Systems and methods for treatment of hepatitis b virus (HBV) infection

Abstract

The present disclosure relates to compositions and methods for excision or inactivation of Hepatitis B Virus (HBV) nucleic acid sequences.

Description

SYSTEMS AND METHODS FOR TREATMENT OF HEPATITIS B VIRUS (HBV) INFECTIONCROSS-REFERENCE

[0001] This application claims the benefit of U. S. Provisional Patent Application No. 63 / 644,384, filed on May 8, 2024, and U. S. Provisional Patent Application No. 63 / 693, 121, filed on September 10, 2024, each of which is incorporated herein by reference in its entirety .BACKGROUND

[0002] Chronic hepatitis B infection, caused by hepatitis B virus (HBV), is one of the most prevalent infectious diseases, with almost 300 million people estimated to be infected. If untreated, chronic hepatitis B infection significantly incr eases the risk of developing liver cirrhosis and hepatocellular carcinoma (HCC). Among a few different forms of HBV DNAs that persist in chronically infected hepatocytes, covalently closed circular DNA (cccDNA) serves as the template of pregenomic RNA (pgRNA), which plays a key role in viral replication .SUMMARY

[0003] Many HBV treatments only slow progression of liver disease by blocking viral replication and / or by modulating the host immune systems. However, a problem with these therapies is that they are chronically administered to reduce viral load in a patient to a sub stantially undetectable level, which is characterized by sustained undetectable circulating HBsAg and HBV DNA. Targeting HBV DNA, including cccDNA, by gene editing is a promising approach for in activating HBV in a patient, thereby providing a promising lifelong (e.g., curative) treatment of chronic HBV infection without requiring chronic dosing. Small insertions and / or deletions (indels), such as edits introduced by Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 system with a single guide RNA (gRNA), may notbe sufficient to suppress viral replication without requiring chronic dosing. In contrast, using two or more gRNAs and simultaneously cutting HBV DNA at multiple sites can give rise to structural variants (SVs), such as excision, that disrupt the viral life cycle by preventing or inhibiting HBV DNA replication.

[0004] Lipid nanoparticles (LNPs) are an in-vivo delivery platform that can be used to deliver nucleic acid payloads to a patient. LNPs can be efficiently targeted to the liver through a natural lipid uptake pathway. Administration of LNPs that encapsulate mRNA encoding a CRISPR-Cas system with multiple guide RNAs leads to multiplex geneediting only in a short time window (e.g., compared to viral vector delivery platforms) due to transient expression of the payload in the cytoplasm of hepatocytes. Using such LNP delivery platforms mitigates the risk of off-target effects, such as by reducing prolonged gene editing. In addition, when using such LNP delivery platforms, the mRNA-derived sequence is not integrated into the chromosomal DNA, and low immunogenicity can provide for repeated dosing to augment gene editing efficacy. For these reasons, multiplex editing by transient delivery of a CRISPR -Cas system formulated in LNP potentially offers an efficacious and safe option for treatment of chronic HBV infection, such as without requiring chronic dosing.

[0005] Provided herein are lipid nanoparticles for the use of inactivating viral nucleic acids, reducing serum viral DNA, and / or treating a viral infection (e.g., hepatitis B virus [HBV]). In some embodiments, provided herein is a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a polynucleotide. In some embodiments, the polynucleotide comprises a sequence encoding a CRISPR-Cas endonuclease. In some embodiments, the lipid nanoparticle comprises a firstguide ribonucleic acid (gRNA) . In some embodiments, the first gRNA hybridizes to a first target sequence . In some embodiments, the first target sequence is within a viral nucleic acid molecule. In some embodiments, the first target sequence is within an HBV nucleic acid molecule. In some embodiments, the lipid nanoparticle comprises a second gRNA. In some embodiments, the second gRNA hybridizes to a second target sequence. In some embodiments, the second target sequence is within a viral nucleic acid molecule. In some embodiments, the second target sequence is within an HBV nucleic acid molecule. In some embodiments, the second target sequence is different from the first target sequence. In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the first gRNA cleaves the viral nucleic acid molecule within or proximal to the first target sequence, generating a first cleaved site. In some embodiments, expression of th e CRISPR-Cas endonuclease in combination with the second gRNA cleaves the viral nucleic acid molecule within or proximal to the second target sequence, generating a second cleaved site. In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the first gRNA cleaves the HBV nucleic acid molecule within or proximal to the first target sequence, generating a first cleaved site. In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the second gRNA cleaves the HBV nucleic acid molecule within or proximal to the second target sequence, generating a second cleaved site. In some embodiments, the cleaved site proximal to the first target sequence is within a distance of about 1 , 2, 3 , 4, 5 , 10, 15, or20 nucleobase positions of the first target sequence. In some embodiments, the cleaved site proximal to the second target sequence is within a distance of about 1 , 2, 3 , 4, 5, 10, 15 , or 20 nucleobase positions of the second target sequence. In some embodiments, generating the first cleaved site and the second cleaved site results in excising a region of the viral nucleic acid molecule (e.g., HBV viral nucleic acid molecule) between the first cleaved site and the second cleaved site.

[0006] Provided herein are LNP-CRISPR-Cas systems useful for inactivating HBV, reducing serum HBV DNA, and / or treating HBV. For example, in come embodiments, provided herein are lipid nanoparticles (LNPs) comprising: (1 ) a polynucleotide comprising a sequence encoding a CRISPR-Cas endonuclease; (2) a first guide ribonucleic acid (gRNA) that hybridizes to a first target sequence within an HBV nucleic acid molecule; and (3 ) a second gRNA that hybridizes to a second target sequence within the HBV nucleic acid molecule, wherein: (i) the second target sequence is differentfrom the first target sequence; and (ii) expression of the CRISPR-associated nuclease in combination with the first gRNA cleaves the HBV nucleic acid molecule within or proximal (e.g. , within a distance of about 1 , 2, 3 , 4, 5 , 10, 15 or 20 nucleobase positions) to the first target sequence, generating a first cleaved site; and (iii) expression of the CRISPR-associated nuclease in combination with the second gRNA cleaves the HBV nucleic acid molecule within or proximal (e.g., within a d istance of about 1 , 2, 3, 4, 5, 10, 15 or 20 nucleobase positions) to the second target sequence, generating a second cleaved site. Also provided herein are methods of treating HBV, wherein the methods comprise administering the LNPs comprising the CRISPR-Cas systems described herein to the individual. In certain embodiments, treating comprises reducing the amount of HBV serum biomarkers (e.g. , compared to an untreated individual or a prior measurement of HBV serum biomarkers). In such embodiments, the HBV serum biomarkers comprise HBV DNA, HBsAg, HBeAg, or a combination thereof. In certain embodiments, treating comprises reducing intrahepatic HB V DNA (e.g. , compared to an untreated individual or a prior measurement of HBV serum biomarkers).

[0007] In some embodiments, an LNP provided herein, which comprises a polynucleotide encoding a CRISPR-Cas endonuclease, a first guide ribonucleic acid (gRNA) that hybridizes to a first target sequence within a viral nucleic acid molecule, and a second gRNA that hybridizes to a second target sequence within the viral nucleic acid molecule, can lead to suppression of viral persistence in a cell through a wide range of DNA repair outcomes, including, but not limited to, indels, excisions, and inversions. In some embodiments, the LNP comprising the polynucleotide encoding a C RISPR-Casendonuclease, a first gRNA, and a second gRNA can be used to induce excision of a region of the viral nucleic acid. In some embodiments, the LNP comprising the polynucleotide encoding a CRISPR-Cas endonuclease, a first gRNA, and a second gRNA can be used to induce inversion of a region of the viral nucleic acid. In some embodiments, the LNP comprising the polynucleotide encoding a CRISPR-Cas endonuclease, a first gRNA, and a second gRNA can be used to induce indels within a region of the viral nucleic acid. In some embodiments, the LNP comprising the polynucleotide encoding a CRISPR-Cas endonuclease, a first gRNA, and a second gRNA can be used to induce concatemerization of a region of the viral nucleic acids. In some embodiments, the LNP comprising the polynucleotide encoding a CRISPR-Cas endonuclease, a first gRNA, and a second gRNA can be used to induce one or more of: excision of a region of the viral nucleic acid, inversion of a region of the viral nucleic acid, indels within a region of the viral nucleic acid, and concatemerization of two or more viral nucleic acid s.

[0008] In some embodiments, an LNP provided herein, which comprises a polynucleotide encoding a CRISPR-Cas endonuclease, a first guide ribonucleic acid (gRNA) that hybridizes to a first target sequence within a hepatitis B viral (HB V) nucleic acid molecule, and a second gRNA that hybridizes to a second target sequence within the HB V nucleic acid molecule, can lead to suppression of HB V persistence in a cell through a wide range of DNA repair outcomes, including, but not limited to, indels, excisions, and inversions . In some embodiments, the LNP comprising the polynucleotide encoding a CRISPR-Cas endonuclease, a first gRNA, and a second gRNA can be used to induce excision of a region of the HB V nucleic acid. In some embodiments, the LNP comprising the polynucleotide encoding a CRISPR-Cas endonuclease, a first gRNA, and a second gRNA can be used to induce inversion of a region of the HBV nucleic acid. In some embodiments, the LNP comprising the polynucleotide encoding a CRISPR-Cas endonuclease, a first gRNA, and a second gRNA can be used to induce indels within a region of the HBV nucleic acid. In some embodiments, the LNP comprising the polynucleotide encoding a CRISPR-Cas endonuclease, a first gRNA, and a second gRNA can be used to induce concatemerization of a region of the HBV nucleic acids. In some embodiments, the LNP comprising the polynucleotide encoding a CRISPR-Cas endonuclease, a first gRNA, and a second gRNA can be used to induce on e or more of: excision of a region of the HBV nucleic acid, inversion of a region of the HBV nucleic acid, indels within a region of the HBV nucleic acid, and concatemerization of a region of the HBV nucleic acid .BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which :

[0010] FIG. 1 provides a schematic of antiviral therapy with multiple guide ribonucleic acids (gRNAs) for treatment of hepatitis B virus (HBV);

[0011] FIG. 2 provides a schematic of the search strategy for guide RNA target sites on consensus HBV sequences;

[0012] FIG. 3 illustrates the positions of guide RNA target (gRNA) sites;

[0013] FIG. 4A provides a schematic of a cell line with integrated HBV target sites transfected with SaCas9 mRNA and guide RNA pair;

[0014] FIG. 4B provides a schematic of excision and indels;

[0015] FIG. 5A provides the excision rate of the integrated HBV reporter construct in the cell line;

[0016] FIG. 5B provides the indel rate at the unexcised target sites of the cell line ;

[0017] FIG. 6A provides a schematic of an experiment in which the reduction of intracellular HBV DNA in a cell transfected with SaCas9 and a pair of gRNAs is measured;

[0018] FIG. 6B shows the reduction in intracellular HBV DNA in HBV-infected cells transfected with SaCas9 mRNA and a pair of gRNAs;

[0019] FIG. 7A provides a schematic of an experiment in which the suppressed secretion of Hepatitis B e-Antigen (HbeAg) and Hepatitis B surface Antigen (HbsAg) from a cell transfected with SaCas9 mRNA and a pair of gRNAs is measured;

[0020] FIG. 7B shows the suppression of secretion of HBeAg in HBV-infected cells transfected with SaCas9 mRNA and a pair of gRNAs;

[0021] FIG. 7C shows the suppression of secretion of HBsAg in HBV-infected cells transfected with SaCas9 mRNA and a pair of gRNAs;

[0022] FIG. 8A provides a schematic of an experiment in which the lowering of serum HBV biomarkers following lipid nanoparticle (LNP)-mediated delivery of SaCas9 mRNA and a pair of gRNAs to an adeno-associated virus-Hepatitis B virus (AAV-HBV) mouse model is measured;

[0023] FIG. 8B shows the reduction of HBsAg in the serum of AAV-HBV mice following LNP-mediated delivery of SaCas9 mRNA and a pair of gRNAs;

[0024] FIG. 8C shows the reduction of HBeAg in the serum of AAV-HBV mice following LNP-mediated delivery of SaCas9 mRNA and a pair of gRNAs;

[0025] FIG. 8D shows the reduction of HB V DNA in the serum of AAV-HBV mice following LNP-mediated delivery of SaCas9 mRNA and a pair of gRNAs;

[0026] FIG. 9A provides a schematic of an experiment in which the degradation of intrahepatic HBV DNA following lipid nanoparticle (LNP) -mediated delivery of SaCas9 mRNA and a pair of gRNAs to an adeno-associated virus-Hepatitis B virus (AAV-HBV) mouse model is measured;

[0027] FIG. 9B shows the reduction of intrahepatic HBV DNA in AAV-HBV mice following LNP-mediated delivery of SaCas9 mRNA and a pair of gRNAs ;

[0028] FIG. 10A d emonstrates the editing efficiency and specificity of SaCas9 protein complexed with gRNA 1 (SEQ ID NO : 601 ) at on-target viral sites following transfection with SaCas9-encoding mRNA and the chemically synthesized gRNA 1 (SEQ ID NO : 601 ), as compared to human genomic sites nominated for off-target analysis (nominated sites);

[0029] FIG. 10B demonstrates the editing efficiency and specificity of SaCas9 protein complexed with gRNA 2 (SEQ ID NO : 603) at on -target viral sites following transfection with SaCas9-encoding mRNA and the chemical synthesized gRNA 2 (SEQ ID NO: 603), as compared to human genomic sites nominated for off-target analysis (nominated sites);

[0030] FIG. 11 A provides a schematic of the time-course of an experiment in which the reduction of intracellular HBV DNA and of secreted HbeAg and Hb sAg in HBV- infected, primary human hepatocytes transfected with SaCas9 mRNA and a pair of gRNAs (g RNA 1 and g RNA 2) is measured;

[0031] FIG. 11B provides a schematic of an experiment in which the reduction of secreted HbeAg and Hb sAg (short dashed box) and of intracellular HBV DNA (long dashed box) in HBV-infected, primary human hepatocytes transfected with SaCas9 mRNA and a pair of gRNAs (gRNA 1 and gRNA 2) is measured;

[0032] FIG. 11C shows the suppression of secretion of HBeAg in HBV-infected primary human hepatocytes transfected with SaCas9 mRNA and a pair of gRNAs;

[0033] FIG. HD shows the suppression of secretion of HBsAg in HBV-infected primary human hepatocytes transfected with SaCas9 mRNA and a pair of gRNAs;

[0034] FIG. HE shows the reduction in intracellular HBV DNA in HBV-infected primary human hepatocytes transfected with SaCas9 mRNA and a pair of gRNAs;

[0035] FIG. 12A provides a second schematic (see also FIG. 8A) of an experiment in which the lowering of serum HBV biomarkers following lipid nanoparticle (LNP)- mediated delivery of SaCas9 mRNA and a pair of gRNAs to an adeno-associated virus- Hepatitis B virus (AAV-HBV) mouse model is measured;

