Engineered meganucleases with specificity for recognition sequences in the genome of hepatitis b virus

Abstract

This disclosure covers engineered nucleases that recognize and cleave recognition sequences within the hepatitis B virus (HBV) genome. When compared to previously described HBV macronucleases, the engineered macronucleases described herein may exhibit improved properties, such as enhanced specificity and / or indel formation efficiency. Furthermore, this disclosure covers pharmaceutical compositions comprising engineered macronuclease proteins, nucleic acids encoding engineered macronucleases, and uses of such compositions to treat HBV infection or diseases associated with HBV infection.

Description

Technical Field

[0001] This disclosure relates to the fields of virology, molecular biology, and recombinant nucleic acid technology. Specifically, this disclosure relates to optimized engineered broad-spectrum nucleases with specificity for recognition sequences within the genome of the hepatitis B virus AG genotype. Such engineered broad-spectrum nucleases can be used in methods for treating hepatitis B virus infection and diseases caused by hepatitis B virus.

[0002] Reference to the sequence list submitted electronically as an XML file This application includes a sequence list submitted in XML format through the USPTO Patent Centre, which is incorporated herein by reference in its entirety. The XML copy was created on October 2, 2024, and is named “P89339_2030USP3.xml”, with a size of 108,321 bytes. Background Technology

[0003] Hepatitis B virus (HBV) is a major health problem worldwide, with over 350 million people chronically carrying the virus. HBV infection is a serious and common infectious disease of the liver. Chronic infection is associated with an increased risk of developing serious liver diseases, including cirrhosis and hepatocellular carcinoma (HCC), one of the most common forms of cancer in humans. The estimated risk of HCC in chronic HBV carriers is approximately 100 times higher than in uninfected individuals. About one-third of the world's population will be infected at some point in their lives, including 240 million to 350 million people with chronic infection. More than 750,000 people die from hepatitis B each year. Of these, approximately 300,000 die from liver cancer. Currently available anti-HBV drugs have limitations. For example, interferon-alpha administration is associated with serious adverse reactions. Nucleoside analogs inhibit viral growth but require long-term administration.

[0004] The HBV genome exhibits genetic variability, with 1.4–3.2 × 10⁻⁶ cells / year per locus. -5 The estimated variation rate of nucleotide substitutions. Numerous viral variants arise during replication as a result of nucleotide misincorporation in the absence of any proofreading ability of viral polymerase. This variability leads to recognized viral subtypes. HBV has been classified into well-defined genotypes based on 8% or higher inter-group differences in the complete genome sequence, each with a distinct geographical distribution. For example, genotype A is widespread in sub-Saharan Africa, Northern Europe, and West Africa; genotypes B and C are common in Asia; genotype C is mainly observed in Southeast Asia; genotype D is dominant in Africa, Europe, Mediterranean countries, and India; genotype G has been reported in France, Germany, and the United States; and genotype H is frequently encountered in Central and South America. Genotype I has recently been reported in Vietnam and Laos. The most recent HBV genotype, genotype J, has been identified in the Ryukyu Islands of Japan.

[0005] HBV belongs to the family Hepatotropic DNA Virusidae ( Hepadnaviridae HBV is an enveloped DNA virus. It contains a small, partially double-stranded (DS), relaxed circular DNA (rcDNA) genome, which replicates via reverse transcription using an RNA intermediate (pregenomic RNA (pgRNA)). The circular DNA genome of HBV is unusual because the DNA is not fully double-stranded. One end of the full-length strand is linked to the viral DNA polymerase. The genome is approximately 3020–3320 nucleotides long (for the full-length strand) and 1700–2800 nucleotides long (for the short-length strand). The negative-sense (non-coding) receptor is complementary to the viral mRNA.

[0006] There are four known genes encoded by the genome, called C, X, P, and S. The core protein is encoded by gene C (HBcAg), and its start codon is preceded by the upstream in-frame AUG start codon (from which the precore protein is produced). HBeAg is produced through the proteolytic processing of the precore protein. DNA polymerase is encoded by gene P. Gene S encodes the surface antigen (HBsAg). The HBsAg gene is a long open reading frame but contains three in-frame "start" (ATG) codons that divide the gene into three segments: pre-S1, pre-S2, and S. Due to the presence of multiple start codons, three polypeptides of different sizes are produced, called large (in the order from surface to interior: preS1 / pre-S2 / S), medium (pre-S2 / S), and small (S). The function of the protein encoded by gene X is not fully understood, but it is associated with the development of liver cancer. It stimulates genes that promote cell growth and inactivates growth-regulating molecules.

[0007] Shortly after cell infection, viral DNA is found in the cell nucleus. Partially double-stranded DNA becomes fully double-stranded by completing the (+) sense strand and removing protein molecules from the (-) sense strand and short RNA sequences from the (+) sense strand. Non-coding bases are removed from the ends of the (-) sense strand, and the ends are rejoined.

[0008] The HBV life cycle begins when the virus attaches to and is internalized by a host cell. Recent studies have demonstrated that sodium taurocholate cotransport polypeptide (NTCP) is a functional receptor in HBV infection. The virus particle relaxes its circular DNA (rcDNA) and delivers it to the nucleus, where it repairs itself to form a covalently closed circular DNA (cccDNA). Free cccDNA serves as a template for transcription via host RNA polymerase II of pregenomic RNA (pgRNA) and other viral mRNAs. The transcript is then exported to the cytoplasm, where viral protein translation occurs. Reverse transcriptase (RT) binds to pgRNA and triggers the assembly of core proteins into an immature RNA-containing nucleocapsid. The immature nucleocapsid then undergoes maturation, whereby pgRNA is reverse transcribed by RT to produce mature rcDNA. A unique feature of reverse transcription in hepatotropic DNA viruses is the initiation of negative-strand DNA synthesis induced by RT, leading to a covalent attachment of RT to the 5' end of the negative-strand DNA.

[0009] Then, the mature nucleocapsid containing rcDNA is encapsulated by viral surface proteins and secreted as a viral particle (secretion pathway), or alternatively, it is recycled back to the nucleus to further amplify the cccDNA pool (recirculation pathway). The persistence of cccDNA in hepatocytes plays a key role in viral persistence, reactivation of viral replication after cessation of antiviral therapy, and treatment resistance.

[0010] Homing endonucleases are a group of naturally occurring nucleases that recognize 15-40 base pair cleavage sites common in plant and fungal genomes. They frequently bind to parasitic DNA elements, such as group 1 self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by creating double-strand breaks on chromosomes, thus recruiting cellular DNA repair mechanisms (Stoddard (2006) Q. Rev. Biophys. 38:49-95). Homing endonucleases are generally grouped into four families: the LAGLIDADG (SEQ ID NO: 2) family, the GIY-YIG family, the His-Cys box family, and the HNH family. These families are characterized by structural motifs (which influence catalytic activity) and recognition sequences. For example, members of the LAGLIDADG (SEQ ID NO: 2) family are characterized by having one or two copies of the conserved LAGLIDADD motif (see, Chevalier et al., (2001) Nucleic Acids Res. 29(18):3757-3774). LAGLIDADD homing endonucleases with a single copy of the LAGLIDADG motif form homodimers, while members with two copies of the LAGLIDADG motif are found to be monomers. Methods for generating homing endonucleases are known in the art.

[0011] I-CreI (SEQ ID NO: 1) is a member of the homing endonuclease family LAGLIDADG, which recognizes and cleaves the algae Chlamydomonas reinhardtii (…). Chlamydomonas reinhardtii The 22-base-pair recognition sequence in chloroplast chromosomes. Genetic selection techniques have been used to modify the preference of wild-type I-CreI cleavage sites (Sussman et al., (2004) J. Mol. Biol. 342:31-41; Chames et al., (2005) Nucleic Acids Res. 33:e178; Seligman et al., (2002) Nucleic Acids Res. 30:3870-9; Arnould et al., (2006) J. Mol. Biol. 355:443-58). Methods for the rational design of single LAGLIDADG homing endonucleases have been described, which enable comprehensive redesign of I-CreI and other homing endonucleases to target a wide variety of DNA sites, including sites in mammalian, yeast, plant, bacterial, and viral genomes (see, for example, WO 2007 / 047859).

