.VQR.1 (PAM sequence: NGAN), SpCas9.VQR.2 (PAM sequence: NGNG), SpCas9.EQR (PAM sequence: NGAG), SpCas9.VRER (PAM sequence: NGCG), SaCas9WT (PAM sequence: NNGRRT), SpRY (PAM sequence: NRN or NYN), NmCas9WT (PAM sequence: NNNNGATT), Cpf1 (PAM sequence: TTTV), or JeCas9WT (PAM sequence: NNNVRYM). Each RNA molecule of the present invention is designed to form a complex with one or more different CRISPR nucleases and to target a polynucleotide sequence of interest using one or more different PAM sequences for each of the CRISPR nucleases used.

[0135] [Table 1]

[0136] To facilitate a more complete understanding of the present invention, the following examples are provided. The following examples illustrate representative modes of making and practicing the invention. However, the scope of the invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only. EXAMPLES

[0137] Experimental details Example 1 Screening of HBV guide sequence portion To identify optimal guide molecules targeting conserved HBV sequences, HeLa cells were stably infected with lentivirus carrying synthetic sequences of relevant regions of the HBV genome (Figure 1). In hepatocyte nuclei, the Hepatitis B virus (HBV) genome is present as an episome in the form of covalently closed circular DNA (cccDNA). Because the copy number of HBV cccDNA molecules varies from cell to cell (usually up to 10 copies per cell), HeLa cells were infected with lentivirus carrying synthetic sequences of HBV at three different multiplicities of infection (MOI), i.e., 2, 5 or 10 copies per cell. Stably infected cells were selected based on puromycin resistance.

[0138] Four different guide molecules (Table 2) targeting distinct regions of HBV DNA were screened for high on-target activity with WT OMNI-79 nuclease and OMNI-79 V5570 variant nuclease in HBV-infected HeLa cells. Briefly, WT OMNI-79 or OMNI-79 V5570 nuclease (64 ng) and the respective guide (20 ng) were co-transfected and screened in a 96-well format using JetOPTIMUS reagent (Polyplus). Cells were harvested 72 hours after DNA transfection. To measure on-target activity by next-generation sequencing (NGS), cell lysis and genomic DNA extraction were performed with Quick Extract (Lucigen) and endogenous genomic regions were amplified using specific primers (Figure 2, Table 2).

[0139] [Table 2]

[0140] [Table 3]

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Claims

Claim 1 A method for modifying a hepatitis B virus (HBV) sequence in a cell, comprising: at least one CRISPR nuclease, or a nucleotide sequence encoding a CRISPR nuclease; and a first RNA molecule comprising a guide sequence portion of 17 to 50 nucleotides, or a nucleotide sequence encoding the first RNA molecule introducing a composition comprising into the cell, a complex of the CRISPR nuclease and the first RNA molecule double-strand breaks the HBV sequence, wherein the guide sequence portion of the first RNA molecule comprises 17 to 50 consecutive nucleotides within the sequence shown in any one of SEQ ID NOs: 1 to 18936. Claim 2 The method according to claim 1, wherein the double-strand break is effected on an HBV gene or a portion thereof, an HBV coding sequence or a portion thereof, an HBV control sequence or a portion thereof, and / or a sequence of a conserved HBV sequence. Claim 3 The method according to claim 1, further comprising introducing into the cell a second RNA molecule comprising a guide sequence portion of 17 to 50 nucleotides, or a nucleotide sequence encoding the second RNA molecule, wherein a complex of the second RNA molecule and the CRISPR nuclease further double-strand breaks the hepatitis B virus sequence. Claim 4 The method according to claim 3, wherein the guide sequence portion of the second RNA molecule comprises 17 to 50 consecutive nucleotides within the sequence shown in any one of SEQ ID NOs: 1 to 18936 other than the sequence of the first RNA molecule. Claim 5 The method according to claim 3 or 4, wherein a nucleotide sequence is excised from a molecule comprising the HBV sequence. Claim 6 The method according to claim 5, wherein the nucleotide sequence excised from the HBV sequence comprises an HBV gene or a portion thereof, an HBV coding sequence or a portion thereof, an HBV control sequence or a portion thereof, and / or a conserved HBV sequence. Claim 7 The method according to any one of claims 1 to 4, wherein the first or second RNA molecule comprises a guide sequence portion targeting a sequence located up to 500 base pairs from an HBV gene, an HBV coding sequence, an HBV control sequence, and / or a conserved HBV sequence excised by the first and second RNA molecules. Claim 8 The method according to any one of claims 1 to 4, wherein the cell is a liver cell or a hepatocyte. Claim 9 The method according to any one of claims 1 to 4, wherein the HBV sequence is located in an HBV covalently closed circular DNA (cccDNA) molecule.

10. The method according to claim 1 or 2, wherein the HBV sequence is located in a genomic DNA molecule into which the HBV sequence has been integrated.

11. A composition comprising a first RNA molecule, wherein the first RNA molecule comprises a guide sequence portion of 17 to 50 consecutive nucleotides within the sequence shown in any one of SEQ ID NOs: 1 to 18936.

12. The composition according to claim 11, further comprising at least one CRISPR nuclease.

13. The composition according to claim 12, further comprising a second RNA molecule comprising a guide sequence portion of 17 to 50 consecutive nucleotides, wherein the second RNA molecule targets the HBV gene and the guide sequence portion of the second RNA molecule is a sequence different from the sequence of the guide sequence portion of the first RNA molecule, and optionally, the guide sequence portion of the second RNA molecule comprises 17 to 50 consecutive nucleotides within the sequence shown in any one of SEQ ID NOs: 1 to 18936 other than the sequence of the first RNA molecule.

14. A method for inactivating hepatitis B virus in a cell, comprising delivering the composition according to any one of claims 11 to 13 to the cell.

15. Use of the composition according to any one of claims 11 to 13 for inactivating hepatitis B virus in a cell, optionally comprising delivering the composition according to any one of claims 11 to 13 to the cell.

16. A medicament comprising the composition according to any one of claims 11 to 13 for use in inactivating hepatitis B virus in a cell, wherein the medicament is administered by delivering the composition according to any one of claims 11 to 13 to the cell.

17. A composition according to any one of claims 11 to 13 or a medicament comprising the composition according to any one of claims 11 to 13 for use in the treatment, remission or prevention of hepatitis B, wherein the medicament is administered by delivering the composition according to any one of claims 11 to 13 to the cells of a subject having or at risk of having hepatitis B.

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