[0036] FIG. 12B shows the reduction of intrahepatic HBV DNA in AAV-HBV mice that were dosed with LNPs encapsulating SaCas9 mRNA and a pair of gRNAs;

[0037] FIG. 13A illustrates the results of the sequencing analysis to detect the frequency of indels at the intended target site of HBV DNA recovered from HBV-infected PHHs transfected with the SaCas9 mRNA and either non -targeting gRNA or SaCas9 gRNA 1 and SaCas9 gRNA 2;

[0038] FIG. 13B illustrates the frequency of structural variant reads that can be generated by intended excisions (left) and inversions (right) of the viral sequences between the two intended cut sites;

[0039] FIG. 14A illustrates an analysis of HBV DNA insertion sites in chromosomal DNA and the positions of HBV DNA connected to human chromosomes identified in chimeric sequence reads mapped on the HBV reference genome (GenBank ID : U9555 1 );

[0040] FIG. 14B illustrates the number of HBV-human chromosome chimeric reads, normalized with the average sequence coverage of the human GAPDH gene, to estimate the relative fraction of chromosomal HBV DNA insertions sites;

[0041] FIG 15A illustrates the quantification of sequencing analysis of total HBV DNA in liver samples from three representative AAV-HBV mice receiving PBS as a negative control or LNPs encapsulating the SaCas9-encoding mRNA and the pair of SaCas9 gRNA 1 and SaCas9 gRNA 2 at the high-dose level;

[0042] FIG. 15B illustrates the calculated frequency of indels at the intended target sites on HBV DNA extracted from the control and edited liver samples of AAV-HBV mice;

[0043] FIG 15C illustrates the calculated frequency of excisions (left) and inversions (right) of the intervening HBV sequence between the two intended target sites for SaCas9 gRNA 1 and SaCas9 gRNA 2;

[0044] FIG. 16A illustrates the analysis of HBV DNA insertion sites in chromosomal DNA and the identified junctions of HBV DNA and mouse chromosomal DNA identified in chimeric reads, which were mapped on the HBV reference genome (GenBank ID: U9555 1 );

[0045] FIG 16B illustrates the number of HB V-chromosome chimeric reads normalized with the average sequence depth of mouse Gapdh to estimate the relative frequency of chromosomal HBV DNA insertions;

[0046] FIG 17A illustrates an exemplary schematic of the transgenic-HB V (Tg-HBV) mouse model, wherein the Tg-HBV sequence is stably inserted in a single chromosomal location and predicted to serve as a template of all viral transcripts required for viral replication in hepatocytes;

[0047] FIG. 17B illustrates the reduction in serum HBsAg in the Tg-HBV mice dosed with LNPs to eliminate HBV DNA by multiplex gene editing to assess the in vivo editing against chromosomally integrated HBV DNA;

[0048] FIG. 17C i llustrates the reduction of HBV DNA in the Tg-HBV mice dosed with LNPs to eliminate HBV DNA by multiplex gene editing to assess the in vivo editing against chromosomally integrated HBV DNA;

[0049] FIG. 17D illustrates the quantification via digital PCR of total HBV DNA in liver samples harvested from the Tg-HBV mice five weeks after LNP administration;

[0050] FIG. 18A illustrates the quantification via digital PCR of total HBV DNA present in the stomach of three Tg-HBV mice;

[0051] FIG. 18B illustrates an exemplary schematic of the Tg-HBV configuration determined by long-read hybridization sequencing;

[0052] FIG. 18C illustrates the calculated frequency of structural variants introduced by anti-HBV multiplex gene editing quantified by long-read hybridization capture sequencing;

[0053] FIG. 19A illustrates an exemplary schematic of hypothetical steps in de novo random HBV DNA integration;

[0054] FIG. 19B illustrates the analysis of de novo HBV DNA insertions sites;

[0055] FIG. 20A illustrates three exemplary hypothetical mechanisms that lead to chromosomal translocations associated with de novo HBV DNA fragments; and

[0056] FIG. 20B illustrates the number of sequence reads derived from chromosomal translocations associated with de novo HBV DNA fragments normalized with the average sequence coverage of the mouse Gapdh gene.DETAILED DESCRIPTIONLNP CRISPR-Cas systems

[0057] Provided herein are lipid nanoparticles for the use of treating a viral infection .In some embodiments, the viral infection is a hepatitis B virus [HBV] infection. In someembodiments, provided herein is a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a polynucleotide. In some embodiments, the polynucleotide comprises a sequence encoding a CRISPR-Cas endonuclease. In some embodiments, the lipid nanoparticle comprises a first guide ribonucleic acid (gRNA.) In some embodiments, the first gRNA hybridizes to a first target sequence . In some embodiments, the first target sequence is within a viral nucleic acid molecule. In some embodiments, the first target sequence is within an HB V nucleic acid molecule. In some embodiments, the lipid nanoparticle comprises a second gRNA. In some embodiments, the second gRNA hybridizes to a second target sequence. In some embodiments, the second target sequence is within a viral nucleic acid molecule. In some embodiments, the second target sequence is within an HBV nucleic acid molecule. In some embodiments, the second target sequence is different from the first target sequence. In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the first gRNA cleaves the viral nucleic acid molecule within or proximal to the first target sequence, generating a first cleaved site. In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the second gRNA cleaves the viral nucleic acid molecule within or proximal to the second target sequence, generating a second cleaved site . In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the first gRNA cleaves the HBV nucleic acid molecule within or proximal to the first target sequence, generating a first cleaved site. In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the seco nd gRNA cleaves the HBV nucleic acid molecule within or proximal to the second target sequence, generating a second cleaved site. In some embodiments, the cleaved site proximal to the first target sequence is within a distance of about 1 , 2, 3 , 4, 5 , 10, 1 5 , or 20 nucleobase positions of the first target sequence. In some embodiments, the cleaved site proximal to the second target sequence is within a distance of about 1 , 2, 3 , 4, 5 , 10, 15 , or 20 nucleobase positions of the second target sequence. In some embodiments, generating the first cleaved site and the second cleaved site results in excising a region of the viral nucleic acid molecule (e g. , HBV viral nucleic acid molecule) between the first cleaved site and the second cleaved site.

[0058] Provided herein are lipid nanoparticles (LNPs) comprising: (1) a polynucleotide comprising a sequence encoding a CRISPR-Cas endonuclease; (2) a first guide ribonucleic acid (gRNA) that hybridizes to a first target sequence within an HBV nucleic acid molecule; and (3) a second gRNA that hybridizes to a second target sequence within the HBV nucleic acid molecule, wherein: (i) the second target sequence isdifferent from the first target sequence; and (ii) expression of the CRISPR-associated nuclease in combination with the first gRNA cleaves the HBV nucleic acid molecule within or proximal (e.g. , within a distance of about 1 , 2, 3 , 4, 5 , 10, 15 or 20 nucleobase positions) to the first target sequence, generating a first cleaved site; and (iii) expression of the CRISPR-associated nuclease in combination with the second gRNA cleaves the HBV nucleic acid molecule within or proximal (e.g. , within a distance of about 1 , 2, 3, 4, 5 , 10, 15 or 20 nucleobase positions) to the second target sequence, generating a second cleaved site.

[0059] In certain instances, the LNP-CRISPR-Cas systems described herein are advantageous in that targeting and cutting of the HBV nucleic acid (e.g. , genome or cccDNA) results in excision (e.g., larger deletions) of a region of the HBV nucleic acid. In certain emb odiments, generating the first cleaved site and the second cleaved site results in excising a region of the HBV nucleic acid molecule between the first cleaved site and the second cleaved site.

[0060] Provided herein are methods of treating a viral infection (e.g . , HBV infection) in an individual. In some embodiments, provided herein is a method of treating a viral infection comprising administering a lipid nanoparticle to the individual. In some embodiments, the lipid nanoparticle comprises a polynucleotide. In s ome embodiments, the polynucleotide comprises a sequence encoding a CRISPR-Cas endonuclease. In some embodiments, the lipid nanoparticle comprises a first guide ribonucleic acid (gRNA.) In some embodiments, the first gRNA hybridizes to a first target sequence In some embodiments, the first target sequence is within a viral nucleic acid molecule. In some embodiments, the first target sequence is within an HBV nucleic acid molecule. In some embodiments, the lipid nanoparticle comprises a second gRNA. In some embodiments, the second gRNA hybridizes to a second target sequence. In some embodiments, the second target sequence is within a viral nucleic acid molecule. In some embodiments, the second target sequence is within an HBV nucleic acid molecule. In some embodiments, the second target sequence is different from the first target sequence. In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the first gRNA cleaves the viral nucleic acid molecule within or proximal to the first target sequence, generating a first cleaved site. In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the second gRNA cleaves the viral nucleic acid molecule within or proximal to the second target sequence, generating a second cleaved site. In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the first gRNA cleaves the HBV nucleic acid molecule within orproximal to the first target sequence, generating a first cleaved site. In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the second gRNA cleaves the HBV nucleic acid molecule within or proximal to the second target sequence, generating a second cleaved site. In some embodiments, the cleaved site proximal to the first target sequence is within a distance of about 1 , 2, 3 , 4, 5 , 10, 15, or 20 nucleobase positions of the first target sequence. In some embodiments, the cleaved site proximal to the second target sequence is within a distance of about 1 , 2, 3 , 4, 5, 10, 15 , or 20 nucleobase positions of the second target sequence. In some embodiments, generating the first cleaved site and the second cleaved site results in excising a region of the viral nucleic acid molecule (e.g., HBV viral nucleic acid molecule) between th e first cleaved site and the second cleaved site.

[0061] Provided herein are methods of inactivating a viral nucleic acid molecule (e.g., HBV nucleic acid molecule) or excising (e.g. , deleting) a region of a viral nucleic acid molecule (e.g., HBV nucleic acid molecule) in a cell. In some embodiments, provided herein is a method of inactivating a viral nucleic acid molecule (e.g. , HBV nucleic acid molecule) or excising (e.g., deleting) a region of a viral nucleic acid molecule (e.g., HBV nucleic acid molecule) comprising contacting the cell with a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a polynucleotide. In some embodiments, the polynucleotide comprises a sequence encoding a CRISPR-Cas endonuclease. In some embodiments, the lipid nanoparticle comprises a firstguide ribonucleic acid (gRNA.) In some embodiments, the first gRNA hybridizes to a first target sequence . In some embodiments, the first target sequence is within a viral nucleic acid molecule. In some embodiments, the first target sequence is within an HBV nucleic acid molecule. In some embodiments, the lipid nanoparticle comprises a second gRNA. In some embodiments, the second gRNA hybridizes to a second target sequence. In some embodiments, the second target sequence is within a viral nucleic acid molecule. In some embodiments, the second target sequence is within an HBV nucleic acid molecule. In some embodiments, the second target sequence is different from the first target sequence. In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the first gRNA cleaves the viral nucleic acid molecule within or proximal to the first target sequence, generating a first cleaved site. In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the second gRNA cleaves the viral nucleic acid molecule within or proximal to the second target sequence, generating a second cleaved site. In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the first gRNA cleaves the HBV nucleic acid molecule within orproximal to the first target sequence, generating a first cleaved site. In some embodiments, expression of the CRISPR-Cas endonuclease in combination with the second gRNA cleaves the HBV nucleic acid molecule within or proximal to the second target sequence, generating a second cleaved site. In some embodiments, the cleaved site proximal to the first target sequence is within a distance of about 1 , 2, 3 , 4, 5 , 10, 15, or 20 nucleobase positions of the first target sequence . In some embodiments, the cleaved site proximal to the second target sequence is within a distance of about 1 , 2, 3 , 4, 5, 10, 15 , or 20 nucleobase positions of the second target sequence. In some embodiments, generating the first cleaved site and the sec ond cleaved site results in excising a region of the viral nucleic acid molecule (e.g., HBV viral nucleic acid molecule) between the first cleaved site and the second cleaved site.

[0062] The CRISPR-Cas systems for excision described herein are generally advantageous over commonly used multiplexed CRISPR-Cas systems that aimed for and / or were limited to the generation of multiple, independent indels. To this effect, the first target sequence and the second target sequence can be within different genes. In certain embodiments, the firsttarget sequence and the second target sequence are within different genes and separated by at least 250, at least 500, at least 1 ,000, at least 1,100, at least 1 ,200, at least 1 ,300, at least 1 ,400, at least 1 ,500, or at least 1 ,600 nucleotides. In such instances, the excised the region of the HBV nucleic acid molecule between the first cleaved site and the second cleaved site comprises at least 250, at least 500, at least 1 ,000, at least 1 , 100, at least 1 ,200, at least 1 ,300, at least 1 ,400, at least 1 ,500, or at least 1 ,600 nucleobase positions. Targeting at least one sequence within a region of the HBV nucleic acid molecule encoding two genes is of additional advantage for inactivating HBV. In certain embodiments, at least one of the first target sequence and the second target sequence are within a region of the HBV nu cleic acid molecule encoding two genes. In certain embodiments, both of the first target sequence and the second target sequence are within a region of the HBV nucleic acid molecule encoding two genes.

[0063] Target sequences can be within HBV gene regions selected from pol, preSl, preS2, S, x, preC, and C. In certain embodiments, the first target sequence is within the pol gene region of the HBV nucleic acid molecule. In certain embodiments, the first target sequence is within the pol and S gene region of the HB V nucleic acid molecule. In certain embodiments, the second target sequence is within the preC gene region of the HBV nucleic acid molecule. In certain embodiments, combinations of targeted genes are selected from the gene regions targeted in Table 7 .

[0064] Target sequences (e.g., as defined by a protospacer sequence) of particular benefit are included in Table 1 or Table 4 or Table 7. In certain embodiments, the first target sequence and the second target sequence each independently comprise a protospacer sequence selected from Table 1 or Table 4 or Table 7. Additional guides can include those described in Tables 2 -3 and 5. In certain embodiments, the first target sequence and the second target sequence each independently comprise a protospacer sequence having at least 90% sequence identity or at least 95% sequence identity to a protospacer sequence selected from Table 1 or Table 4 or Table 7.CRISPR-Cas endonucleases

[0065] CRISPR systems refer to and include elements involved in the expression of or directing the activity of a CRISPR-associated (Cas) endonuclease, including guide RNA sequences and components thereof, such as a tracr (trans -activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a spacer sequence (also referred to as a guide sequence), or other sequences and transcripts from a CRISPR locus. In general, a CRISPR system is characterized by such elements that promote the formation of a CRISPR complex atthe site of a target sequence. In the context of formation of a CRISPR complex, a target sequence (protospacer sequence) refers to a sequence to which a spacer sequence is designed to hybridize to, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.

[0066] The CRISPR-Cas systems include Type I CRISPR-Cas system, Type II CRISPR-Cas system, Type III CRISPR-Cas system, and derivatives thereof. CRISPR- Cas systems include engineered and / or programmed nuclease systems derived from naturally occurring CRISPR-Cas systems. CRISPR-Cas systems may contain engineered and / or mutated Cas proteins. In certain embodiments, nucleases generally refer to enzymes capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. In certain embodiments, endonucleases are generally capable of cleaving the phosphodiester bond within a polynucleotide chain. Nickases refer to endonucleases that cleave only a single strand of a DNA duplex.