[0012] As first described in WO 2009 / 059195, I-CreI and its engineered derivatives are typically dimers, but can be fused into single polypeptides using short peptide linkers that connect the C-terminus of the first subunit to the N-terminus of the second subunit (see also Li et al., (2009) Nucleic Acids Res. 37:1650-62; Grizot et al., (2009) Nucleic Acids Res. 37:5405-19). Therefore, a wide range of functional “single-stranded” nucleases can be expressed from a single transcript.

[0013] The use of engineered large-scale nucleases for the treatment of HBV infection has been proposed. For example, WO 2010 / 136841 proposes the use of engineered large-scale nucleases to cleave the genome of non-genomic integrated viruses. Such large-scale nucleases include I-CreI variants that target 22-base-pair large-scale nuclease recognition sequences, which are different from those described herein and are present only in a few HBV genotypes.

[0014] The applicant previously disclosed a number of engineered wide-ranging nucleases specific for recognition sequences present in the HBV genome, including the HBV 11-12 recognition sequence (SEQ ID NO: 3), in PCT / US2017 / 56638, PCT / US2019 / 27203 and PCT / US2020 / 063479.

[0015] This disclosure improves upon engineered large-scale nucleases previously described in the art in several respects. When generating endonucleases for therapeutic administration to patients, enhanced (i.e., increased) target specificity while simultaneously reducing or eliminating off-target cleavage within the target cell genome is crucial. Here, the applicant has developed an additional engineered large-scale nuclease targeting the HBV 11-12 recognition sequence. The large-scale nuclease of this disclosure has a novel and unique sequence generated through extensive experimentation. Furthermore, the large-scale nuclease described herein exhibits numerous improved and unexpected properties when compared to previously disclosed engineered large-scale nucleases, including a significant reduction in off-target cleavage within the host cell genome. In particular, the engineered large-scale nuclease described herein shows a significant enhancement (i.e., an increase) in the formation of indels (i.e., insertions or deletions at cleavage sites within the HBV genome, indicating targeted cleavage) in cell lines containing an integrated copy of the HBV genome. Therefore, the large-scale nuclease of this disclosure further advances the technology in several ways, which is essential for developing clinical products targeting HBV infection and HBV-associated hepatocellular carcinoma. Summary of the Invention

[0016] In one aspect, this disclosure provides an engineered large-scale nuclease that binds to and cleaves a recognition sequence comprising or consisting of SEQ ID NO: 3 within the hepatitis B virus (HBV) genome, wherein the engineered large-scale nuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and wherein the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region.

[0017] In some embodiments, the HVR1 region contains an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 204-259 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0018] In some embodiments, the HVR1 region contains an amino acid sequence that has at least 97% sequence identity with residues 204-259 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0019] In some embodiments, the HVR1 region contains one or more residues corresponding to residues 204, 206, 208, 210, 212, 213, 218, 220, 222, 224, 226, 248, 250, 255 and 257 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0020] In some implementations, the HVR1 region contains residues 204, 206, 208, 210, 212, 213, 218, 220, 222, 224, 226, 248, 250, 255 and 257 corresponding to SEQ ID NO: 5 or SEQ ID NO: 6.

[0021] In some implementations, the HVR1 region contains residue 237 corresponding to residue SEQ ID NO: 5 or SEQ ID NO: 6.

[0022] In some implementations, the HVR1 region contains residue 241 corresponding to residue SEQ ID NO: 5 or SEQ ID NO: 6.

[0023] In some implementations, the HVR1 region contains residue 251 corresponding to residue SEQ ID NO: 5 or SEQ ID NO: 6.

[0024] In some implementations, the HVR1 region contains residue 252 corresponding to residue SEQ ID NO: 5 or SEQ ID NO: 6.

[0025] In some implementations, the HVR1 region contains residues corresponding to SEQ ID NO: 5 or SEQ ID NO: 6 and residue 253.

[0026] In some embodiments, the HVR1 region contains Y, R, K, or D at residue 246 corresponding to residue SEQ ID NO: 5 or SEQ ID NO: 6.

[0027] In some implementations, the HVR1 region contains residues 204-259 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0028] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 187-333 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0029] In some embodiments, the first subunit comprises an amino acid sequence having at least 99% sequence identity with residues 187-333 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0030] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 185-343 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0031] In some embodiments, the first subunit comprises an amino acid sequence having at least 99% sequence identity with residues 185-343 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0032] In some implementations, the first subunit contains residue 260 corresponding to SEQ ID NO: 6.

[0033] In some embodiments, the first subunit contains G, S, or A at residue 199 corresponding to residue SEQ ID NO: 5 or SEQ ID NO: 6.

[0034] In some embodiments, the first subunit contains E, Q, or K at residue 260 corresponding to residue SEQ ID NO: 5 or SEQ ID NO: 6.

[0035] In some embodiments, the first subunit comprises residues 187-333 of either SEQ ID NO: 5 or SEQ ID NO: 6.

[0036] In some embodiments, the first subunit comprises residues 185-343 of either SEQ ID NO: 5 or SEQ ID NO: 6.

[0037] In some embodiments, the HVR2 region contains an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 24-79 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0038] In some implementations, the HVR2 region contains one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75 and 77 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0039] In some implementations, the HVR2 region contains residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75 and 77 corresponding to SEQ ID NO: 5 or SEQ ID NO: 6.

[0040] In some implementations, the HVR2 region contains residue 51 corresponding to residue 51 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0041] In some embodiments, the HVR2 region contains Y, R, K or D at residue 66 corresponding to residue SEQ ID NO: 5 or SEQ ID NO: 6.

[0042] In some implementations, the HVR2 region contains residues 24-79 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0043] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 7-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0044] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 7-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0045] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 6-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0046] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 6-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0047] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 5-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0048] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 5-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0049] In some embodiments, the second subunit is an N-terminal subunit and comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 4-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0050] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 4-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0051] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 3-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0052] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 3-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0053] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 2-153 of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the second subunit comprises residues other than M at position 1 corresponding to position 1 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0054] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 2-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0055] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 1-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0056] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 1-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0057] In some embodiments, the second subunit contains residue 19 corresponding to residue SEQ ID NO: 5 or SEQ ID NO: 6.

[0058] In some embodiments, the second subunit contains residue 80 corresponding to residue 80 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0059] In some embodiments, the second subunit contains residue 96 corresponding to residue 96 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0060] In some embodiments, the second subunit contains residue 99 corresponding to residue 99 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0061] In some embodiments, the second subunit comprises residue 100 corresponding to residue SEQ ID NO: 5 or SEQ ID NO: 6.

[0062] In some embodiments, the second subunit contains G, S, or A at residue 19 corresponding to residue SEQ ID NO: 5 or SEQ ID NO: 6.

[0063] In some embodiments, the second subunit contains E, Q, or K at residue 80 corresponding to residue SEQ ID NO: 5 or SEQ ID NO: 6.

[0064] In some embodiments, the subunit contains residues 7-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0065] In some embodiments, the second subunit comprises residues 6-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0066] In some embodiments, the second subunit comprises residues 5-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0067] In some embodiments, the second subunit comprises residues 4-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0068] In some embodiments, the second subunit comprises residues 3-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0069] In some embodiments, the second subunit comprises residues 2-153 of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the second subunit comprises residues other than M at position 1 corresponding to SEQ ID NO: 5 or SEQ ID NO: 6.

[0070] In some embodiments, the second subunit comprises residues 1-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0071] In some implementations, the engineered large-scale nuclease is a single-stranded large-scale nuclease containing a linker, wherein the linker covalently binds to a first subunit and a second subunit.

[0072] In some embodiments, the adapter comprises the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16.

[0073] In some embodiments, the adapter comprises the amino acid sequence shown in SEQ ID NO: 15.

[0074] In some embodiments, the N-terminus of the connector is fused with a residue (i.e., a D residue) corresponding to residue 153 of SEQ ID NO: 5 or SEQ ID NO: 6, and the C-terminus of the connector is fused with a residue (i.e., a Y residue) corresponding to residue 185 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0075] In some embodiments, the engineered wide-range nuclease comprises a first subunit containing an amino acid sequence having at least 99% sequence identity with residues 187-333 of SEQ ID NO: 5 or SEQ ID NO: 6; a linker containing the amino acid sequence shown in SEQ ID NO: 15, wherein the linker covalently binds to the first and second subunits; and a second subunit containing an amino acid sequence having at least 99% sequence identity with residues 7-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0076] In some embodiments, the engineered wide-range nuclease comprises a first subunit containing an amino acid sequence having at least 99% sequence identity with residues 185-343 of SEQ ID NO: 5 or SEQ ID NO: 6; a linker containing the amino acid sequence shown in SEQ ID NO: 15, wherein the linker covalently binds the first subunit and the second subunit; and a second subunit containing an amino acid sequence having at least 99% sequence identity with residues 1-153 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0077] In some embodiments, the engineered wide range of nucleases comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 4-343 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0078] In some implementations, the engineered wide-range nuclease comprises an amino acid sequence having at least 92% sequence identity with residues 4-343 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0079] In some embodiments, the engineered wide range of nucleases comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 3-343 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0080] In some implementations, the engineered wide-range nuclease comprises an amino acid sequence having at least 92% sequence identity with residues 3-343 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0081] In some embodiments, the engineered broad-spectrum nuclease comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 2-343 of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the second subunit comprises residues other than M at position 1 corresponding to SEQ ID NO: 5 or SEQ ID NO: 6.

[0082] In some implementations, the engineered wide-range nuclease comprises an amino acid sequence having at least 92% sequence identity with residues 2-343 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0083] In some implementations, the engineered wide range of nucleases comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 5 or SEQ ID NO: 6.

[0084] In some implementations, the engineered wide-range nuclease comprises an amino acid sequence having at least 92% sequence identity with SEQ ID NO: 5 or SEQ ID NO: 6.

[0085] In some implementations, the engineered wide range of nucleases comprises the amino acid sequence of residues 4-343 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0086] In some implementations, the engineered wide range of nucleases comprises the amino acid sequence of residues 3-343 of SEQ ID NO: 5 or SEQ ID NO: 6.

[0087] In some embodiments, the engineered broad-spectrum nuclease comprises the amino acid sequence of residues 2-343 of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the second subunit comprises residues other than M at position 1 corresponding to SEQ ID NO: 5 or SEQ ID NO: 6.

[0088] In some embodiments, the engineered broad-spectrum nuclease comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

[0089] In some implementations, the engineered wide-range nuclease is encoded by a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0090] In some implementations, the engineered wide-range nuclease is encoded by the nucleic acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0091] In some implementations, engineered large-scale nucleases contain nuclear localization signals.

[0092] In some implementations, the nuclear localization signal is located at the N-terminus of engineered large-scale nucleases.

[0093] In some implementations, the nuclear localization signal is located at the C-terminus of engineered large-scale nucleases.

[0094] In some implementations, engineered wide-range nucleases include a first nuclear localization signal at the N-terminus and a second nuclear localization signal at the C-terminus.

[0095] In some embodiments, the nuclear localization signal comprises an amino acid sequence having at least 80% or at least 90% sequence identity with SEQ ID NO: 17. In some embodiments, the nuclear localization signal comprises SEQ ID NO: 17.

[0096] In some embodiments, the nuclear localization signal comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 65. In some embodiments, the nuclear localization sequence comprises SEQ ID NO: 65.

[0097] In some embodiments, the engineered wide-range nuclease comprises an N-terminal nuclear localization sequence containing an amino acid sequence having at least 80% or at least 90% sequence identity with SEQ ID NO: 17; and a C-terminal nuclear localization sequence containing an amino acid sequence having at least 80% or at least 90% sequence identity with SEQ ID NO: 17.

[0098] In some embodiments, the engineered wide-range nuclease comprises an N-terminal nuclear localization sequence containing the amino acid sequence shown in SEQ ID NO: 17; and a C-terminal nuclear localization sequence containing the amino acid sequence shown in SEQ ID NO: 17.

[0099] In some embodiments, the engineered wide-range nuclease comprises an N-terminal nuclear localization sequence containing an amino acid sequence having at least 80% or at least 90% sequence identity with SEQ ID NO: 17; and a C-terminal nuclear localization sequence containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 65.

[0100] In some embodiments, the engineered wide-range nuclease comprises an N-terminal nuclear localization sequence comprising the amino acid sequence shown in SEQ ID NO: 17; and a C-terminal nuclear localization sequence comprising the amino acid sequence shown in SEQ ID NO: 65.

[0101] In some embodiments, the engineered wide-range nuclease comprises an N-terminal nuclear localization sequence containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 65; and a C-terminal nuclear localization sequence containing an amino acid sequence having at least 80% or at least 90% sequence identity with SEQ ID NO: 17.

[0102] In some embodiments, the engineered wide-range nuclease comprises an N-terminal nuclear localization sequence comprising the amino acid sequence shown in SEQ ID NO: 65; and a C-terminal nuclear localization sequence comprising the amino acid sequence shown in SEQ ID NO: 17.

[0103] In some embodiments, the engineered wide-range nuclease comprises an N-terminal nuclear localization sequence containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 65; and a C-terminal nuclear localization sequence containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 65.

[0104] In some embodiments, the engineered wide-range nuclease comprises an N-terminal nuclear localization sequence containing the amino acid sequence shown in SEQ ID NO: 65; and a C-terminal nuclear localization sequence containing the amino acid sequence shown in SEQ ID NO: 65.

[0105] On the other hand, this disclosure provides engineered, wide-ranging nucleases described herein for use as pharmaceuticals.

[0106] On the other hand, this disclosure provides polynucleotides comprising nucleic acid sequences encoding the engineered wide range of nucleases described herein.

[0107] In some embodiments, the polynucleotide includes a 5' ALB untranslated region (UTR) containing a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO:18 or SEQ ID NO:82. In some embodiments, the polynucleotide includes a 5' ALB untranslated region (UTR) containing the nucleic acid sequence shown in SEQ ID NO:18 or SEQ ID NO:82.

[0108] In some embodiments, the polynucleotide comprises a 3' SNRPB UTR containing a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 19 or SEQ ID NO: 83. In some embodiments, the polynucleotide comprises a 3' SNRPB UTR containing the nucleic acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 83.

[0109] In some embodiments, the polynucleotide includes a termination sequence. In some embodiments, the polynucleotide includes a polyA termination sequence. In some embodiments, the polyA termination sequence includes a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 20. In some embodiments, the polyA termination sequence includes the nucleic acid sequence shown in SEQ ID NO: 20.

[0110] In some embodiments, the polynucleotide comprises a Kozak sequence. In some embodiments, the polynucleotide comprises a Kozak sequence containing the nucleic acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 84.

[0111] In some implementations, the nucleic acid sequence encoding an engineered wide-range nuclease is thymine or uracil depleted.

[0112] In some implementations, the nucleic acid sequence encoding an engineered wide range of nucleases is optimized for liver expression codons.

[0113] In some implementations, the polynucleotide is mRNA.

[0114] In some embodiments, the mRNA comprises: (a) a 5' ALBUTR containing the nucleic acid sequence shown in SEQ ID NO: 82; (b) a nucleic acid sequence encoding an engineered wide-ranging nuclease, wherein the nucleic acid sequence is uracil-depleted and optimized for liver expression codons; (c) a 3' SNRPB UTR containing the nucleic acid sequence shown in SEQ ID NO: 83; and (d) a polyA termination sequence.

[0115] In some embodiments, the mRNA comprises: (a) a 5' ALBUTR containing the nucleic acid sequence shown in SEQ ID NO: 82; (b) a Kozak sequence containing the nucleic acid sequence shown in SEQ ID NO: 84; (c) a nucleic acid sequence encoding an engineered wide-ranging nuclease, wherein the nucleic acid sequence is uracil-depleted and optimized for liver expression codons; (d) a 3' SNRPB UTR containing the nucleic acid sequence shown in SEQ ID NO: 83; and (e) a polyA termination sequence.