[0067] In some embodiments, the CRISPR-Cas system used herein can be a type I, a type II, or a type III system. Non-limiting examples of suitable CRISPR-Cas endonucleases include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a l, Cas8a2, Cas8b, Cas8c, Cas9, Cas lO, Cas lOd, CasF, CasG, CasH, CasX, CasO, Csy l , Csy2, Csy3, Cse l (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC),Csc l , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5 , Csm6, Cmrl , Cmr3, Cmr4, Cmr5, Cmr6, Csb l, Csb2, Csb3, Csx l7, Csx l4, Csxl O, Csxl6, CasX, Csx3, Cszl , Csxl 5, Csfl, Csf2, Csf3, Csf4, and Cu l 966. In certain embodiments, the CRISPR-Cas protein or endonuclease is Cas9. In certain embodiments, the CRISPR-Cas protein or endonuclease is Cas l 2. In certain embodiments, the CRISPR-Cas protein or endonuclease is CasX. In certain embodiments, the CRISPR-Cas protein or endonuclease is CasO.

[0068] In some embodiments, the Cas9 protein can be from or derived from: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp . , Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp . , Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp ., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Fine goldia magna, Natranaerobius thermophilus, Pelotomaculum the rmopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp ., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp ., Arthrospira maxima, Arthrospira platensis, Arthrospira sp . , Lyngbya sp ., Microcoleus chthonoplastes, Oscillatoria sp . , Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina.

[0069] In some embodiments, the gene editing system comprises a CRISPR- associated (Cas) protein, or functional fragment or derivative thereof. In certain embodiments, the Cas protein is an endonuclease, including but not limited to the Cas9 nuclease. In some embodiments, the Cas9 protei n comprises an amino acid sequence identical to the wild-type Streptococcus pyogenes or Staphylococcus aureus Cas9 amino acid sequence. In some embodiments, the Cas protein may comprise the amino acid sequence of a Cas protein from other species, for example other Streptococcus species, such as therm ophilus; Pseudomonas aeruginosa, Escherichia coli, or other sequenced bacteria genomes and archaea, or other prokaryotic microorganisms. Other Cas proteins, useful for the present disclosure, known or can be ide ntified, using methods known in the art (see e.g., Esvelt et al. , 2013 , Nature Methods, 10 : 1 1 16 - 1121). In certainembodiments, the Cas protein may comprise a modified amino acid sequence, as compared to its natural source.

[0070] CRISPR-Cas proteins comprise at least one RNA recognition and / or RNA binding domain. RNA recognition and / or RNA binding domains interact with guide RNAs (gRNAs). CRISPR-Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNase domains, proteinprotein interaction domains, dimerization domains, as well as other domains.

[0071] In some embodiments, the CRISPR-Cas-like protein can be a wild type CRISPR-Cas protein, a modified CRISPR-Cas protein, or a fragment of a wild type or modified CRISPR-Cas protein. In some embodiments, the CRISPR-Cas-like protein can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein. For example, nuclease (i.e., DNase, RNase) domains of the CRISPR-Cas-like protein can be modified, deleted, or inactivated. Alternatively, in some embodiments, the CRISPR-Cas-like protein can be truncated to remove domains that are not essential forthe function of the Cas protein. In some embodiments, the CRISPR-Cas-like protein can also be truncated or modified to optimize the activity of the effector domain of the Cas protein.

[0072] In some embodiments, the CRISPR-Cas-like protein can be derived from a wild-type Cas protein or fragment thereof. In certain embodiments, the CRISPR-Cas-like protein is a modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, etc.) of the protein relative to wild-type or another Cas protein. Alternatively, in some embodiments, domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild-type Cas9 protein.

[0073] In some embodiments, the CRISPR-Cas protein may comprise an amino acid sequence of: KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLE RLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTY YEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNN LVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKP EFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEI EQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIP TTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFN YEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHIL NLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYF RVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWK KLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRV DKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMY HHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYG NKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKEN YYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEV NMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKK G (SEQ ID NO : 621 ) or a variant thereof. In certain instances, amino acid sub stitutions can be made in the sequence of any of the peptides described herein, without necessarily decreasing or ablating its activity (as measured by, e.g. , the binding or functional assays described herein). In some embodiments, a variant of an amino acid sequence includes one or more amino acid residues that comprise conservative sub stitutions (e.g., a replacement of one or more amino acids with others that have similar biochemical properties, including, but not limited to, charge, hydrophobicity, or size). In some embodiments, a variant of an amino acid sequence comprises one or more amino acid residues that have been substituted with non-naturally occurring amino acids. In some embodiments, the variant sequence comprises one or more amino acid sub stitutions. In certain embodiments, the variant sequence comprises one amino acid sub stitution. In certain embodiments, the variant sequence comprises two amino acid sub stitutions. In certain embodiments, the variant sequence comprises three amino acid substitutions. In certain instances, sub stitutions include conservative sub stitutions (e.g. , sub stitutions with amino acids of comparable chemical characteristics). In certain instances, a non polar amino acid can be sub stituted and replaced with another non-polar amino acid, wherein non-polar amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan and methionine. In certain instances, a neutrally charged polar amino acids can be substituted and replaced with another neutrally charged polar amino acid, wherein neutrally charged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In certain instances, a positively charged amino acid can be sub stituted and replaced with another positively charged amino acid, wherein positively charged amino acids include arginine, lysine and histidine. In certain instances, a negatively charged amino acid can be substituted and replaced with another negatively charged amino acid, wherein negatively charged amino acids include asparticacid and glutamic acid. Examples of amino acid substitutions also include substituting an L-amino acid for its corresponding D -amino acid, sub stituting cysteine for homocysteine or other non-natural amino acids.

[0074] In some embodiments, the disclosed CRISPR-Cas compositions should also be construed to include any form of a protein having substantial homology to a Cas protein (e.g. , Cas9, saCas9, Cas9 protein) disclosed herein. Preferably, a protein which is “ sub stantially homologous” is about 50% homol ogous, more preferably about 70% homologous, even more preferably about 80% homologous, more preferably about 90% homologous, even more preferably, about 95% homologous, and even more preferably about 99% homologous to amino acid sequence of a Cas protein disclosed herein. In certain embodiments, the CRISPR-Cas endonuclease comprises one or more nuclear localization signals.

[0075] The CRISPR-Cas endonucleases described herein can be encoded on a nucleic acid (e.g., an mRNA) using methods known in the art. In some embodiments, the CRISPR-Cas endonuclease is encoded on a messenger RNA polynucleotide comprising a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NO : 621 or a variant thereof.

[0076] For example, the CRISPR-Cas endonuclease can be encoded on a messenger RNA polynucleotide comprising a 5 ' cap structure, a 5 ' UTR, the sequence encoding the CRISPR-Cas endonuclease, a 3 ' UTR, and a polyAtail. 5 ' cap structures, 5 ' and 3 ' UTR elements, and polyA tail sequences are both provided herein and known in the art. For example, the 5 ’ UTR may comprise a nucleotide sequence of GGGAGACUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGCCACC (SEQ ID NO : 622). Additionally, for example, the 3 ’ UTR m ay comprise a nucleotide sequence ofUGACUCGAGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUU GUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGC AUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGC (SEQ ID NO: 623).

[0077] In some embodiments, the messenger RNA polynucleotide comprises the sequence encoding the CRISPR-Cas endonuclease and one or more nuclear localization signals (NLSs). In some embodiments, the messenger RNA poly nucleotide can comprise a nucleotide sequence ofAUGGCGCCGAAGAAGAAGAGGAAGGUGGGAAGCAAAAGAAACUACAUCCUG GGGCUGGACAUCGGCAUCACAAGCGUGGGCUACGGCAUAAUCGACUACGAA ACCAGAGACGUCAUCGACGCGGGGGUACGGCUAUUCAAGGAAGCGAACGUAGAGAACAACGAGGGCCGGCGGAGCAAGAGAGGGGCGCGAAGACUGAAAAGAAGAAGAAGACACAGGAUCCAGAGAGUGAAAAAACUGCUGUUCGACUACAACCUCCUGACCGACCACAGCGAACUGAGCGGAAUCAACCCCUACGAGGCAAGAGUGAAAGGGCUGAGCCAGAAGCUGAGCGAGGAAGAGUUCAGCGCCGCGCUCCUGCACCUGGCAAAGCGGCGGGGCGUACACAACGUGAACGAGGUGGAAGAAGACACCGGCAACGAACUGAGCACAAAGGAACAAAUCAGCCGAAACAGCAAAGCACUGGAGGAAAAGUACGUGGCAGAACUGCAGCUCGAACGGCUGAAGAAAGACGGCGAGGUGAGGGGGAGCAUCAACAGGUUCAAGACGAGCGACUACGUGAAGGAAGCGAAACAGCUGCUAAAAGUGCAGAAGGCGUACCACCAACUAGACCAGAGCUUCAUCGACACCUACAUCGACCUGCUGGAGACACGGCGGACCUACUACGAAGGCCCCGGGGAGGGAAGCCCAUUCGGAUGGAAGGACAUCAAAGAAUGGUACGAAAUGCUGAUGGGACACUGCACAUACUUCCCAGAGGAGCUGAGAAGCGUGAAGUACGCAUACAACGCGGACCUCUACAACGCGCUGAACGACCUGAACAACCUCGUGAUCACAAGAGACGAGAACGAAAAGCUGGAAUACUACGAAAAAUUCCAAAUCAUCGAGAAUGUGUUCAAGCAGAAGAAAAAGCCGACACUGAAGCAGAUCGCCAAAGAGAUCCUGGUGAACGAGGAAGACAUCAAGGGCUACAGAGUGACCAGCACCGGCAAGCCGGAAUUCACCAACCUGAAGGUGUACCACGACAUCAAGGACAUCACGGCACGGAAGGAAAUAAUAGAGAACGCGGAGCUCCUGGACCAGAUCGCCAAGAUACUGACGAUAUACCAGAGCAGCGAGGACAUACAGGAGGAGCUGACCAACCUGAACAGCGAACUGACCCAAGAGGAAAUAGAACAGAUCAGCAACCUCAAAGGGUACACCGGAACCCACAACCUCAGCCUGAAGGCGAUAAACCUGAUUCUCGACGAGCUGUGGCACACAAACGACAACCAGAUCGCCAUAUUCAACAGACUGAAGCUAGUGCCAAAAAAAGUCGACCUCUCACAGCAAAAGGAGAUACCCACCACGCUGGUGGACGACUUCAUACUGAGCCCCGUCGUGAAAAGAAGCUUCAUACAGAGCAUAAAGGUAAUCAACGCCAUCAUAAAGAAGUACGGGCUGCCCAACGACAUAAUCAUCGAGCUGGCCAGAGAAAAAAACAGCAAGGACGCCCAAAAGAUGAUCAAUGAAAUGCAGAAGAGGAACAGACAGACGAACGAACGGAUAGAGGAGAUCAUCCGGACGACAGGAAAGGAAAACGCGAAGUACCUCAUAGAAAAGAUCAAGCUGCACGACAUGCAAGAGGGGAAGUGCCUCUACAGCCUAGAAGCAAUACCCCUGGAGGACCUGCUCAACAACCCGUUCAACUACGAGGUAGACCACAUUAUCCCUCGAAGCGUAAGCUUCGACAACAGCUUCAACAACAAAGUGCUGGUGAAACAGGAAGAGAACAGCAAGAAAGGGAACAGAACCCCCUUCCAGUACCUCUCCAGCAGCGACUCGAAGAUAUCCUACGAGACCUUCAAAAAACACAUCCUGAACCUAGCCAAGGGAAAGGGCCGGAUAAGCAAGACCAAAAAGGAGUACCUGCUCGAAGAGAGGGACAUCAACAGAUUCAGCGUGCAGAAGGACUUCAUCAACCGAAACCUGGUCGACACAAGAUACGCCACCAGAGGCCU GAUGAACCUGCUCAGAAGCUACUUCAGAGUGAACAACCUGGACGUAAAGGU CAAGAGCAUCAACGGCGGGUUCACAAGCUUCCUGAGACGAAAAUGGAAAUU CAAAAAGGAACGGAACAAAGGCUACAAACACCACGCAGAGGACGCCCUGAU AAUCGCGAACGCGGACUUCAUCUUCAAGGAGUGGAAGAAACUCGACAAGGC GAAAAAAGUGAUGGAGAACCAGAUGUUCGAGGAAAAGCAAGCGGAGAGCAU GCCGGAAAUCGAGACAGAGCAGGAAUACAAAGAGAUCUUCAUAACGCCGCA CCAGAUAAAACACAUCAAGGACUUCAAGGACUACAAGUACAGCCACAGGGU GGACAAAAAGCCAAACCGCGAACUGAUAAACGACACGCUGUACAGCACAAG AAAGGACGACAAAGGAAACACCCUGAUCGUGAACAACCUGAACGGAUUAUA CGACAAGGACAACGACAAGCUGAAGAAGCUGAUCAACAAAAGCCCCGAGAA ACUGCUCAUGUACCACCACGACCCACAGACCUACCAAAAGCUCAAACUGAUC AUGGAGCAGUACGGGGACGAAAAAAACCCCCUCUACAAAUACUACGAGGAG ACGGGAAACUACCUGACAAAAUACAGCAAAAAGGACAACGGCCCGGUCAUC AAAAAGAUCAAGUACUACGGAAACAAACUCAACGCGCACCUGGACAUCACA GACGACUACCCAAACAGCAGGAACAAGGUGGUGAAGCUGAGCCUGAAACCC UACAGAUUCGAUGUGUACCUAGACAACGGGGUGUACAAGUUCGUCACGGUG AAGAACCUGGACGUCAUAAAGAAGGAGAACUACUACGAAGUGAACAGCAAG UGCUACGAAGAAGCGAAGAAGCUGAAGAAAAUAAGCAACCAGGCCGAGUUC AUAGCAAGCUUCUACAACAACGACCUAAUCAAAAUCAACGGAGAACUGUAC AGAGUAAUCGGGGUGAACAACGACCUGCUGAACCGGAUCGAAGUAAACAUG AUCGACAUCACCUACCGGGAGUACCUCGAAAACAUGAACGACAAGCGCCCCC CAAGGAUAAUAAAGACGAUCGCGAGCAAGACCCAGAGCAUCAAGAAGUACU CGACCGACAUACUGGGCAACCUGUACGAGGUAAAGAGCAAGAAGCACCCCC AGAUCAUAAAAAAGGGCAAGCGGCCAGCAGCCACCAAGAAAGCAGGGCAAG CCAAAAAGAAGAAAGCCUAA (SEQ ID NO: 624).Guide RNAs (gRNAs)

[0078] The gRNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and targeting sequence (also referred to as a spacer sequence) that defines the template nucleic acid target to be modified. The gRNA functions, in part, by hybridizing to a template nucleic acid molecule (e.g. , at a targeted site or protospacer).