[0116] In some embodiments, the mRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 75. In some embodiments, the mRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 75. In some embodiments, the mRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 77. In some embodiments, the mRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 77. In some embodiments, the mRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 79. In some embodiments, the mRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 79.In some embodiments, the mRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 81.

[0117] In some embodiments, the mRNA comprises the nucleic acid sequence shown in SEQ ID NO: 74. In some embodiments, the mRNA comprises the nucleic acid sequence shown in SEQ ID NO: 75. In some embodiments, the mRNA comprises the nucleic acid sequence shown in SEQ ID NO: 76. In some embodiments, the mRNA comprises the nucleic acid sequence shown in SEQ ID NO: 77. In some embodiments, the mRNA comprises the nucleic acid sequence shown in SEQ ID NO: 78. In some embodiments, the mRNA comprises the nucleic acid sequence shown in SEQ ID NO: 79. In some embodiments, the mRNA comprises the nucleic acid sequence shown in SEQ ID NO: 80. In some embodiments, the mRNA comprises the nucleic acid sequence shown in SEQ ID NO: 81.

[0118] On the other hand, this disclosure provides a polynucleotide comprising a nucleic acid sequence encoding an engineered wide range of nucleases described herein, which is used as a drug.

[0119] On the other hand, this disclosure provides a recombinant DNA construct comprising the polynucleotides described herein (i.e., comprising a nucleic acid sequence encoding the engineered wide range of nucleases described herein).

[0120] In some implementations, the recombinant DNA construct is plasmid DNA.

[0121] In some embodiments, the polynucleotide comprises: (a) a 5' ALB untranslated region (UTR) containing the nucleic acid sequence shown in SEQ ID NO: 18; (b) a nucleic acid sequence encoding an engineered wide-ranging nuclease; (c) a 3' SNRPB UTR containing the nucleic acid sequence shown in SEQ ID NO: 19; and (d) a polyA termination sequence. Preferably, the nucleic acid sequence is thymine-depleted and optimized for liver expression codons.

[0122] In some embodiments, the polynucleotide comprises: (a) a 5' ALB UTR comprising the nucleic acid sequence shown in SEQ ID NO: 18; (b) a Kozak sequence comprising the nucleic acid sequence shown in SEQ ID NO: 21; (c) a nucleic acid sequence encoding an engineered wide-ranging nuclease; (d) a 3' SNRPB UTR comprising the nucleic acid sequence shown in SEQ ID NO: 19; and (e) a polyA termination sequence. Preferably, the nucleic acid sequence is thymine-depleted and optimized for liver expression codons.

[0123] In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 67. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 67. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 69. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 69. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 71.In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 73. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 73.

[0124] In some embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 66. In some embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 67. In some embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 68. In some embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 69. In some embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 70. In some embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 71. In some embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 72. In some embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 73. In some embodiments, the recombinant DNA construct encodes a recombinant virus comprising the polynucleotide. In some embodiments, the recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant adeno-associated virus (AAV). In some embodiments, the recombinant virus is a recombinant AAV.

[0125] In some embodiments, the polynucleotide includes a promoter operatively linked to a nucleic acid sequence encoding a wide range of engineered nucleases. In some embodiments, the promoter is a liver-specific promoter.

[0126] On the other hand, this disclosure provides a recombinant DNA construct comprising the polynucleotides described herein (i.e., comprising nucleic acid sequences encoding the engineered wide range of nucleases described herein), which is used as a drug.

[0127] On the other hand, this disclosure provides a recombinant virus comprising the polynucleotides described herein (i.e., comprising a nucleic acid sequence encoding the engineered wide range of nucleases described herein).

[0128] In some implementations, the recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant AAV. In some implementations, the recombinant virus is a recombinant AAV.

[0129] In some embodiments, the polynucleotide includes a promoter operatively linked to a nucleic acid sequence encoding a wide range of engineered nucleases. In some embodiments, the promoter is a liver-specific promoter.

[0130] In another aspect, this disclosure provides a recombinant virus comprising the polynucleotides described herein (i.e., comprising a nucleic acid sequence encoding an engineered wide range of nucleases described herein), which is used as a drug.

[0131] In another aspect, this disclosure provides a lipid nanoparticle composition comprising lipid nanoparticles containing the polynucleotides described herein (i.e., containing nucleic acid sequences encoding the engineered wide range of nucleases described herein).

[0132] On the other hand, this disclosure provides the lipid nanoparticle compositions described herein for use as pharmaceuticals.

[0133] In another respect, this disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an engineered, wide-ranging nuclease as described herein.

[0134] In another respect, this disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the polynucleotide described herein (i.e., comprising a nucleic acid sequence encoding the engineered wide range of nucleases described herein).

[0135] In another respect, this disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant DNA construct as described herein (i.e., comprising the polynucleotides described herein).

[0136] In another respect, this disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant virus as described herein (i.e., comprising the polynucleotides described herein).

[0137] In another respect, this disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the lipid nanoparticle composition described herein.

[0138] On the other hand, this disclosure provides pharmaceutical compositions described herein for use as medicines.

[0139] On the other hand, this disclosure provides a host cell that contains the polynucleotides described herein (i.e., contains a nucleic acid sequence encoding the engineered wide range of nucleases described herein).

[0140] In another aspect, this disclosure provides a method for inactivating a polymerase (pol) gene of an HBV genome or an HBV genome fragment, the method comprising introducing into a eukaryotic cell containing an HBV genome or an HBV genome fragment: (a) a polynucleotide comprising a nucleic acid sequence encoding the engineered large-scale nuclease described herein, wherein the engineered large-scale nuclease is expressed in a eukaryotic cell; or (b) the engineered large-scale nuclease described herein; wherein the engineered large-scale nuclease generates a cleavage site within the pol gene at a recognition sequence comprising SEQ ID NO: 3 or thereof, wherein the pol gene is inactivated by introducing an insertion or deletion (indel) at the cleavage site, or by eliminating the HBV genome or the HBV genome fragment.

[0141] In some embodiments, the HBV genome or HBV genome fragment is contained via covalently closed circular DNA (cccDNA). In some embodiments, the cccDNA is eliminated after generation at the cleavage site. In some embodiments, the pol gene in the cccDNA is inactivated by introducing an indel at the cleavage site. In some embodiments, the indel is introduced via non-homologous end joining (NHEJ). In some embodiments, the inactivated pol gene does not encode active and / or full-length HBV polymerase protein.

[0142] In some embodiments, the HBV genome or HBV genome fragments are included in the genome of a eukaryotic cell. In some embodiments, the genome is a nuclear genome. In some embodiments, the genome is a mitochondrial genome. In some embodiments, the indel is introduced via NHEJ. In some embodiments, the inactivated pol gene does not encode active and / or full-length HBV polymerase protein.

[0143] In some implementations, the method inactivates the HBV S antigen (HBsAg) gene in the HBV genome or HBV genome fragments.

[0144] In some embodiments, the eukaryotic cells are human cells. In some embodiments, the human cells are liver cells. In some embodiments, the liver cells are hepatocytes.

[0145] In some implementations, the polynucleotide is the polynucleotide described herein (i.e., a nucleic acid sequence comprising an engineered wide range of nucleases described herein).

[0146] In some implementations, polynucleotides are introduced into eukaryotic cells via mRNA or recombinant viruses.

[0147] In some embodiments, the mRNA is the mRNA described herein. In some embodiments, the mRNA is introduced into eukaryotic cells by contacting eukaryotic cells with lipid nanoparticles containing the mRNA.

[0148] In some embodiments, the recombinant virus is the recombinant virus described herein. In some embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the recombinant AAV has the AAV8 serotype.