[0079] Hybridization, as used herein, generally refers to and includes the capacity and / or ability of a first nucleic acid molecule to non -covalently bind (e.g., form Watson- Crick-base pairs and / or G / U nucleobase pairs), anneal, and / or hybridize to a second nucleic acid molecule under the appropriate or certain in vitro and / or in vivo conditionsof temperature, pH, and / or solution ionic strength. Generally, standard Watson -Crick nucleobase pairing includes: adenine (A) pairing with thymidine (T); adenine (A) pairing with uracil (U); and guanine (G) pairing with cytosine (C). In some embodiments, hybridization comprises at least two nucleic acids comprising complementary sequences (e.g. , fully complementary, substantially complementary, or partially complementary). In certain embodiments, hybridization comprises at least two nucleic acids comprising fully complementary sequences. In certain embodiments, hybridization comprises at least two nucleic acids comprising substantially complementary sequences (e.g., greater than about 75%, greaterthan about 80%, greater than about 85%, greaterthan about 90%, or greater than about 95% complementary). In certain embodiments, hybridization comprises at least two nucleic acids comprising partially complementary sequences (e.g., greater than about40%, greater than about 50%, greater than about 60%, or greaterthan about 70% complementary). In certain embodiments, partially complementary sequences comprise one or more regions of fully or sub stantially complementary sequences. In certain embodiments, partially complementary sequences comprise one or more regions of fully or substantially complementary sequences, even if an overall complementarity is low (e.g., a total complementarity lower than about 50%, lowerthan about 40%, lower than about 30%, or lower than about 20%). The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementation, variables well known in the art. For example, the greater the degree of complementation between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations between nucleic acids with short stretches of complementarity (e.g. , com plementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides) the position of mismatches becomes important (see Sambrook et al. , supra, 1 1 .7 - 1 1 .8).

[0080] Complementary or complementarity, as used herein, generally refers to a polynucleotide that includes a nucleotide sequence capable of selectively annealing to an identifying region of a target polynucleotide under certain conditions. As used herein, the term substantially complementary and grammatical equivalents is in tended to mean a polynucleotide that includes a nucleotide sequence capable of specifically annealing to an identifying region of a target polynucleotide under certain conditions. Annealing refers to the nucleotide nucleobase -pairing interaction of one nucleic acid with another nucleic acid that results in the formation of a duplex, triplex, or other high er -ordered structure. The primary interaction is typically nucleotide nucleobase specific, e.g. , A:T, A:U, and G:C, by Watson-Crick and Hoogsteen-type hydrogen bonding. In certainembodiments, nucleobase-stacking and hydrophobic interactions can also contribute to duplex stability. Conditions under which a polynucleotide anneals to complementary or sub stantially complementary regions of target nucleic acids are well known in the art, e.g. , as described in Nucleic Acid Hybridization, A Practical Approach, Hames and Higgins, eds., IRL Press, Washington, D.C. (1985) and Wetmur and Davidson, Mol. Biol. 3 1 :349 (1968). Annealing conditions will depend upon the particular application and can be routinely determined by persons skilled in the art, without undue experimentation. Hybridization generally refers to process in which two single-stranded polynucleotides bind non-covalently to form a stable double -stranded polynucleotide.

[0081] The temperature and solution salt concentration are generally recognized as factors facilitating hybridization and may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementarity. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E: F. and Maniatis, T. Molecular Cloning: A Laboratory Manual - Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11 .1 therein; and Sambrook, J. and Russell, W ., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor (2001). The conditions of temperature and ionic strength determine the stringency of the hybridization. In some embodiments, hybridization is measured a under physiological temperature (e.g., 37 degrees Celsius) and salt concentrations (e.g., 0.15 molar or 0.9% salt in solution).

[0082] Target specificity can be used in reference to a guide RNA, or a crRNA specific to a target polynucleotide sequence or region and further includes a sequence of nucleotides capable of selectively annealing / hybridizing to a target (sequence or region) of a target polynucleotide, e.g., a target DNA. Target specific nucleotides can have a single species of oligonucleotide, or it can include two or more species with different sequences. Thus, the target specific nucleotide can be two or more sequences, including 3 , 4, 5, 6, 7, 8, 9 or 10 or more different sequences. In certain embodiments, a crRNA or the derivative thereof contains a target-specific nucleotide region complementary to a region of the target DNA sequence. In certain embodiments, a crRNA or the derivative thereof may contain other nucleotide sequences besides a target-specific nucleotide region. In certain embodiments, the other nucleotide sequences may be from a tracrRNA sequence.

[0083] gRNAs are generally supported by a scaffold, wherein a scaffold refers to the portions of gRNA or crRNA molecules comprising sequences which are sub stantiallyidentical or are highly conserved across natural biological species (e.g. , not conferring target specificity). Scaffolds include the tracrRNA segment and the portion of the crRNA segment otherthan the polynucleotide-targeting guide sequence at or near the 5 ' end of the crRNA segment, excluding any unnatural portions comprising sequences not conserved in native crRNAs and tracrRNAs. In some embodiments, the gRNA comprises a CRISPR RNA (crRNA):trans activating cRNA (tracrRNA) duplex. In some embodiments, the gRNA comprises a stem-loop that mimics the natural duplex between the crRNA and tracrRNA. In some embodiments, the stem-loop comprises a nucleotide sequence comprising non-naturally occurring sequence. For example, in some embodiments, the composition comprises a synthetic or chimeric guide RNA comprising a crRNA, stem, and tracrRNA.

[0084] Generally, a protospacer adj acent motif (PAM) is also an important sequence element mediating enzymatic activity of a Cas nuclease. A PAM sequence or element also refers to and includes an approximately 2 -6 nucleobase pair DNA sequence that is an important targeting component of a Cas nuclease. The PAM sequence further comprises, in certain instances, a DNA sequence that may be required for a Cas / sgRNA to form an R-loop to interrogate a specific DNA sequence through Watson-Crick pairing of its guide RNA with the genome. In certain instances, the PAM specificity can be a function of the DNA-binding specificity of the Cas protein (e.g. , a PAM recognition domain of a Cas), wherein, a protospacer adj acent motif recognition domain refers to a Cas amino acid sequence that comprises a binding site to a DNA target PAM sequence.

[0085] Typically, the PAM sequence is on either strand and is downstream in the 5' to 3 ' direction of Cas9 cleavage site. The commonly used PAM sequence (i. e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5 '-NGG-3' wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases. Exemplary CasX PAM motifs include 5 '-TTCN-3'. Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms. In addition, any given Cas9 nuclease, e.g., SpCas9, may be modified to alter th e PAM specificity of the nuclease such that the nuclease recognizes alternative PAM sequence. In the CRISPR-Cas system derived from S. pyogenes (spCas9), the protospacer region DNA typically immediately precedes a 5 '-NGG or NAG proto-spacer adjacent motif (PAM). Other Cas9 orthologs can have different PAM specificities. For example, Cas9 from S. thermophilus (stCas9) requires 5 '-NNAGAA for CRISPR 1 and 5 '-NGGNG for CRISPR3 and Neisseria meningitidis (nmCas9) requires 5 '-NNNNGATT. Cas9 from Staphylococcus aureus subsp. aureus (saCas9) requires 5 '-NNGRRT (R=A or G). Insome embodiments, Cas9 enzymes from different bacterial species (i.e., Cas9 orthologs) can have varying PAM specificities. For example, Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN. In addition, Cas9 from Neisseria meningitidis (NmCas) recognizes NNNNGATT. In another example, Cas9 from Streptococcus thermophilus (StCas9) recognizes NNAGAAW. In still another example, C as9 from Treponema denticola (TdCas) recognizes NAAAAC. In another example, CasX recognizes TTCN. These examples are not meant to be limiting. It will be further appreciated that non-SpCas9s bind a variety of PAM sequences, which makes them useful when no suitable SpCas9 PAM sequence is present at the desired target cleavage site.

[0086] In some embodiments, the gRNA spacer sequence comprises about 15 nucleotides to about 28 nucleotides. In certain embodiments, the gRNA spacer sequence is 17, 18, 19, 20, 21 , 22, or 23 nucleobases.

[0087] In certain instances, a gRNA (e.g. , a pair of gRNAs) can be encoded on an expression vector (e.g. , using one or more pol III promoters). In such instances, the expression vector also encodes the Cas endonuclease.Template Nucleic Acids

[0088] The LNPs described herein are useful in the delivery of CRSIPR-Cas systems for excising regions of nucleic acids from a template nucleic acid molecule. For example, provided herein are vectors having improved excision outcomes for CRISPR-Cas systems that target two different target sites within an HBV genome (e.g. , an HBV nucleic acid molecule). These examples can be extended and applied to improving excision efficacy on other viral template nucleic acid molecules (e.g. , viral deoxyribonucleic acid molecules). Thus, the LNPs described herein are, in certain instances, useful for excising a target nucleic acid molecule from a template nucleic acid molecule in a cell. In certain embodiments, the template nucleic acid molecule is a viral nucleic acid molecule. In certain embodiments, the viral nucleic acid molecule is an episomal nucleic acid. In certain embodiments, the viral nucleic acid molecule is an integrated nucleic acid. In certain embodiments, the viral nucleic acid molecule is an episomal and an integrated nucleic acid. In certain embodiments, the viral nucleic acid molecule is an HBV nucleic acid .Target Nucleic Acids

[0089] The LNPs described herein are useful for improving ex cision outcomes using CRISPR-Cas systems having two different gRNAs (e.g. , having different spacer sequences) for targeting the CRISPR-Cas system to two different target nucleic acid sequences for cleavage at or near the target nucleic acid sequences withi n a templatenucleic acid molecule (e.g., a template deoxyribonucleic acid molecule). Thus, in certain instances, the targeting of CRISPR-Cas endonucleases by hybridization of the two different gRNAs to the template nucleic acid molecule generates two cleaved regions within the template nucleic acid molecule at, within, or near the two different target nucleic acid sequences and excising a region from the template nucleic acid molecule.

[0090] Compared to indel formation, the excision of larger regions within the viral template nucleic acid molecule, in certain instances, provides for improved viral inactivation. Excision outcomes can readily be determined and / or identified by the in vitro assays described in Examples 1 and 2. In some embodiments, the two different target nucleic acid sequences are separated by a distance of at least 250, at least 500, at least 750, at least 1 ,000, at least 1 , 100, at least 1 ,200, at least 1 ,300, at least 1,400, at least 1 , 500, or at least 1 ,600 nucleobases. In such embodiments, the first target nucleic acid sequence and the second targetnucleic acid sequence are separated by a distance of at least at least 250, at least 500, at least 750, at least 1 ,000, at least 2,000, at least 5,000, or at least 8,000 nucleobases. Accordingly, in certain embodiments, a first cleaved region and a second cleaved region are separated by at least 500, at least 750, at least 1,000, at least 2,000, at least 5 ,000, or at least 8,000 nucleobases. Furthermore, in certain embodiments, the excised regions comprise at least 500, at least 750, at least 1,000, at least 2,000, at least 5 ,000, or at least 8,000 nucleobases.

[0091] As described herein, the two different target nucleic acid sequences can be located within different genes or gene regions. In such embodiments , the first target nucleic acid sequence and the second target nucleic acid sequence are located within different genes (e.g. , a first gene and a second gene). Accordingly, in certain embodiments, a first cleaved region and a second cleaved region within d ifferent genes and the excised regions comprises the nucleobases between the two different genes. In some embodiments, one or more of the two different target nucleic acid sequences are repeated within the template nucleic acid molecule.

[0092] In addition to deletion of larger regions (i. e., excision) of a template nucleic acid molecule, outcomes of simultaneous strand breaks atmultiple sites (e.g., as opposed to indels) also include inversions of the sub strate (template) nucleic acid molecule (e.g., the HBV genome) and / or concatemerization of two or more substrate nucleic acid molecules (e.g. , HBV genomes) . In some instances, such inversions and concatemerization s are provided as DNA repair products from simultaneous cutting of viral DNA copies at two or more sites. Thus, in certain embodiments, generating a first cleaved site and a second cleaved site can result in (i) excising a region of the HBVnucleic acid molecule between the first cleaved site and the second cleaved site, (ii) an inversion (e.g. , one or more inversions) within the HBV genome, and / or (iii) concatemerization of two or more HBV genomes at one or both of the cleaved sites.

[0093] In certain embodiments, generating the first cleaved site and the second cleaved site results in excising a region of the HBV nucleic acid molecule between the first cleaved site and the second cleaved site. In certain embodiments, generating the first cleaved site and the second cleaved site results in an inversion (e.g. , one or more inversions) within the HBV genome one or both of the cleaved sites. In certain embodiments, generating the first cleaved site and the second cleaved site results in a concatemerization of two or more HBV genomes at one or both of the cleaved sites.Microhomology

[0094] The LNPs described herein are also advantageous for delivering multiplexed CRISPR-Cas systems that can achieve MMEJ-mediated excision between two cleavage sites and / or over large distances (e.g., >500 base pairs, >1,000 base pairs, etc.) separating the two cleaved regions through the delivery of multiplexed CRISPR-Cas systems having two different gRNAs. Generally, MMEJ-mediated deletions are considered to be limited to indels at single cleavage sites having smaller distances (e.g., <15 nucleotides) between microhomologous sequences. Moreover, MMEJ prediction algorithms generally reduce MMEJ predictions as a function of the distance between microhomologous sequences (e.g. , reducing predicted MMEJ frequencies as the distance between microhomologous sequences increases). However, as described herein, excision can be achieved, in certain instances, by generating at least two cleaved regions at different target sites and having microhomology .

[0095] In some embodiments, the sequences surrounding or within cleaved regions comprises sequences having microhomology. In such instances, the target sites are chosen / selected to target sites that will generate cleaved regions having microhomology . For example, the first cleaved region comprises a sequence having microhomology to a sequence within the second cleaved region. In such embodiments, cutting the template nucleic acid molecule at the first cleaved region and cutting the template nucleic acid molecule at the second cleaved region activates microhomology -mediated end j oining (MMEJ) for rejoining template nucleic acid molecule, thereby excising a region of the target nucleic acid molecule.

[0096] In some embodiments, a cleaved region comprises about 5 nucleobase pairs 5' and 3 ' of a cleavage site to about25 nucleobase pairs 5 ' and 3 ' of a cleavage site. In someembodiments, a cleaved or cleavable region comprises about 10 nucleobase pairs 5' and 3 ' of a cleavage site to about 20 nucleobase pairs 5 ' and 3 ' of a cleavage site.

[0097] Microhomology -mediated end j oining (MMEJ), as used herein, generally refers to and includes the mechanism for double stranded breaks in a template nucleic acid molecule (e.g. , within a genome), which relies on exposed microhomologous sequences (i.e., sequences having microhomology) flanking broken junction to fix DSBs in a Ku- and ligase IV-independent manner. MMEJ generally involves five steps for repairing a double stranded break: resection of the DSB ends (generally 5 ' to 3 ' resection), annealing of region / sequences having microhomology, removal of heterologous flaps, fill-in synthesis (i.e. , polymerase extension), and ligation. Additional pathways for repair of the cleaved or cleavable regions described herein .

[0098] In some embodiments, microhomology can be determined by various known methods, such as Microhomology-Predictor (Bae, S. , Kweon, J. , Kim, H. et al. Microhomology -based choice of Cas9 nuclease target sites. Nat Methods 1 1 , 705-706 (2014) and MENTHU (Robust Activation of Microhomology -mediated End Joining for Precision Gene Editing Applications. Ata H, Ekstrom TL, Martinez -Galvez G, Mann CM, Dvornikov AV, Schaefbauer KJ, Ma AC, Dobbs D, Clark KJ, Ekker SC. PLOS Genetics 14(9): e l 007652), inDelphi (Max W. Shen, Mandana Arbab, Jonathan Y. Hsu, Daniel Worstell, Sannie J. Culbertson, Olga Krabbe, Christopher A. Cassa, David R. Liu, David K. Gifford, and Richard I. Sherwood. "Predictable and precise template-free editing of pathogenic variants." Nature, 2018), ForCasT (Elrick H, Nelakuditi V, Clark G, Brudno M, Ramani AK, Nutter LM. FORCAST: a fully integrated and open source pipeline to design Cas-mediated mutagenesis experiments) Lindel, and MENdel (Gabriel Martinez- Galvez, Parnal Joshi, Iddo Friedberg, Armando Manduca, Stephen C Ekker, Deploying MMEJ using MENdel in precision gene editing applications for gene therapy and functional genomics, Nucleic Acids Research, Volume 49, Issue 1 , 1 1 January 2021), each of which are herein incorporated by reference for the application of determining and / or identifying microhomology .

[0099] In some embodiments, sequences having microhomology comprise about 3 to about 20 nucleotides. In certain embodiments, the sequences having microhomology comprise greater than 2, greater than 3 , greater than 4, greater than 5 , greater than 10, or greater than 15 nucleotides.

[0100] In some embodiments, sequences having microhomology comprise 3 to about 20 complementary nucleotides. In certain embodiments, the sequences havingmicrohomology comprise greater than 2, greater than 3 , greater than 4, greater than 5, greater than 10, or greater than 15 complementary nucleotides.

[0101] In some embodiments, sequences having microhomology comprise 3 to about 20 nucleotides capable of annealing. In certain embodiments, the sequences having microhomology comprise greater than 2, greater than 3 , greater than 4, greater than 5, greater than 10, or greater than 15 nucleotides capable of annealing.

[0102] In some embodiments, the first and second sequences having microhomology are located in different genes. In some embodiments, the first and second sequences having microhomology located in coding regions of different genes. In certain embodiments, the first and second sequences having microhomology are separated by a distance of at least 250, at least 500, at least 750, at least 1 ,000, at least 1 , 100, at least 1 ,200, at least 1 ,300, at least 1 ,400, at least 1 ,500, or at least 1 ,600 nucleobase pairs.

[0103] In some embodiments, the microhomology comprises three or more complementary nucleotides (e.g., in a contiguous sequence) having a GC (guanine or cytosine) content greater than or equal to 50%. In some embodiments, the microhomology comprises at least 3 (e.g., at least 5, at least 10, at least 15, or at least 20) complementary nucleotides. In some embodiments, sequences within (e.g., internal to) the first cleaved region lack microhomology; and sequences within (e.g., internal to) the second cleaved region lack microhomology. In some embodiments, microhomology of sequences within (e.g., internal to) the first cleaved region is less (e.g., in number or degree) than the microhomology of first nucleic acid sequence and the second sequence; and microhomology of sequences within (e.g., internal to) the second cleaved region is less (e.g., in number or degree) than the microhomology of first nucleic acid sequence and the second sequence.

[0104] In certain embodiments, microhomologous sequences are capable of hybridizing to one another. In certain embodiments, hybridization comprises at least two nucleic acids comprising substantially complementary sequences (e.g., is greater than about75%, greaterthan about 80%, greater than about 85%, greaterthan about90%, or greater than about 95% complementary). In certain embodiments, hybridization comprises at least two nucleic acids comprising partially complementary sequence s (e.g., greater than about40%, greater than about 50%, greater than about 60%, or greaterthan about70% complementary). In certain embodiments, partially complementary sequences comprise one or more regions of fully or substantially complementary sequences. In certain embodiments, partially complementary sequences comprise one or more regions of fully or substantially complementary sequences, even if an overall complementarityis low (e.g., a total complementarity lower than about 50%, lowerthan about 40%, lower than about 30%, or lower than about 20%). The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementation, variables well known in the art. For example, the greater the degree of complementation between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations between nucleic acids with short stretches of complementarity (e .g. , complementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides) the position of mismatches becomes important (see Sambrook et al. , supra, 1 1 .7 - 1 1 .8).CRISPR-Cas Systems Targeting HBV

[0105] Provided herein are CRISPR-Cas Systems comprising: (1 ) a CRISPR-Cas endonuclease or a polynucleotide comprising a sequence encoding the CRISPR -Cas endonuclease; (2) a first guide ribonucleic acid (gRNA) that hybridizes to a first target sequence within an HBV nucleic acid molecule or nucleic acid encoding the guide; and (3 ) a second gRNA that hybridizes to a second target sequence within the HBV nucleic acid molecule or a nucleic acid encoding the guide. Of notable advantage, (i) the second target sequence is different from the first target sequence; and (ii) expressing the CRISPR-associated nuclease in combination with the first gRNA cleaves the HBV nucleic acid molecule within or proximal to (e.g., within a distance of about 1 , 2, 3, 4, 5, 10, 15 or 20 nucleobase positions) the first target sequence, generating a first cleaved site; and (iii) expression of the CRISPR-associated nuclease in combination with the second gRNA cleaves the HBV nucleic acid molecule within or proximal (e.g., within a distance of about 1 , 2, 3 , 4, 5 , 10, 15 or 20 nucleobase positions) to the second target sequence, generating a second cleaved site.

[0106] In certain instances, the CRISPR-Cas systems described herein are advantageous in that targeting and cutting of the HBV nucleic acid (e.g. , genome or cccDNA) results in excision (e.g., larger deletions) of a region of the HBV nucleic acid. In certain embodiments, generating the first cleaved site and the second cleaved site results in excising a region of the HBV nucleic acid molecule between the first cleaved site and the second cleaved site.

[0107] In certain embodiments, the first target sequence and the second target sequence are within different genes and separated by at least at least 250, at least 500, at least 1 ,000, at least 1 , 100, at least 1 ,200, at least 1 ,300, at least 1 ,400, at least 1,500, or at least 1 ,600 nucleotides. In such instances, the excised the region of the HBV nucleic acid molecule between the first cleaved site and the second cleaved site comprises atleast 250, at least 500, at least 1 ,000, at least 1 , 100, at least 1 ,200, at least 1 ,300, at least 1 ,400, at least 1 , 500, or at least 1 ,600 nucleobase positions. Targeting at least a sequence within a region of the HBV nucleic acid molecule encoding two genes is of additional advantage for inactivating HBV. In certain embodiments, at least one of the first target sequence and the second target sequence are within a region of the HBV nucleic acid molecule encoding two genes. In certain embodiments, both of the first target sequence and the second target sequence are within a region of the HBV nucleic acid molecule encoding two genes.

[0108] Target sequences can be within HBV gene regions selected from pol, preSl, preS2, S, x, preC, and C. In certain embodiments, the first target sequence is within the pol gene region of the HBV nucleic acid molecule. In certain embodiments, the first target sequence is within the pol and S gene region of the HBV nucleic acid molecule. In certain embodiments, the second target sequence is within the preC gene region of the HBV nucleic acid molecule. In certain combinations of targeted genes are selected from the gene regions targeted in Table 7.

[0109] Targets sequences (e.g., as defined by a protospacer sequence) of particular benefit are included in Table 1 or Table 4 or Table 7. In certain embodiments, the first target sequence and the second target sequence each independently comprise a protospacer sequence selected from Table 1 or Table 4 or Table 7. Additional guides can include those described in Tables 2 -3 and 5. In certain embodiments, the first target sequence and the second target sequence each independently comprise a protospacer sequence having at least 90% sequence identity or at least 95% sequence identity to a protospacer sequence selected from Table 1 or Table 4 or Table 7.

[0110] In certain embodiments, the Cas endonuclease is a Cas9 endonuclease. In certain embodiments, the Cas9 endonuclease is saCas9. In certain embodiments, the Cas endonuclease is a CasX endonuclease .Lipid Nanoparticles (LNPs)[OHl] LNPs and method of formulating lipids for the generation of the LNPs described herein are known in the art, for example, as described in Kazemi an et al , Lipid- Nanoparticle-Based Delivery of CRISPR / Cas9 Genome-Editing Components. Mol Pharm. 2022 Jun 6; 19(6):1669- 1686. doi : 10. 1021 / acs.molpharmaceut. l c00916. Epub 2022 May 20. PMID : 35594500 or Chen et al. Strategies for nonviral nanoparticle -based delivery of CRISPR / Cas9 therapeutics. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2020 May; 12(3):el609. doi : 10.1002 / wnan. l609. Epub 2019 Dec 2. PMID : 31797562 or Huang et al. The landscape of mRNA nanomedicine. Nat Med. 2022 Nov;28(l 1):2273 -2287. doi : 10. 1038 / s41591-022-02061-1. Epub 2022 Nov 10. PMID : 36357682 or Yang et al. Recent Advances in Lipid Nanoparticles for Delivery of mRNA. Pharmaceutics. 2022 Dec 1 ; 14(12):2682. doi: 10.3390 / pharmaceuticsl4122682. PMID : 36559175; PMCID : PMC9787894.

[0112] LNP generally refers to particles having at least one dimension on the order of nanometers (e.g. , 1 - 1 ,000 nm) that include one or more lipid c ompounds. In some embodiments, lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the l ike). Such lipid nanoparticles also typically comprise a one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, can be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response. In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 1 10 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, l OO nm, 105 nm, l l O nm, 1 15 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non -toxic. In certain embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease.

[0113] Exemplary LNP components are described in Table 9. In some embodiments, the LNP comprises an ionizable lipid having a tertiary amine; a sterol, a phosphatidylcholine lipid, and a pegylated lipid. In certain embodiments, the LNP comprises SM- 102, distearoylphosphatidylcholine (DSPC), cholesterol, and 1 ,2- dimyristoyl-rac-glycero-3 -methoxypolyethylene gly col-2000 (DMG-PEG2000). In certain embodiments, the LNP comprises LP01, DSPC, cholesterol, and DMG-PEG2000. Exemplary suitable molar ratios for LNP formulation are also provided in Table 9.Methods

[0114] Provided herein is a CRISPR-Cas system containing LNPs useful for methods of editing a region of a viral nucleic acid (e.g. , genome and / or cccDNA). In someembodiments, the editing is excision of a region of the viral nucleic acid. In some embodiments, the editing is inversion of a region of the viral nucleic acid. In some embodiments, the editing is generation of one or more indels within a region of the viral nucleic acid. In some embodiments, the editing is concatemerization of two or more viral nucleic acids. In some embodiments, the editing includes one or more of: excision of a region of the viral nucleic acid, inversion of a region of the viral nucleic acid, generation of one or more indels within a region of the viral nucleic acid, and concatemerization of a region of the viral nucleic acids. In some embodiments, the editing of the region of the viral nucleic acid results in inactivation of the viral nucleic acid. In some embodiments, the viral nucleic acid is a hepatitis B viral (HBV) nucleic acid.

[0115] Provided herein are CRISPR-Cas system containing LNPs useful for methods of excising a region of an HBV nucleic acid (e.g., genome and / or cccDNA). Thus, in certain instances, also provided are methods of inactivating an HBV virus that include use the LNPs for excising a viral nucleic acid (e.g., viral genome). According, in some embodiments, provided herein are methods of inactivating an HBV nucleic acid molecule in a cell, the method comprising: contacting the cell with the LNPs comprising the CRISPR-Cas systems described herein. Also provided are methods of excising (e.g., deleting) a region of an HBV nucleic acid molecule in a cell, the method comprising: contacting the cell with the LNPs comprising the CRISPR-Cas systems described herein. In certain embodiments, the cell is in a human.

[0116] Provided herein are CRISPR-Cas system containing LNPs useful for methods of generating an inversion of a region of an HBV nucleic acid (e.g. , genome and / or cccDNA). Thus, in certain instances, also provided are methods of i nactivating an HBV virus that include use the LNPs for generating an inversion of a viral nucleic acid (e.g., viral genome). Accordingly, in some embodiments, provided herein are methods of inactivating an HBV nucleic acid molecule in a cell, the method co mprising: contacting the cell with the LNPs comprising the CRISPR-Cas systems described herein. Also provided are methods of generating an inversion of a region of an HBV nucleic acid molecule in a cell, the method comprising: contacting the cell with the LNPs comprising the CRISPR-Cas systems described herein. In certain embodiments, the cell is in a human.

[0117] Provided herein are CRISPR-Cas system containing LNPs useful for methods of generating an indel within a region of an HBV nucleic acid (e.g. , genome an d / or cccDNA). Thus, in certain instances, also provided are methods of inactivating an HBV virus that include use the LNPs for generating an indel within a viral nucleic acid (e.g.,viral genome). Accordingly, in some embodiments, provided herein are methods of inactivating an HB V nucleic acid molecule in a cell, the method comprising: contacting the cell with the LNPs comprising the CRISPR-Cas systems described herein. Also provided are methods of generating an indel within a region of an HBV nucleic acid molecule in a cell, the method comprising: contacting the cell with the LNPs comprising the CRISPR-Cas systems described herein. In certain embodiments, the cell is in a human.

[0118] Provided herein are CRISPR-Cas system containing LNPs useful for methods of generating a concatemer of two or more HBV nucleic acids (e.g. , genome and / or cccDNA and / or a region of a genome and / or cccDNA). Thus, in certain instances, also provided are methods of inactivating an HBV virus that include use the LNPs for generating a concatemer of a region of the viral nucleic acid (e.g., viral genome or region of a viral genome). Accordingly, in some embodiments, provided herein are methods of inactivating an HBV nucleic acid molecule in a cell, the method comprising: contacting the cell with the LNPs comprising the CRISPR-Cas systems described herein. Also provided are methods of generating a concatemer of a region of the HBV nucleic acid molecule in a cell, the method comprising: contacting the cell with the LNPs comprising the CRISPR-Cas systems described herein. In certain embodiments, the cell is in a human.