[0149] In another aspect, this disclosure provides a method for inactivating the pol gene of an HBV genome or an HBV genome fragment in target cells of a subject, the method comprising delivering to a target cell containing the HBV genome or an HBV genome fragment: (a) a polynucleotide comprising a nucleic acid sequence encoding the engineered large-scale nuclease described herein, wherein the engineered large-scale nuclease is expressed in the target cell; or (b) the engineered large-scale nuclease described herein; wherein the engineered large-scale nuclease generates a cleavage site within the pol gene at a recognition sequence comprising SEQ ID NO: 3 or thereof, wherein the pol gene is inactivated by introducing an indel at the cleavage site, or by eliminating the HBV genome or the HBV genome fragment.

[0150] In some embodiments, the HBV genome or HBV genome fragments are contained in cccDNA. In some embodiments, the cccDNA is eliminated after generation at the cleavage site. In some embodiments, the pol gene in the cccDNA is inactivated by introducing an indel at the cleavage site. In some embodiments, the indel is introduced via NHEJ. In some embodiments, the inactivated pol gene does not encode active and / or full-length HBV polymerase protein.

[0151] In some embodiments, the HBV genome or HBV genome fragments are included in the genome of the target cell. In some embodiments, the genome is a nuclear genome. In some embodiments, the genome is a mitochondrial genome. In some embodiments, the indel is introduced via NHEJ. In some embodiments, the inactivated pol gene does not encode active and / or full-length HBV polymerase protein.

[0152] In some implementations, the method inactivates the HBsAg gene in the HBV genome or HBV genome fragments. In some implementations, serum HBsAg concentrations are reduced in the subject.

[0153] In some embodiments, the target cells are liver cells. In some embodiments, the liver cells are hepatocytes.

[0154] In some implementations, the polynucleotide is the polynucleotide described herein (i.e., a nucleic acid sequence comprising an engineered wide range of nucleases described herein).

[0155] In some implementations, the polynucleotide is mRNA or is contained in the genome of the recombinant virus.

[0156] In some embodiments, the mRNA is the mRNA described herein. In some embodiments, the mRNA is delivered to target cells using lipid nanoparticles containing the mRNA. In some embodiments, the polynucleotides are delivered to target cells using a recombinant virus described herein that contains polynucleotides in its genome. In some embodiments, the recombinant virus is the recombinant virus described herein. In some embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the recombinant AAV has the AAV8 serotype.

[0157] In another aspect, this disclosure provides a method for treating HBV infection or diseases associated with hepatitis B virus infection, the method comprising delivering to target cells in a subject: (a) a therapeutically effective amount of a polynucleotide comprising a nucleic acid sequence encoding the engineered macronuclease described herein, wherein the engineered macronuclease is expressed in the target cells; or (b) a therapeutically effective amount of the engineered macronuclease described herein; wherein the target cells comprise an HBV genome or an HBV genome fragment containing a pol gene, wherein the engineered macronuclease generates a cleavage site within the pol gene at a recognition sequence comprising SEQ ID NO: 3 or thereof, wherein the pol gene is inactivated by introducing an indel at the cleavage site, or wherein the pol gene is inactivated by eliminating the HBV genome or an HBV genome fragment.

[0158] In some implementations, the disease is chronic hepatitis B. In some implementations, the disease is hepatocellular carcinoma. In some implementations, the disease is cirrhosis.

[0159] In some embodiments, the HBV genome or HBV genome fragments are contained in cccDNA. In some embodiments, the cccDNA is eliminated after generation at the cleavage site. In some embodiments, the pol gene in the cccDNA is inactivated by introducing an indel at the cleavage site. In some embodiments, the indel is introduced via NHEJ. In some embodiments, the inactivated pol gene does not encode active and / or full-length HBV polymerase protein.

[0160] In some embodiments, the HBV genome or HBV genome fragment is contained in the genome of the target cell. In some embodiments, the genome is a nuclear genome. In some embodiments, the genome is a mitochondrial genome. In some embodiments, the indel is introduced via NHEJ. In some embodiments, the inactivated pol gene does not encode active and / or full-length HBV polymerase protein. In some embodiments, the method inactivates the HBsAg gene in the HBV genome or HBV genome fragment.

[0161] In some implementations, serum HBsAg concentrations in subjects are reduced.

[0162] In some embodiments, the target cells are liver cells. In some embodiments, the liver cells are hepatocytes.

[0163] In some embodiments, the polynucleotide is a polynucleotide as described herein (i.e., a nucleic acid sequence comprising an engineered wide-ranging nuclease as described herein). In some embodiments, the polynucleotide is mRNA or is contained in the genome of a recombinant virus. In some embodiments, the mRNA is mRNA as described herein. In some embodiments, mRNA is delivered to target cells using lipid nanoparticles containing mRNA. In some embodiments, the polynucleotide is delivered to target cells using a recombinant virus as described herein that contains a polynucleotide in its genome. In some embodiments, the recombinant virus is a recombinant virus as described herein. In some embodiments, the recombinant virus is recombinant AAV. In some embodiments, the recombinant AAV has the AAV8 serotype.

[0164] In some implementations, the subject is also administered one, two, three, four or more additional therapeutic agents selected from the following: HBV combination therapy, HBV vaccine, HBV DNA polymerase inhibitor, immunomodulator, Toll-like receptor (TLR) modulator, interferon alpha receptor ligand, hyaluronidase inhibitor, HBV antigen inhibitor (e.g., HBV core antigen (HBcAg) inhibitor, HBV surface antigen (HBsAg) inhibitor, HBx inhibitor, HBV E antigen inhibitor), anti-HBV antigen antibody, HBV-targeting inhibitory nucleic acid (e.g., antisense oligonucleotide, short interfering RNA (siRNA), DNA-directed RNA interference (ddRNAi)), HBsAg secretion or assembly inhibitor, HBV virus entry inhibitor, immune checkpoint inhibitor, cytotoxic T lymphocyte-associated protein 4 (CTLA4) inhibitor, cyclin inhibitor, endonuclease modulator, ribonucleotide reductase inhibitor, covalently closed circular DNA. (cccDNA) inhibitors, farnesoid X receptor (FXR) agonists, STING agonists, anti-HBV antibodies, CCR2 chemokine antagonists, thymosin agonists, cytokines, nucleoprotein regulators, retinoic acid-induced gene 1 stimulators, NOD2 stimulators, phosphatidylinositol 3-kinase (PI3K) inhibitors, indoleamine-2,3-dioxygenase (IDO) pathway inhibitors, PD-1 inhibitors, PD-L1 inhibitors, recombinant thymosin α-1, Bruton's tyrosine kinase (BTK) inhibitors, KDM inhibitors, HBV replication inhibitors, arginase inhibitors, gene therapy and cell therapy, gene editors, cell therapy and TCR-T cell therapy.

[0165] In some implementations, the subject is also administered one, two, three, four, or more additional therapeutic agents selected from the following: 3-dioxygenase (IDO) inhibitors, apolipoprotein A1 modulators, arginase inhibitors, B and T lymphocyte attenuator inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, CCR2 chemokine antagonists, CD137 inhibitors, CD160 inhibitors, CD305 inhibitors, CD4 agonists and modulators, compounds targeting hepatitis B core antigen (HBcAg), core protein allosteric modulators, covalently closed circular DNA (cccDNA) inhibitors, cyclophilin inhibitors, cytotoxic T lymphocyte-associated protein 4 (CTLA4) inhibitors, DNA polymerase inhibitors, endonuclease modulators, epigenetic modifiers, farnesol X receptor (FXR) agonists, HBV DNA polymerase inhibitors, HBV replication inhibitors, and HBV... RNase inhibitors, HBV virus entry inhibitors, HBx inhibitors, hepatitis B large envelope protein modulators, hepatitis B large envelope protein stimulators, hepatitis B structural protein modulators, hepatitis B surface antigen (HBsAg) inhibitors, hepatitis B surface antigen (HBsAg) secretion or assembly inhibitors, hepatitis B virus e antigen inhibitors, hepatitis B virus replication inhibitors, hepatitis virus structural protein inhibitors, HIV-1 reverse transcriptase inhibitors, hyaluronidase inhibitors, inhibitors of apoptosis protein family (IAPs) proteins, IL-2 agonists, IL-7 agonists, immunomodulators, indoleamine-2 inhibitors, ribonucleotide reductase inhibitors, interleukin-2 ligands, ipi4 inhibitors, lysine demethylase inhibitors, histone demethylase inhibitors, KDM1 inhibitors, KDM5 inhibitors, cytotoxic cell lectin-like receptor G subfamily member 1 inhibitors, lymphocyte activation gene 3 inhibitors, etc. Toxin β receptor activator, Axl modulator, B7-H3 modulator, B7-H4 modulator, CD160 modulator, CD161 modulator, CD27 modulator, CD47 modulator, CD70 modulator, GITR modulator, HEVEM modulator, ICOS modulator, Mer modulator, NKG2A modulator, NKG2D modulator, OX40 modulator, SIRPα modulator, TIGIT modulator, Tim-4 modulator, Tyro modulator, Na+-taurocholate cotransporter (NTCP) inhibitor, natural killer cell receptor 2B4 inhibitor, NOD2 gene stimulator, nucleoprotein inhibitor, nucleoprotein modulator, OX-40 receptor agonist, PD-1 inhibitor, PD-L1 inhibitor, peptidylprolyl isomerase inhibitor, phosphatidylinositol-3 kinase (PI3K) inhibitor, retinoic acid-induced gene 1 stimulator, reverse transcriptase inhibitor, ribonuclease inhibitor, RNADNA polymerase inhibitors, SLC10A1 gene inhibitors, SMAC mimics, Src tyrosine kinase inhibitors, interferon gene stimulators (STING) agonists, NOD1 stimulators, T cell surface glycoprotein CD28 inhibitors, T cell surface glycoprotein CD8 regulators, thymosin agonists, thymosin α1 ligands, Tim-3 inhibitors, TLR-3 agonists, TLR-7 agonists, TLR-9 agonists, TLR9 gene stimulators, Toll-like receptor (TLR) regulators, and viral ribonucleotide reductase inhibitors.

[0166] In some implementations, the subject is also administered an antiviral drug or an immunomodulatory agent. In some implementations, the subject is also administered one or more drugs selected from the following: tenofovir disoproxil fumarate, tenofovir alafenamide, entecavir, buleviride, telbivudine, adefovir dipivoxil, lamivudine, pegylated interferon, and interferon-alpha. Attached Figure Description

[0167] Figure 1 The HBV 11-12 recognition sequence in the HBV gene. The HBV 11-12 recognition sequence targeted by the engineered wide-ranging nuclease disclosed herein contains two recognition halves. Each recognition halves contains nine base pairs separated by a four-base-pair central sequence. The HBV 11-12 recognition sequence (SEQ ID NO: 3) contains two recognition halves referred to as HBV11 and HBV12.

[0168] Figure 2 The engineered wide-range nuclease disclosed herein comprises two subunits, including a first subunit comprising an HVR1 region that binds to a first recognition half-site (e.g., HBV11), and a second subunit comprising an HVR2 region that binds to a second recognition half-site (e.g., HBV12). In embodiments in which the engineered wide-range nuclease is a single-stranded wide-range nuclease, the first subunit comprising the HVR1 region may be positioned as an N-terminal subunit or a C-terminal subunit. Similarly, the second subunit comprising the HVR2 region may be positioned as an N-terminal subunit or a C-terminal subunit.

[0169] Figure 3 Comparison of engineered large-scale nucleases of HBV 11-12L.1090QQ adapter 1923(1 / 2), HBV 11-12L.1090QE adapter 1923(1 / 2) and HBV 11-12L.1090QQ adapter 1.

[0170] Figures 4A-4E Evaluation of a wide range of engineered nucleases from parental HBV 11-12L.1090, including the linker 1923. Figure 4A ), connector 1766 ( Figure 4B), connector 1771 ( Figure 4C ), connector 1808 ( Figure 4D ) and connector 1814 ( Figure 4E The results of CHO reporter cell assays of HBV 11-12L.1090 variants with and without combinations of various amino acid modifications in the N-terminal and C-terminal subunits were compared with those of the mock and CHO 23-24 controls.

[0171] Figures 5A-5B The study provided data showing that HBV 11-12L.1090QQ adapter 1923 (1 / 2) and HBV 11-12L.1090QQ adapter 1 engineered wide-range nucleases were used in HepG2-sAg cells for 2 days. Figure 5A ) and 6 days ( Figure 5B The bar chart formed by indel when )

[0172] Figure 6 Bar graphs showing the percentage of indels of HBV11-12L.1090 broad-spectrum nucleases with adapter 1 or adapter 1923 on days 2 and 6, as measured by ddPCR, are provided at 100 ng and 10 ng doses. Broad-spectrum nucleases labeled Max1.0 utilize the pRNA6 vector and do not include uridine depletion in the RNA coding sequence; while those containing Max2.0 utilize the pRNA8 vector, which includes uridine depletion.

[0173] Figures 7A-7F Line graphs showing the percentage of indel and HBsAg inhibition by the indicated macronuclease at doses of 1 ng, 10 ng, or 100 ng at days 2, 6, and 9 post-transfection are provided. Four different macronucleases were tested in each graph. The first macronuclease was the HBV 11-12L.1090 macronuclease labeled QQ-linker 1923(1 / 2) with linker 1923. The second macronuclease tested was the HBV 11-12L.1090 macronuclease labeled QQ-linker 1 with linker 1. The third macronuclease tested was the HBV 11-12L.1090 macronuclease labeled QE-linker 1923(1 / 2) with linker 1923, which has a glutamine (Q) to glutamate (E) mutation at position 80 of the C-terminal subunit corresponding to I-CreI. The fourth type of broad-spectrum nuclease tested was the HBV11-12L.1090 broad-spectrum nuclease, labeled QE-linker 1, which has the same QE mutation but carries linker 1. An mCherry control at a 100 ng transfection dose is also shown. Figure 7A , Figure 7C and Figure 7EThe percentage of indels in HepG2-sAg cells transfected with 1 ng, 10 ng, or 100 ng of RNA for each indicated wide range of nucleases is provided. Figure 7B , Figure 7D and Figure 7F The percentage of HBsAg inhibition in HepG2-sAg cells transfected with RNA of each of the indicated wide range of nucleases is provided, at 1 ng, 10 ng, or 100 ng.

[0174] Figure 8A and Figure 8B The percentage of indels in HepG2-sAg cells transfected with HBV11-12L.1090 QQ-connector 1923(1 / 2) macronuclease (labeled QQ-connector 1923(1 / 2)) or HBV11-12L.1090 QE-connector 1923(1 / 2) macronuclease (labeled QE-connector 1923(1 / 2)) is provided. Figure 8A ) or HBsAg inhibition ( Figure 8B The dose-response curves are provided. EC50 and EC90 values ​​for indel or HBsAg are also provided for each of these curves based on a wide range of nucleases.

[0175] Figure 9 The results of an oligonucleotide capture assay identifying off-target cleavage induced by HBV11-12L.1090 QE-linker 1923(1 / 2) macronuclease (labeled QE-linker 1923(1 / 2)), HBV11-12L.1090 QQ-linker 1923(1 / 2) macronuclease (labeled QQ-linker 1923(1 / 2)), or an oligonucleotide control are provided in graphs. Experiments were performed in triplicate, and circles indicate target sites.

[0176] Figures 10A-10D A bar graph showing the results of MTA assays performed on HepG2-sAg cells transfected with the indicated wide range of nucleases is provided. Figure 10A MTA results are provided for HepG2-sAg cells transfected with a wide range of nucleases using HBV11-12L.1090QQ adapter 1. Figure 10B MTA results are provided for HepG2-sAg cells transfected with the HBV11-12L.1090QQ adapter 1923(1 / 2) wide range of nucleases. Figure 10C MTA results are provided for HepG2-sAg cells transfected with the HBV11-12L.1090QE adapter 1923(1 / 2) wide range of nucleases. Figure 10DComprehensive results of MTA assays for each wide range of nucleases are provided, showing the percentage edit at intermediate target sites compared to top off-target sites. In each figure, gray bars highlight the percentage edit at intermediate target sites, and black bars indicate off-target sites with a limit of detection (LOD) > 0.2%.