[0119] The CRISPR-Cas systems and methods described herein are useful for treating HBV (e.g., an infection). Provided herein are methods of treating HBV, wherein the methods comprise administering the LNPs comprising the CRISPR-Cas systems described herein to the individual. In certain embodiments, treating comprises reducing the amount of HBV serum biomarkers (e.g., compared to an untreated individual or a prior measurement of HBV serum biomarkers). In such embodiments, the HBV serum biomarkers comprise HBV DNA, HBsAg, HBeAg, or a combination thereof. In certain embodiments, treating comprises reducing intrah epatic HBV DNA (e.g. , compared to an untreated individual or a prior measurement of HBV serum biomarkers). Suitable routes of administrating the LNPs include intravenous administration.

[0120] The LNPs comprising the CRISPR-Cas systems are also useful when combined with an antiviral therapy. Accordingly, in some embodiments, the methods further comprise administering an antiviral . Exemplary antivirals include Bulevirtide, Entecavir (Baraclude), Tenofovir disoproxil fumarate (Viread), Tenofovir alafenamide (Vemlidy), Lamivudine (Epivir-HBV), or Telbivudine (Tyzeka). In certain embodiments, themethods further comprise administering the HBV antiviral (e.g., before, after, or concurrently with LNP).Exemplary Definitions

[0121] The determination of percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Set. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Set. USA 90 :5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215 :403-410. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm utilized for th e comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 :2444- 8. Alternatively, sequence alignment may be carried out using the CLUSTAL algorithm (e.g. , as provided in the program Clustal-omega), as described by Higgins et al., 1996, Methods Enzymol. 266 :383 -402.

[0122] As used herein, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps. As also used herein, in any instance or embodiment described herein, “comprising” may be replaced with “consisting essentially of’ and / or “consisting of’ , used herein, in any instance or embodiment described herein, “comprises” may be replaced with “consists essentially of” and / or “consists of” .

[0123] As used herein, the term “about” in the context of a given value or range includes and / or refers to a value or range that is within 20%, within 10%, and / orw ithin 5% of the given value or range.

[0124] As used herein, the term “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each were set out individually herein.

[0125] As used herein, a “ sample” includes and / or refers to any fluid or liquid sample which is being analyzed in order to detect and / or quantify an analyte. In some embodiments, a sample is a biological sample. Examples of samples include without limitation a bodily fluid, an extract, a solution containing proteins and / or DNA, a cell extract, a cell lysate, or a tissue lysate. Non -limiting examples of bodily fluids include urine, saliva, blood, serum, plasma, cerebrospinal fluid, tears, semen, sweat, pleural effusion, liquified fecal matter, and lacrimal gland secretion.EXAMPLESExample 1 :

[0126] Combinations of genome editing nucleases (editors) and paired guide RNAs (gRNAs) that effectively inactivate replication of hepatitis B virus (HBV) by specifically cleaving the viral DNA molecules that persist in HBV -infected cells were identified (FIG. 1) Editors complexed with each of paired gRNAs were expressed in hepatocytes for removal of HBV DNA by LNPs encapsulating editor mRN As and pairs of chemically synthesized gRNAs. HBV DNA copies cut with multiple gRNAs eliminated replication- competent virus particles. Replication -competent virus with cleavage-resistant mutations at multiple target sites was also less likely to emerge.Materials and methods

[0127] In-silico analysis was used to identify target sites that are well conserved in a broad range of HBV genotypes. Consensus sequences were built for each genotype of the HBV genome using sequences deposited in the public database and gRNA target sites on the consensus viral genomes were found (FIG. 2). Sequence conservation of each gRNA target site was calculated and the sites where the protospacers (sequences hybridized with gRNAs) and protospacer-adjacent motifs (PAMs) were highly conserved in genotypes of HBV genomic sequences were selected (FIG. 3). Sequences of SaCas9 gRNAs targeting highly conserved target sites across HBV genotypes and the calculated percentage of perfectly matched guide RNA target site in each HBV genotype are provided in Table 1.Table 1

[0128] Sequences of SaCas9 gRNAs targeting additional target sites across HBV genotypes and the calculated percentage of perfectly matched guide RNA target site in each HBV genotype are provided in Table 2.Table 2

[0129] Sequences of SaCas9 gRNAs targeting distinct sites across HBV genotypes and the calculated percentage of perfectly matched guide RNA target site in each HBV genotype are provided in Table 3.Table 3i SE i Protospacer sequence i PAM i Stra [ A [ B i C [ D i E [ F [ G i H Q [ IUM M

[0130] Sequences of CasX gRNAs targeting highly conserved target sites across HBV genotypes and the calculated percentage of perfectly matched guide RNA target site in each HBV genotype are provided in Table 4.Table 4>

[0131] Sequences of CasX gRNAs targeting additional sites across HBV genotypes and the calculated percentage of perfectly matched guide RNA target site in each HBV genotype are provided in Table 5.Table 5

[0132] The reference human genome (hg38) was scanned for sites homologous to the guide RNA target sites to mitigate the risk of off-target effects when choosing gRNAs(Table 6)Table 6

[0133] Genes targeted by selected gRNAs are provided in Table 7, and FIG. 3 provides a schematic of the corresponding gRNA target cites.Table 7

[0134] All guide RNAs used in experiments were chemically synthesized and purified by HPLC. On both ends of the gRNA, 3 nucleotides and theirphosphodiester bonds were modified to protect them from degradation by cellular ribonucleases (Table 8): “m” = 2'-O-methyl modification; = phosphorothioate.Table 8

[0135] The sequence encoding SaCas9 with the N-terminal SV40 NLS and the C- terminal nucleoplasmin NLS (SEQ ID NO: 624) was modified to improve protein expression in human cells and to reduce immunogenicity by reducing the number of thymines. 5'UTR (SEQ ID NO: 622) and 3 'UTR (SEQ ID NO: 623) derived from human beta-globin (HBB) mRNA were added. Additionally, approximately 160 adenine nucleotides were added to the terminus of the 3 'UTR, GCT (underlined) was inserted between the 43 rd and 44 th adenines from the 5 ' end of the poly A (SEQ ID NO: 612):AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0136] The SaCas9 mRNA sequence was produced by in vitro transcription (IVT) with the T7 RNA polymerase and the plasmid pL-1042 (TCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGA TCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACC AGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAAC TGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTT AGCCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAAT CCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGG ACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGG TTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACC TACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGA CAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTT CCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGA CTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAA CGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAG TGAGCGAGGAAGCGGAAGGCGAGAGTAGGGAACTGCCAGGCATCAAACTAAG CAGAAGGCCCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTCTGTGTT GTAAAACGACGGCCAGTCTTAAGCTCGGGCCCCCTGGGCGGTTCTGATAACGA GTAATCGTTAATCCGCAAATAACGTAAAAACCCGCTTCGGCGGGTTTTTTTATG GGGGGAGTTTAGGGAAAGAGCATTTGTCAGAATATTTAAGGGCGCCTGTCACT TTGCTTGATATATGAGAATTATTTAACCTTATAAATGAGAAAAAAGCAACGCA CTTTAAATAAGATACGTTGCTTTTTCGATTGATGAACACCTATAATTAAACTAT TCATCTATTATTTATGATTTTTTGTATATACAATATTTCTAGTTTGTTAAAGAGA ATTAAGAAAATAAATCTCGAAAATAATAAAGGGAAAATCAGTTTTTGATATCA AAATTATACATGTCAACGATAATACAAAATATAATACAAACTATAAGATGTTA TCAGTATTTATTATGCATTTAGAATAAATTTTGTGTCGCCCTTTACACGTACTT AGTCGCTGAAGGGGTAATACGACTCACTATAGGGAGACTTCTGACACAACTGT GTTCACTAGCAACCTCAAACAGCCACCATGGCGCCGAAGAAGAAGAGGAAGG TGGGAAGCAAAAGAAACTACATCCTGGGGCTGGACATCGGCATCACAAGCGT GGGCTACGGCATAATCGACTACGAAACCAGAGACGTCATCGACGCGGGGGTA CGGCTATTCAAGGAAGCGAACGTAGAGAACAACGAGGGCCGGCGGAGCAAGAGAGGGGCGCGAAGACTGAAAAGAAGAAGAAGACACAGGATCCAGAGAGTGAAAAAACTGCTGTTCGACTACAACCTCCTGACCGACCACAGCGAACTGAGCGGAATCAACCCCTACGAGGCAAGAGTGAAAGGGCTGAGCCAGAAGCTGAGCGAGGAAGAGTTCAGCGCCGCGCTCCTGCACCTGGCAAAGCGGCGGGGCGTACACAACGTGAACGAGGTGGAAGAAGACACCGGCAACGAACTGAGCACAAAGGAACAAATCAGCCGAAACAGCAAAGCACTGGAGGAAAAGTACGTGGCAGAACTGCAGCTCGAACGGCTGAAGAAAGACGGCGAGGTGAGGGGGAGCATCAACAGGTTCAAGACGAGCGACTACGTGAAGGAAGCGAAACAGCTGCTAAAAGTGCAGAAGGCGTACCACCAACTAGACCAGAGCTTCATCGACACCTACATCGACCTGCTGGAGACACGGCGGACCTACTACGAAGGCCCCGGGGAGGGAAGCCCATTCGGATGGAAGGACATCAAAGAATGGTACGAAATGCTGATGGGACACTGCACATACTTCCCAGAGGAGCTGAGAAGCGTGAAGTACGCATACAACGCGGACCTCTACAACGCGCTGAACGACCTGAACAACCTCGTGATCACAAGAGACGAGAACGAAAAGCTGGAATACTACGAAAAATTCCAAATCATCGAGAATGTGTTCAAGCAGAAGAAAAAGCCGACACTGAAGCAGATCGCCAAAGAGATCCTGGTGAACGAGGAAGACATCAAGGGCTACAGAGTGACCAGCACCGGCAAGCCGGAATTCACCAACCTGAAGGTGTACCACGACATCAAGGACATCACGGCACGGAAGGAAATAATAGAGAACGCGGAGCTCCTGGACCAGATCGCCAAGATACTGACGATATACCAGAGCAGCGAGGACATACAGGAGGAGCTGACCAACCTGAACAGCGAACTGACCCAAGAGGAAATAGAACAGATCAGCAACCTCAAAGGGTACACCGGAACCCACAACCTCAGCCTGAAGGCGATAAACCTGATTCTCGACGAGCTGTGGCACACAAACGACAACCAGATCGCCATATTCAACAGACTGAAGCTAGTGCCAAAAAAAGTCGACCTCTCACAGCAAAAGGAGATACCCACCACGCTGGTGGACGACTTCATACTGAGCCCCGTCGTGAAAAGAAGCTTCATACAGAGCATAAAGGTAATCAACGCCATCATAAAGAAGTACGGGCTGCCCAACGACATAATCATCGAGCTGGCCAGAGAAAAAAACAGCAAGGACGCCCAAAAGATGATCAATGAAATGCAGAAGAGGAACAGACAGACGAACGAACGGATAGAGGAGATCATCCGGACGACAGGAAAGGAAAACGCGAAGTACCTCATAGAAAAGATCAAGCTGCACGACATGCAAGAGGGGAAGTGCCTCTACAGCCTAGAAGCAATACCCCTGGAGGACCTGCTCAACAACCCGTTCAACTACGAGGTAGACCACATTATCCCTCGAAGCGTAAGCTTCGACAACAGCTTCAACAACAAAGTGCTGGTGAAACAGGAAGAGAACAGCAAGAAAGGGAACAGAACCCCCTTCCAGTACCTCTCCAGCAGCGACTCGAAGATATCCTACGAGACCTTCAAAAAACACATCCTGAACCTAGCCAAGGGAAAGGGCCGGATAAGCAAGACCAAAAAGGAGTACCTGCTCGAAGAGAGGGACATCAACAGATTCAGCGTGCAGAAGGACTTCATCAACCGAAACCTGGTCGACACAAGATACGCCACCAGAGGCCTGATGAACCTGCTCAGAAGCTACTTCAGAGTGAACAACCTGGACGTAAAGGTCAAGAGCATCAACGGCGGGTTCACAAGCTTCCTGAGACGAAAATGGAAATTCAAAAAGGAACGGAACAAAGGCTACAAACACCACGCAGAGGACGCCCTGATAATCGCGAACGCGGACTTCATCTTCAAGGAGTGGAAGAAACTCGACAAGGCGAAAAAAGTGATGGAGAACCAGATGTTCGAGGAAAAGCAAGCGGAGAGCATGCCGGAAATCGAGACAGAGCAGGAATACAAAGAGATCTTCATAACGCCGCACCAGATAAAACACATCAAGGACTTCAAGGACTACAAGTACAGCCACAGGGTGGACAAAAAGCCAAACCGCGAACTGATAAACGACACGCTGTACAGCACAAGAAAGGACGACAAAGGAAACACCCTGATCGTGAACAACCTGAACGGATTATACGACAAGGACAACGACAAGCTGAAGAAGCTGATCAACAAAAGCCCCGAGAAACTGCTCATGTACCACCACGACCCACAGACCTACCAAAAGCTCAAACTGATCATGGAGCAGTACGGGGACGAAAAAAACCCCCTCTACAAATACTACGAGGAGACGGGAAACTACCTGACAAAATACAGCAAAAAGGACAACGGCCCGGTCATCAAAAAGATCAAGTACTACGGAAACAAACTCAACGCGCACCTGGACATCACAGACGACTACCCAAACAGCAGGAACAAGGTGGTGAAGCTGAGCCTGAAACCCTACAGATTCGATGTGTACCTAGACAACGGGGTGTACAAGTTCGTCACGGTGAAGAACCTGGACGTCATAAAGAAGGAGAACTACTACGAAGTGAACAGCAAGTGCTACGAAGAAGCGAAGAAGCTGAAGAAAATAAGCAACCAGGCCGAGTTCATAGCAAGCTTCTACAACAACGACCTAATCAAAATCAACGGAGAACTGTACAGAGTAATCGGGGTGAACAACGACCTGCTGAACCGGATCGAAGTAAACATGATCGACATCACCTACCGGGAGTACCTCGAAAACATGAACGACAAGCGCCCCCCAAGGATAATAAAGACGATCGCGAGCAAGACCCAGAGCATCAAGAAGTACTCGACCGACATACTGGGCAACCTGTACGAGGTAAAGAGCAAGAAGCACCCCCAGATCATAAAAAAGGGCAAGCGGCCAGCAGCCACCAAGAAAGCAGGGCAAGCCAAAAAGAAGAAAGCCTAATGACTCGAGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATGAAGAGCCGTCAATCGAGTTCGTACCTAAGGGCGACACCCCCTAATTAGCCCGGGCGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCAGTTCCCTACTCTCGCATGGGGAGTCCCCACACTACCATCGGCGCTACGGCGTTTCACTTCTGAGTTCGGCATGGGGTCAGGTGGGACCACCGCGCTACTGCCGCCAGGCAAACAAGGGGTGTTATGAGCCATATTCAGGTATAAATGGGCTCGCGATAATGTTCAGAATTGGTTAATTGGTTGTAACACTGACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATG AGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAATATGA GCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCT GATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGAC AATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATG GCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGG CTGACGGAATTTATGCCACTTCCGACCATCAAGCATTTTATCCGTACTCCTGAT GATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCGTTCCAGGTATT AGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCC TGCGCCGGTTGCACTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCG TATTTCGCCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCG AGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGA AATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTT CTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGT TGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACT GCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTA TTGATAATCCTGATATGAATAAATTGCAATTTCATTTGATGCTCGATGAGTTTT TCTAAGCAGAGCATTACGCTGACTTGACGGGACGGCGCAAGCTCATGACCAAA ATCCCTTAACGTGAGTTACGCGCGCG; SEQ ID NO: 625) linearized with BspQI. A Cap l structure was enzymatically added following IVT .