[0177] Figures 11A-11C The results of MTA assays performed on HepG2sAg or primitive PHH cells transfected with HBV 11-12L.1090QQ adapter 1 (labeled QQ adapter 1), HBV11-12L.1090QQ adapter 1923(1 / 2) (labeled QQ adapter 1923(1 / 2)), or HBV11-12L.1090QE adapter 1923(1 / 2) (labeled QE adapter 1923(1 / 2)) are provided, along with bar graphs showing the percentage of editing at target sites (black bars) or off-target sites (gray bars). Figure 11A The results show the on-target and off-target effects of the indicated wide-range nucleases transfected in HepG2sAg cells at limiting doses of 1 ng, 10 ng, and 100 ng. Figure 11B The results show the on-target and off-target effects of the indicated broad-spectrum nucleases transfected at EC50 or EC90 doses in HepG2sAg cells using Off-amp MTA assays or On-amp MTA assays. Figure 11C The results show the target and off-target effects of the indicated wide-range nucleases when transfected with limiting dose curves of 5 ng, 50 ng, or 500 ng in native PHH cells without HBV 11-12 target sites six days post-transfection.

[0178] Figure 12A and Figure 12B The results show that Huh-1 Dex cells transfected with HBV11-12L.1090QQ adapter 1923(1 / 2) macronuclease (labeled as HBV11-12L.1090QQ) at doses of 100 ng, 10 ng, or 1 ng and 10 ng are indicative of the results. Figure 12A ) or HepG2 cells ( Figure 12B Bar graphs showing the percentage of indels measured by ddPCR on days 3 and 6. Simulated and mCherry controls are also shown.

[0179] Figure 13A and Figure 13B The results show the effects of transfection of Huh-1 Dex cells (100 ng, 10 ng, or 1 ng) with the HBV11-12L.1090QE adapter 1923(1 / 2) macronuclease (labeled as HBV11-12L.1090 QE). Figure 13A ) or HepG2 cells ( Figure 13B The bar graph shows the percentage of indels measured by ddPCR on days 3 and 6. Simulated and mCherry controls are also shown.

[0180] Figure 14A and Figure 14B The results show that Huh-1 Dex cells transfected with HBV11-12L.1090QQ adapter 1923(1 / 2) macronuclease (labeled as HBV11-12L.1090 QQ) at doses of 100 ng, 10 ng, or 1 ng are indicative of the results. Figure 14A ) or HepG2 cells ( Figure 14B The image shows bar graphs of HBsAg levels (in IU / mL) on days 3 and 6. Simulated and mCherry controls are also shown.

[0181] Figure 15A and Figure 15B The results show the effects of HBV11-12L.1090QE adapter 1923(1 / 2) macronuclease (labeled as HBV11-12L.1090 QE) transfected into Huh-1 Dex cells at doses of 100 ng, 10 ng, or 1 ng and 10 ng. Figure 15A ) or HepG2 cells ( Figure 15B The image shows bar graphs of HBsAg levels (in IU / mL) on days 3 and 6. Simulated and mCherry controls are also shown.

[0182] Figures 16A to 16F The percentage of HBsAg inhibition is shown in PHH cells transfected with HBV serotype B infected with 800 GE / cell and on days 3 and 6 post-infection with 1.0 µg or 0.1 µg of engineered macronuclease encoding HBV 11-12L.1090QQ adapter 1923(1 / 2) mRNA (labeled as QQ adapter 1923(1 / 2) 1.0 µg or QQ adapter 1923(1 / 2) 0.1 µg). Figure 16A ), HBV DNA inhibition percentage ( Figure 16B ), HBV RNA inhibition percentage ( Figure 16C HBeAg inhibition percentage () Figure 16D ), percentage of cccDNA inhibition ( Figure 16E ) and percentage of cell viability ( Figure 16FThe bar graph shows the results. HBsAg, HBV DNA, HBV RNA, and HBeAg were measured on days 6, 9, 12, and 15 post-infection. cccDNA and cell viability levels were measured on days 9, 12, and 15 post-infection.

[0183] Figures 17A to 17F The percentage of cell viability shown is presented in PHH cells transfected with mRNA encoding a wide range of nucleases, ranging from 0.001 µg to 1 µg / mL, at 800 GE / cell and on days 3 and 6 post-infection, HBV serotype B. Figure 17A ), HBV DNA inhibition percentage ( Figure 17B ), HBV RNA inhibition percentage ( Figure 17C ), HBsAg inhibition percentage ( Figure 17D HBeAg inhibition percentage () Figure 17E ) and cccDNA inhibition percentage ( Figure 17F A bar chart.

[0184] Figure 18A and Figure 18B The code HBV 11-12L.1090QQ adapter 1923(1 / 2) (labeled QQ) is provided for HBV serotype B infection with 800 GE / cells and administered on days 3 and 6 post-infection at doses ranging from 0.001 µg to 1 µg. Figure 18A ) or HBV 11-12L.1090QE connector 1923(1 / 2) (marked as QE; Figure 18B DNA blotting at the cccDNA level in PHH cells transfected with mRNA from engineered large-scale nucleases. Figure 18A and Figure 18B In the diagram, each lane labeled TA1 or TA2 corresponds to a lane in which cells were transfected with the indicated dose of QQ or QE macronuclease, with or without the indicated dose of LAM. Lanes 11-13 provide additional controls: culture medium only, LAM 6nM control, and HBV-targeted siRNA control, respectively. HBV fragment DNA is present in... Figure 18A and Figure 18B Lanes 1 and 14 are available. Figure 18A An additional cccDNA positive loading control was found in lane 15.

[0185] Figure 19Bar graphs are provided showing the percentage of cell viability in PHH cells infected with HBV serotype B at 800 GE / cell and transfected on days 3 and 6 post-infection with mRNA encoding HBV 11-12L.1090QQ adapter 1923(1 / 2) (labeled QQ) or HBV 11-12L.1090QE adapter 1923(1 / 2) (labeled QE) engineered wide range of nucleases, alone or in combination with the nucleoside analog LAM.

[0186] Figures 20A to 20D The percentage of HBV DNA inhibition is shown in PHH cells transfected with mRNA of either HBV serotype B, 800 GE / cell, and engineered with 0.1 µg of HBV 11-12L.1090QQ adapter 1923(1 / 2) (labeled QQ) or HBV 11-12L.1090QE adapter 1923(1 / 2) (labeled QE), on days 3 and 6 post-infection. Figure 20A ), HBsAg inhibition percentage ( Figure 20B HBeAg inhibition percentage () Figure 20C ) and HBV RNA inhibition percentage ( Figure 20D The line graph shows that some cells were also treated with nucleoside analogues LAM or HBV-targeted siRNA.

[0187] Brief description of the sequence SEQ ID NO: 1 shows the amino acid sequence of the wild-type I-CreI large-scale nuclease from Chlamydomonas reinhardtii.

[0188] SEQ ID NO: 2 shows the amino acid sequence of the LAGLIDADG motif.

[0189] SEQ ID NO: 3 shows the nucleic acid sequence of the sense strand of the HBV 11-12 recognition sequence.

[0190] SEQ ID NO: 4 shows the nucleic acid sequence of the antisense strand of the HBV 11-12 recognition sequence.

[0191] SEQ ID NO: 5 shows the amino acid sequence of the HBV 11-12L.1090QQ adapter 1923(1 / 2) wide range nuclease.

[0192] SEQ ID NO: 6 shows the amino acid sequence of the HBV 11-12L.1090QE adapter 1923(1 / 2) wide range nuclease.

[0193] SEQ ID NO: 7 shows the amino acid sequence of the HBV11 subunit of the HBV 11-12L.1090QQ linker 1923(1 / 2) wide range nuclease.

[0194] SEQ ID NO: 8 shows the amino acid sequence of the HBV11 subunit of the HBV 11-12L.1090QE linker 1923(1 / 2) wide range nuclease.