[0137] In experiments with cultured cells, liposomes were formulated using Lipofectamine MessengerMAX for transfection with SaCas9 mRNA and gRNAs comprising synthetic SaCas9 gRNA 1 (SEQ ID NO: 601) and SaCas9 gRNA 2 (SEQ ID NO: 603)

[0138] Lipid nanoparticles (LNPs) encapsulating the SaCas9 mRNA and synthetic gRNAs comprising SaCas9 gRNA 1 and SaCas9 gRNA 2 were prepared as summarized in Table 9. First, SM-102 ionizable lipid, DSPC, cholesterol and DMG-PEG2000 were mixed and dissolved in ethanol at the molar ratio of 50 : 10:38.5: 1.5 (Table 1). Next, the SaCas9 mRNA, SaCas9 gRNA 1 (SEQ ID NO: 601) and SaCas9 gRNA2 (SEQ ID NO: 603 ) were mixed at 2 : 1 : 1 in weight (e.g. , 1 mg + 0.5 mg + 0.5 mg; Table 1). Finally, the LNP was formulated by mixing the SaCas9 mRNA and paired SaCas9 gRNAs with the lipids at a ratio of positively-charged polymer amine (N = nitrogen) groups to negatively- charged nucleic acid phosphate (P) group s N / P ratio of 6 (Table 9).Table 9PrescriptionSM-102 Molar ratio: 50Distearoylphosphatidylcholine Molar ratio: 10(DSPC) | |Cholesterol Molar ratio :38.5LNP formula( N / P ratio: 6RNA [ Concentration in aqueous phase: 0.463 mg / mL, 4.32 ml ]1 (Img SaCas9 mRNA+0.5 mg SaCas9 gRNA 1 + SaCas9_ [ gRNA 2) jLipid ( Concentration in organic phase: 50mM [Results

[0139] To assess genome editing activities by selected editors and paired gRNAs, reporter human cell lines carrying parts of the consensus HBV genomic sequence in the predetermined chromosomal location were created and transfected with editor-encoding mRNAs and pairs of chemically synthesized gRNAs (FIG. 4A). The levels of excision and indel creation were quantified (FIG. 4B). The degree of excision introduced in the transfected cell lines was varied by combinations of editors and pairs of gRNAs. One representative of the editors and paired gRNAs examined was subj ected to further analyses. The selected editor was Cas9 from Staphylococcus aureus (SaCas9), and the selected gRNAs were a pair of SaCas9 gRNAs that targeted the hepatitis B virus polymerase- and surface antigen-encoding sequence (SEQ ID NO : 601) and the viral gene encoding the core antigen (SEQ ID NO: 603). Excision was introduced at 60% of the integrated HBV reporter constructs (FIG. 5A). Indels were detected at 45% of the SaCas9 gRNA 1 (SEQ ID NO : 1) and 30% of the SaCas9 gRNA 2 (SEQ ID NO : 2) unexcised target sites (FIG. 5B)

[0140] To test if an editor and pairs of gRNAs could access and degrade HBV DNA that persisted primarily as episomal molecules, experiments using tissue culture were conducted as follows: HepG2 -derived cells that expressed the HBV entry receptor sodium taurocholate co-transporting polypeptide (NTCP) were generated by lentiviral transduction and infected with HBV particles prepared from the HepAD38 cell line (FIG. 6A). HBV-infected cells were transfected with SaCas9-encoding mRNA and paired gRNAs (FIG. 6A; SEQ ID NO: 601 and SEQ ID NO: 603), and the culture medium and cell pellets were harvested separately. Quantification of intrahepatic (intracellular) HBV DNA by digital PCR showed that the copy number of HBV DNA was significantly lowered in the transfected cells (FIG. 6B) Consistent with this ob servation, secretion ofHepatitis B HBsAg and HBeAg (clinical HBV biomarkers) was also suppressed (FIG. 7A, FIG. 7B , and FIG. 7C). Myrcludex B (MyrB) is a synthetic peptide that blocks entry of HBV through interaction with sodium taurocholate co -transporting polypeptide (NTCP). A significantly lower copy number of intracellular HBV DNA and very little HBsAg and HBeAg were detected from cells treated with MyrB prior to HBV infection, indicating that the majority of intracellular HBV DNA and the other two clinical HBV biomarkers were derived from the infected virus, rather than from the residual virus that are present extracellular space without infection. These results suggested that SaCas9 complexed with each of the paired gRNAs cleaved episomal HBV DNA released from the infected HBV.

[0141] To demonstrate that SaCas9 with the paired gRNAs suppressed the viral load in vivo, a lipid nanoparticle (LNP) that encapsulated the SaCas9 mRNA and a pair of gRNAs was administered to an adeno-associated virus-Hepatitis B virus (AAV-HBV) mouse model (FIG. 8A). Each of six mice in the high dose cohort received 0.070 milligrams of the mixture of the SaCas9 mRNA and synthetic gRNAs comprising SaCas9 gRNA 1 (SEQ ID NO : 601) and SaCas9 gRNA 2 (SEQ ID NO: 603), while every animal was dosed with 0.035 milligrams of the nucleic acid payload in the low dose cohort. This model was generated by intravenous injection of a recombinant AAV8 vector carrying a 1 .3 -fold excess length of the HBV genotype D ayw strain genomic sequence. Serum HBV biomarkers (HBV DNA, HBsAg and HBeAg) were significantly lowered in mice receiving the LNP (FIG. 8B, FIG. 8C, and FIG. 8D). In addition, quantification of intrahepatic HBV DNA (FIG. 9 A) by digital PCR indicated thatthe LNP administration reduced the copy number of HBV DNA (FIG. 9B), suggesting that SaCas9 was expressed and complexed with each of the paired guide RNAs in hepatocytes , and that cutting of HBV DNA by the SaCas9 ribonucleoprotein complex led to its degradation in viv o.Example 2 :

[0142] The efficiency and specificity of genome editing at HBV target sites that were on the chromosomally integrated reporter construct was determined by comparing the rates of genomic modification at viral target sites and at “nominated” off-target sites (sites in the human genome with high-sequence identity to the viral target sites).

[0143] Candidate off-target sites (“nominated sites”) for SaCas9 gRNA 1 (SEQ ID NO : 601 ) and SaCas9 gRNA 2 (SEQ ID NO : 603 ) were identified using GUIDE-SEQ in the HBV reporter cell line (FIG. 4A), a cell line with a chromosomally integrated reporter construct carrying a partial HBV genomic sequence . The HBV reporter cell line was transfected using Lonza 4-D nucleofectorto co-deliver: (1) SaCas9 mRNA, (2) eitherSaCas9 gRNA 1 (SEQ ID NO : 601 ) or SaCas9 gRNA 2 (SEQ ID NO : 603 ) , and (3) double-stranded oligonucleotides (dsODN). DsODNs are sequences that integrate into any site in the genome that contains a double -stranded break, DSB; thus, b oth sites cleaved by SaCas9 complexed with gRNA 1 or gRNA 2 and spontaneous double strand breaks were detected. Four to five days post transfection, genomic DNA was extracted from transfected cells and next generation sequencing was performed to identify human genomic sites in which dsODNS were inserted, indicating the sites of DSBs (e.g., sites cleaved by SaCas9 complexed with gRNA 1 or gRNA 2) . Among human genomic sites detected by GUIDE-SEQ, four sites for each guide RNA were identified as having the highest sequence identity with the HBV on -target sites (“nominated sites”; see Table 10) Table 10 provides nucleotide sequences of each nominated site aligned with the on- target sites for the corresponding guide RNAs (“Alignment” column).Table 10

[0144] To assess the rate of sequence modifications (such as indels) at the on-target and nominated sites (of Table 10), the HBV reporter cell line was transfected with (1) SaCas9 mRNA and (2) either SaCas9 gRNA 1 (SEQ ID NO : 601 ) or SaCas9 gRNA 2 (SEQ ID NO : 603 ) using Lonza 4 -D nucleofector. Cells were harvested 5 days post transfection to extract genomic DNA. The flanking regions of the on-target and nominated sites were individually PCR-amplified and analyzed by next generation sequencing. Predicted cut sites were set between 4 and 3 nucleotides from the 3 ’ end of protospacers, and sequence reads containing nucleotide insertions and deletions (indels) within 5 nucleotide positions upstream and downstream of the cut sites were considered “modified reads” .

[0145] On-target sites in cells transfected with either of SaCas9 gRNA 1 or SaCas9 gRNA 2 were modified with high efficiency (mean of 99.3% of SaCas9 gRNA 1 sites and mean of 89.0% of SaCas9 gRNA 2, FIG. 10A and FIG. 10B, specifically). Thus, the HBV-targeting SaCas9 ribonucleoprotein complex was delivered with high efficiency to target sites. In contrast, the percentage of modified sequence reads carrying the nominated sites was 0. 1 % or lower and was not sub stantially different between nontransfected control samples and transfected samples. These results demonstrate that SaCas9 gRNA 1 and SaCas9 gRNA 2 direct SaCas9 to the HBV on-target sites with high specificity .Example 3 :

[0146] Anti-viral activity by multiplex gene editing with for SaCas9 gRNA 1 (SEQ ID NO : 601 ) and SaCas9 gRNA 2 (SEQ ID NO : 603) in HBV-infected primary humanhepatocytes was evaluated as follows (FIG. 11 A): cry op re served, primary human hepatocytes were seeded at 350,000 live cells per well in 24-well collagen -coated plates (Day 0) and infected with HB V (prepared from the HepAD38 cell line) at MOI of 500 on the next day (Day 1 ). Transfection with SaCas9 mRNA, SaCas9 gRNA 1 , and SaCas9 gRNA 2 was performed using the lipofectamine messengerMAX reagent 4 days post HBV infection (Day 5), and the culture medium was replaced on the next day (Day 6). The culture medium and cells were collected separately 8 days or 1 1 days post HBV infection (Day 9 or Day 12). For cells harvested on Day 12, the culture medium was replaced on Day 9. Hepatitis B e-Antigen (HBeAg) and Hepatitis B surface Antigen (HBsAg) in the collected culture media (illustrated in the short-dashed box in FIG. 11B) were analyzed by chemiluminescent immunoassays. Genomic DNA was extracted from the harvested cells (illustrated in the long-dashed box in FIG. 11B) and intrah epatic HBV DNA copy numbers were quantified by digital PCR. The results showed that transfection with SaCas9 mRNA, SaCas9 gRNA 1 , and SaCas9 gRNA 2 reduced HBeAg, HBsAg, and intrahepatic HBV DNA by 75 %, 79 % and 58 %, respectively (FIG. 11 C, FIG. 11D, an d FIG. HE, respectively), in the samples collected 8 days post HBV infection (Day 9). In the samples collected on 1 1 days post HBV infection (Day 12), secretion of HBeAg, HBsAg, and replication of intrahepatic HBV DNA were further suppressed (by 88 %, 85 % and 71 %, FIG. 11 C, FIG. HD, and FIG. HE, respectively).

[0147] These results demonstrate that SaCas9 ribonucleoprotein complexed with either SaCas9 gRNA 1 or SaCas9 gRNA 2 cut HBV DNA that persisted in the nuclei of cultured human hepatocytes, leading to degradation of HBV DNA and suppression of HBeAg and HB sAg secretion in vitro .Example 4 :

[0148] The quantity of HBV DNA in hepatocytes of mice transduced with a recombinant AAV8 vector carrying a 1 .3 -fold excess length of the HBV genome (as described in Example 1 ) was quantified. In short, a lipid nanoparticle (LNP) that encapsulated SaCas9 mRNA, gRNA 1 (SEQ ID NO : 601) and gRNA 2 (SEQ ID NO: 603) was administered to an adeno-associated virus-Hepatitis B virus (AAV-HBV) mouse model (FIG. 8A and FIG. 12A, dashed box).

[0149] DNA was extracted from the mouse liver samples described in Example 1 using MasterPure Complete DNA and RNA Purification Kit and proteinase K following the instructions described in Allweiss, et al., Gut, 2023. Digital PCR showed that approximately 280 HBV DNA copies were present per liver cell in AAV-HBV mice that did not receive lipid nanoparticles encapsulating SaCas9 mRNA, SaCas9 gRNA 1 , andSaCas9 gRNA2. These results indicate that the DNA extraction protocol used in Example 1 led to an underestimation of intrahepatic HBV DNA copy number (>30 fold). However, the ob served LNP-mediated reduction of HBV copy number in mice that were dosed with an LNP encapsulating SaCas9 mRNA, gRNA 1 , and gRNA 2 (up to 80 %, FIG. 12B) was similar to that found using the protocols described in Example 1 (FIG. 9B).

[0150] These results demonstrate that SaCas9 ribonucleoprotein complexed with either SaCas9 gRNA 1 or SaCas9 gRNA 2 cut HBV DNA that persisted in the nuclei of hepatocytes in AAV-HBV mice, leading to degradation of intrahepatic HBV DNA and reduction of serum HBV DNA in vivo.Example 5 :

[0151] The frequency of indels at the intended target sites on HBV DNA recovered from HBV-infected primary human hepatocytes (PHHs) tran sfected with the SaCas9- encoding mRNA and either the non-targeting gRNA or a pair of gRNA (SaCas9 gRNA 1 [SEQ ID NO : 601] and SaCas9 gRNA 2 [SEQ ID NO : 603 ]) was measured via sequencing analysis (FIG. 13A). In the edited cells, indels were observed at 1 .8% and 1 . 1% of the HBV DNA on-target sites for SaCas9 gRNA 1 and SaCas9 gRNA 2, respectively. In contrast, such edits were found at less than 0.1 % of these intended target sites in the samples treated with the non-targeting gRNA. Frequency of structural vari ant reads, including excision and inversion of the intervening sequence between the two HBV on- target sites, were calculated by dividing the number of the corresponding structural variant reads by the number of sequence reads containing the 25 -bp flanking regions of the two intended cut sites regardless of indels, excisions, and inversions ( FIG. 13B). These types of sequence modifications were increased by transfection with the SaCas9 - encoding mRNA, SaCas9 gRNAl , and SaCas9 gRNA 2, indicating that the HBV- targeting SaCas9 edited the viral target sequences (FIG. 13B, excisions on left graph, inversions on right graph).

[0152] To investigate the impact of anti -HBV gene editing treatment on HBV gene integration, the positions of HBV DNA joined to the human genome were identified from the hybridization capture sequencing data and mapped on the HBV reference genome (GenBank ID: U95551) (FIG. 14A). The total number of reads from three replicates were plotted against their corresponding HBV coordinates. The junction s of HBV sequences to human chromosomes were distributed across the entire HBV genome and increased upstream of the core-antigen gene. The positions of the two observed spikes were proximal to the ends of double-stranded linear HBV DNA, which has been known to be the primary source of integrated HBV DNA copies. The relative fraction of HBVintegration was estimated by dividing count of HBV-human chromosome chimeric sequence reads by the average sequence coverage of the parti al human GAPDH gene. HBV DNA insertion sites were reduced by 58% in the edited samples compared to the controls (FIG. 14B), indicating that targeted cleavage of HBV DNA led to suppression of HBV DNA integration.

[0153] To analyze outcomes of multiplex gene editing against HBV DNA in liver samples taken from the AAV-HBV mouse model illustrated in FIG. 12A, DNA was reextracted from small liver pieces of three randomly selected mice in each of the control and high-dose groups. Quantification of intrahepatic HBV DNA by digital PCR verified that HBV DNA was reduced by administration of LNPs encapsulating the HBV -targeting SaCas9 payload (FIG. 15A). Both episomal and integrated HBV DNA copies were sequenced by hybridization capture sequencing as conducted above. Indels were detected at 18% and 19% of the HBV on-target sites for SaCas9 gRNA and SaCas9 gRNA 2, respectively (FIG. 15B). Excisions and inversions of the fragments between the two on- target sites were also observed in 20% and 10% of total sequence reads spanning these sites (FIG. 15C). These results suggest that the HBV -targeting SaCas9 cut and altered HBV DNA sequences to block viral gene expression in vivo .

[0154] The hybridization capture sequencing data was further analyzed to determine if nuclease-induced fragmentation of HBV DNA affected chromosomal integration in vivo . Consistent with the findings in HBV -infected primary human hepatocytes (PHHs) (FIG. 14A), junctions to mouse chromosomal DNA were distributed across the entire HBV genome, and two prominent peaks were found around the 5 ’ terminus of the core antigen-encoding gene (FIG. 16A). This analysis suggested that anti -HBV multiplex gene editing is sufficient to suppress HBV DNA integration by 58% in the liver samples from the AAV-HBV mice (FIG. 16B).Example 6 :

[0155] In vivo activity against a chromosomally integrated HBV DNA sequences was examined using a transgenic mouse model provided by Beijing Vitalstar Biotechnology Co. Ltd. The mouse model was generated through pronuclear microinjection of a linearized DNA fragment carrying the 1 .3 -fold oversized HBV genome (GenBank ID: AF305422. 1, subtype adw2, genotype A) into the fertilized egg of a C57BL / 6 mouse. In the liver of the inj ected mice, viral mRNA was transcribed from the transgenic -HBV (Tg- HBV) construct, generating infectious virions and viral antigens (FIG. 17A). The mice were divided into three groups and treated with PBS and with LNPs comprising LP01, DSPC, cholesterol, and DMG-PEG2000 encapsulating the SaCas9-encoding mRNA anda pair of SaCas9 gRNA 1 and SaCas9 gRNA 2 at one of two different dose levels. Dosedependent reduction in serum HBsAg and HBV DNA was observed and maintained for 5 weeks until the mice were euthanized for tissue harvesting (FIG. 17B and FIG. 17C). Total HBV DNA that had accumulated in the liver of these animals was quantified by digital PCR and found to be decreased significantly by anti -HBV gene editing (FIG.17D)

[0156] To understand the molecular mechanism underlying suppression of HBV replication by HBV-targeting SaCas9, the detailed structure of the Tg-HBV construct was analyzed using DNA extracted from stomach samples from three Tg-HBV mice. Given than HBV replication did not take place in this tissue type, digital PCR indicated that two copies of the target region that could be amplified by the HBV-specific PCR primers and probe were present in each cell (FIG. 18A). Tg-HBV was identified at a single site of mouse chromosome 3 by long-read hybridization capture sequencing.

[0157] As shown in FIG. 18B, the 2.3 -fold oversized genotype-A HBV genome containing 5 total target sites forHBV -targeting paired gRNAs was inserted along with the partial cloning vector and reverse complement of a short (around 240 -bp) HBV fragment (HBV inverted repeat [HBV-IR]). Specifically, the Tg-HBV DNA was composed of 2.3 x tandem repeats of the genotype-A HBV genome (GenBank ID: AF305422. 1) connected to the partial cloning vector and the 240 -bp HBV inverted repeat (HBV-IR; positions 2631 -2870 of the nucleotide sequence of GenBank ID: AF305422.1). The Tg-HBV DNA was located in the reverse orientation between the positions 124,418,553 and 124,418, 556 of mouse chromosome 3. The 2.3 x tandem copies contained two on-target sites for SaCas9 gRNA 1 and 3 for SaCas9 gRNA 2, starting at position 2,860 and ending at position 3 ,758 of the nucleotide sequence of GenBank ID: AF305422. 1. Structural variants on the Tg-HBV construct were examined by long-read hybridization capture sequencing with DNA extracted from the liver samples of Tg-HBV mice. Excisions and inversions of the HBV DNA sequences between a pair of the HBV on-target sites and combinations of these two types of sequence modifications were all detected at significantly higher frequency over the background estimated from the control group treated with PB S (FIG. 18C). The number of sequence reads containing the distal end from HBV-IR (junction of the mouse chromosome and the 2.3 x tandem HBV genomic copies) was used as the denominator to calculate frequency of the structural variants. These results suggested thatthe HBV-targeting SaCas9 could cut and edit chromosomally integrated HBV DNA to disrupt viral gene expression.

[0158] In addition to sequence reads containing Tg-HBV and its flanking chromosomal DNA, those containing HB VDNA inserted in different locations of mouse chromosomes from Tg-HBV were identified in long-read hybridization capture sequencing data. Such chimeric reads were expected to represent de novo random HBV DNA integration as illustrated in FIG. 19A. Without being bound by theory, HBV pregenomic RNA (pgRNA) may be transcribed from the Tg-HBV construct and converted to double-stranded DNA by the viral polymerase during encapsidation. These HBV DNA molecules and Tg-HB V-derived sequences fragmented by the HBV -targeting SaCas9 can be inserted into spontaneous breaks that occur on chromosomes. Consistent with the results of the previous experiments with HBV -infected PHHs and the AAV- HBV mouse model (FIG. 14B and FIG. 16B, respectfully), the number of new HBV integration-derived reads relative to the mouse Gapdh gene decreased in Tg-HBV mice receiving the LNPs (FIG. 19B). The number of HBV-chromosome chimeric reads containing the distinct junctions from those for the Tg-HBV construct was normalized with the average sequence coverage of the internal control gene locus, mouse Gapdh. A small number of reads with sequences derived from regions of two distinct chromosomes interrupted by HBV DNA were also found in the long-read hybrid capture sequencing data. These reads were expected to be derived from chromosomal translocations that occurred through one of the three mechanisms shown in FIG. 20A. The number of such sequence reads relative to the average sequence coverage of the mouse Gapdh gene was examined. No significant difference was found between the control a nd treated samples, suggesting that anti-HBV multiplex gene editing had little impact on chromosomal translocation with de novo HBV DNA fragments (FIG. 20B).

[0159] All publications and patents mentioned in the present application are herein incorporated by reference. Various modifications and variations of the described methods and compositions of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific preferred embodiments, it should be understood that the disclosure should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the relevant field are intended to be within the scope of the following claims.

[0160] Although the present disclosure has been described in considerable detail with references to certain embodiments thereof, other embodiments are possible without departing from the present disclosure. The spirit and scope of the appended claims shouldbe limited to the description of the preferred embodiments contained herein. All embodiments that come within the meaning of the claims, either literally or by equivalence, are intended to be embraced herein.

[0161] Furthermore, the advantages described above are not necessarily the only advantages of the presently described technology, and it is not necessarily expected that all of the described advantages will be achieve d with every embodiment of the disclosure.

Claims

CLAIMSWHAT IS CLAIMED IS:1 . A lipid nanoparticle (LNP) comprising:(1 ) a polynucleotide comprising a sequence encoding a CRISPR-Cas endonuclease;(2) a first guide ribonucleic acid (gRNA) that hybridizes to a first target sequence within a hepatitis B virus (HBV) nucleic acid molecule; and(3) a second gRNAthathybridizes to a second target sequence within the HBV nucleic acid molecule, wherein:(i) the second target sequence is different from the first target sequence; and(ii) expression of the CRISPR-Cas endonuclease in combination with the first gRNA cleaves the HBV nucleic acid molecule within or proximal to the first target sequence, generating a first cleaved site; and(iii) expression of the CRISPR-Cas endonuclease in combination with the second gRNA cleaves the HBV nucleic acid molecule within or proximal to the second target sequence, generating a second cleaved site.

2. The LNP of claim 1 , wherein generating the first cleaved site and the second cleaved site results in excising a region of the HBV nucleic acid molecule between the first cleaved site and the second cleaved site.

3. The LNP of claim 1 , wherein generating the first cleaved site and the second cleaved site results in inversion of a region of the HBV nucleic acid molecule between the first cleaved site and the second cleaved site.

4. The LNP of claim 1, wherein generating the first cleaved site and the second cleaved site results in one or more indels at or proximal to the first cleavage site, at or proximal to the second cleavage site, or a combination thereof.

5. The LNP of claim 1 , wherein generating the first cleaved site and the second cleaved site results in concatemerization of two or more HBV nucleic acid molecules.

6. The LNP of any one of claims 1 -5, wherein the first target sequence and the second target sequence are within different genes.

7. The LNP of any one of claims 1 -6, wherein at least one of the first target sequence and the second target sequence are within a region of the HBV nucleic acid molecule encoding two genes.

8. The LNP of any one of claims 1 -7, wherein both ofthefirsttarget sequence and the second target sequence are within a region of the HBV nucleic acid molecule encodingtwo genes.

9. The LNP of any one of claims 1 -8, wherein the first target and the second target sequence are at least 250, atleast 500, atleast 1,000, atleast 1,100, atleast 1,200, atleast 1,300, at least 1,400, at least 1,500, or at least 1,600 base positions apart.

10. The LNP of any one of claims 1-9, wherein the region of the HBV nucleic acid molecule between the first cleaved site and the second cleaved site comprises atleast 250, at least 500, atleast 1,000, atleast 1, 100, atleast 1,200, atleast 1,300, atleast 1,400, atleast 1,500, or at least 1,600 nucleotides.

11. The LNP of any one of claims 1 -10, wherein the first target sequence is within the pol and S gene region of the HBV nucleic acid molecule.

12. The LNP of any one of claims 1 -11, wherein the second target sequence is within the preC gene region of the HBV nucleic acid molecule.

13. The LNP of any one of claims 1 -12, wherein the firsttarget sequence and the second target sequence each independently comprise a protospacer sequence selected from any one of Tables 1 -5 or Table 7.

14. The LNP of any one of claims 1 -13, wherein the firsttarget sequence and the second target sequence each independently comprise a protospacer sequence selected from Table 1, Table 4, or Table 7.

15. The LNP of any one of claims 1 -14, wherein the CRISPR-Cas endonuclease is a Cas9 endonuclease.

16. The LNP of claim 15, wherein the Cas9 endonuclease is SaCas9.

17. The LNP of claim 16, wherein the SaCas9 comprises SEQIDNO: 621 ora variantthereof.

18. The LNP of any one of claims 1 -17, wherein the first gRNA comprises SEQ ID NO: 601.

19. The LNP of any one of claims 1 -17, wherein the second gRNA comprises SEQ ID NO: 603.

20. The LNP of any one of claims 1 -19, wherein the first gRNA comprises SEQ ID NO: 601 and wherein the second gRNA comprises SEQ ID NO: 603.21 . The LNP of any one of claims 1 -15, wherein the CRISPR-Cas endonuclease is a CasX endonuclease.

22. The LNP of any one of claims 1 -21, wherein the polynucleotide is a messenger RNA polynucleotide comprising a 5' cap structure, a 5' UTR, the sequence encoding the CRISPR-Cas endonuclease, a 3' UTR, and a poly A tail.

23. The LNP of any one of claims 1 -22, wherein the LNP comprises an ionizable lipid having a tertiary amine; a sterol, a phosphatidylcholine lipid, and a pegylated lipid.

24. The LNP of claim 23, wherein the LNP comprise SM-102, distearoylphosphatidylcholine (DSPC), cholesterol, and DMG-PEG2000.

25. The LNP of claim 23, wherein the LNP comprises LP01, distearoylphosphatidylcholine (DSPC), cholesterol, and DMG-PEG2000.

26. The LNP of any one of claims 1 -25, wherein the HBV nucleic acid molecule is an HBV genome or covalently closed circular DNA (cccDNA).

27. A method of treating HBV in an individual, the method comprising: administering the LNP of any one of claims 1 -26 to the individual.

28. The method of claim 27, wherein treating comprises reducing the amount of HBV serum biomarkers.

29. The method of claim 28, wherein the HBV serum biomarkers comprise HBV DNA, hepatitis B surface antigen (HBsAg), Hepatitis B e-antigen (HBeAg), or a combination thereof.

30. The method of any one of claims 27-29, wherein treating comprises reducing intrahepatic HBV DNA.

31. The method of claim 28-30, wherein the reduction of the amount of HBV serum biomarkers is compared to an amount of HBV serum biomarkers from an untreated individual.

32. The method of claim 28-31, wherein the reduction of the amount of HBV serum biomarkers is compared to an amount of HBV serum biomarkers from the individual from a prior measurement.

33. The method of any one of claims 27-32, wherein the method further comprises administering an HBV antiviral to the individual.

34. The method of claim 33, wherein the HBV antiviral is administered before administering the LNP.

35. The method of claim 33, wherein the HBV antiviral is administered after administering the LNP.

36. The method of any one of claims 33-35, wherein the HBV antiviral is Bulevirtide, Entecavir (Baraclude), Tenofovir disoproxil fumarate (Viread), Tenofovir alafenamide (Vemlidy), Lamivudine (Epivir-HBV), or Telbivudine (Tyzeka).

37. A method of inactivating an HBV nucleic acid molecule in a cell, the method comprising: contacting the cell with the LNP of any one of claims 1 -26.

38. A method of excising a region of an HBV nucleic acid molecule in a cell, the method comprising: contacting the cell with the LNP of any one of claims 1 -26.

39. The method of any one of claims 37-38, wherein the cell is in a human.

40. The method of any one of claims 37-39, wherein the method further comprises providing an HBV antiviral to the cell.

41. The method of claim 40, wherein the HBV antiviral is Bulevirtide, Entecavir (Baraclude), Tenofovir disoproxil fumarate (Viread), Tenofovir alafenamide (Vemlidy), Lamivudine (Epivir-HBV), or Telbivudine (Tyzeka).

42. The method of any one of claims 37-41, wherein the HBV nucleic acid molecule is an HBV genome or covalently closed circular DNA (cccDNA).

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