[0195] SEQ ID NO: 9 shows the amino acid sequence of the HBV12 subunit of the HBV 11-12L.1090QQ linker 1923(1 / 2) wide range nuclease.

[0196] SEQ ID NO: 10 shows the amino acid sequence of the HBV12 subunit of the HBV 11-12L.1090QE linker 1923(1 / 2) wide range nuclease.

[0197] SEQ ID NO: 11 shows the nucleic acid sequence of the HBV 11-12L.1090QQ adapter 1923(1 / 2) wide range nuclease.

[0198] SEQ ID NO: 12 shows the nucleic acid sequence of the HBV 11-12L.1090QE adapter 1923(1 / 2) wide range nuclease.

[0199] SEQ ID NO: 13 shows the amino acid sequence of the HBV 11-12L.1090QQ linker 1 wide range of nucleases.

[0200] SEQ ID NO: 14 shows the amino acid sequence of the HBV 11-12L.1090QE linker 1 wide range of nucleases.

[0201] SEQ ID NO: 15 shows the amino acid sequence of linker 1923.

[0202] SEQ ID NO: 16 shows the amino acid sequence of linker 1.

[0203] SEQ ID NO: 17 shows the amino acid sequence of the SV40 nuclear localization signal (NLS).

[0204] SEQ ID NO: 18 shows the nucleic acid sequence of the 5' ALB untranslated region (UTR).

[0205] SEQ ID NO: 19 shows the nucleic acid sequence of the 3' SNRPB UTR.

[0206] SEQ ID NO: 20 shows the nucleic acid sequence of the polyA termination sequence.

[0207] SEQ ID NO: 21 shows the nucleic acid sequence of the Kozak sequence.

[0208] SEQ ID NO: 22 shows the nucleic acid sequence of the probe sequence.

[0209] SEQ ID NO: 23 shows the nucleic acid sequence of the forward primer sequence.

[0210] SEQ ID NO: 24 shows the nucleic acid sequence of the reverse primer sequence.

[0211] SEQ ID NO: 25 shows the nucleic acid sequence of the probe sequence.

[0212] SEQ ID NO: 26 shows the nucleic acid sequence of the forward primer sequence.

[0213] SEQ ID NO: 27 shows the nucleic acid sequence of the reverse primer sequence.

[0214] SEQ ID NO: 28 shows the nucleic acid sequence of the reverse primer sequence.

[0215] SEQ ID NO: 29 shows the nucleic acid sequence of the probe sequence.

[0216] SEQ ID NO: 30 shows the nucleic acid sequence of the forward primer sequence.

[0217] SEQ ID NO: 31 shows the nucleic acid sequence of the reverse primer sequence.

[0218] SEQ ID NO: 32 shows the nucleic acid sequence of the probe sequence.

[0219] SEQ ID NO: 33 shows the nucleic acid sequence of the forward primer sequence.

[0220] SEQ ID NO: 34 shows the nucleic acid sequence of the reverse primer sequence.

[0221] SEQ ID NO: 35 shows the nucleic acid sequence of the probe sequence.

[0222] SEQ ID NO: 36 shows the nucleic acid sequence of the forward primer sequence.

[0223] SEQ ID NO: 37 shows the nucleic acid sequence of the reverse primer sequence.

[0224] SEQ ID NO: 38 shows the nucleic acid sequence of the forward primer sequence.

[0225] SEQ ID NO: 39 shows the nucleic acid sequence of the reverse primer sequence.

[0226] SEQ ID NO: 40 shows the nucleic acid sequence of the forward primer sequence.

[0227] SEQ ID NO: 41 shows the nucleic acid sequence of the reverse primer sequence.

[0228] SEQ ID NO: 42 shows the amino acid sequence of linker 1766.

[0229] SEQ ID NO: 43 shows the amino acid sequence of linker 1771.

[0230] SEQ ID NO: 44 shows the amino acid sequence of linker 1808.

[0231] SEQ ID NO: 45 shows the amino acid sequence of linker 1814.

[0232] SEQ ID NO: 46 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing linker 1923 and without subunit modification.

[0233] SEQ ID NO: 47 shows the amino acid sequence of the HBV11-12L.1090 engineered wide-range nuclease containing the 1923 linker and group 1 (K96A / K57Y, E61T) subunits modified.

[0234] SEQ ID NO: 48 shows the amino acid sequence of the HBV11-12L.1090 engineered wide-range nuclease containing the 1923 adapter and group 2 (Q99A, K100D / none) subunit modifications.

[0235] SEQ ID NO: 49 shows the amino acid sequence of the HBV 11-12L.1090 engineered wide range of nucleases containing the 1923 linker and group 3 (H37Y / W53F) subunit modifications.

[0236] SEQ ID NO: 50 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing linker 1923 and subunits of group 1 (K96A / K57Y, E61T) and group 3 (H37Y / W53F).

[0237] SEQ ID NO: 51 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing adapter 1923 and group 2 (Q99A, K100D / none) and group 3 (H37Y / W53F) subunit modifications.

[0238] SEQ ID NO: 52 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing adapter 1923 and subunits modified with group 1 (K96A / K57Y, E61T), group 2 (Q99A, K100D / none), and group 3 (H37Y / W53F).

[0239] SEQ ID NO: 53 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing linker 1766 and without subunit modification.

[0240] SEQ ID NO: 54 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing adapter 1766 and subunits of group 1 (K96A / K57Y, E61T) and group 2 (Q99A, K100D / none) modified.

[0241] SEQ ID NO: 55 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing adapter 1766 and subunits modified with group 1 (K96A / K57Y, E61T), group 2 (Q99A, K100D / none), and group 3 (H37Y / W53F).

[0242] SEQ ID NO: 56 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing linker 1771 and without subunit modification.

[0243] SEQ ID NO: 57 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing adapter 1771 and subunits of group 1 (K96A / K57Y, E61T) and group 2 (Q99A, K100D / none) modified.

[0244] SEQ ID NO: 58 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing adapter 1771 and subunits modified with group 1 (K96A / K57Y, E61T), group 2 (Q99A, K100D / none), and group 3 (H37Y / W53F).

[0245] SEQ ID NO: 59 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing linker 1808 and without subunit modification.

[0246] SEQ ID NO: 60 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing adapter 1808 and subunits of group 1 (K96A / K57Y, E61T) and group 2 (Q99A, K100D / none) modified.

[0247] SEQ ID NO: 61 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing adapter 1808 and subunits modified with group 1 (K96A / K57Y, E61T), group 2 (Q99A, K100D / none) and group 3 (H37Y / W53F).

[0248] SEQ ID NO: 62 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing linker 1814 and without subunit modification.

[0249] SEQ ID NO: 63 shows the amino acid sequence of the HBV 11-12L.1090 engineered wide range of nucleases containing adapter 1814 and subunits modified with group 1 (K96A / K57Y, E61T) and group 2 (Q99A, K100D / none).

[0250] SEQ ID NO: 64 shows the amino acid sequence of an HBV 11-12L.1090 engineered wide range of nucleases containing adapter 1814 and subunits modified with group 1 (K96A / K57Y, E61T), group 2 (Q99A, K100D / none), and group 3 (H37Y / W53F).

[0251] SEQ ID NO: 65 shows the amino acid sequence of the SV40 nuclear localization signal (NLS).

[0252] SEQ ID NO: 66 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QQ adapter 1923(1 / 2) wide range of nucleases.

[0253] SEQ ID NO: 67 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QQ adapter 1923(1 / 2) wide range of nucleases.

[0254] SEQ ID NO: 68 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QQ adapter 1923(1 / 2) wide range of nucleases.

[0255] SEQ ID NO: 69 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QQ adapter 1923(1 / 2) wide range of nucleases.

[0256] SEQ ID NO: 70 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QE adapter 1923(1 / 2) wide range of nucleases.

[0257] SEQ ID NO: 71 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QE adapter 1923(1 / 2) wide range of nucleases.

[0258] SEQ ID NO: 72 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QE adapter 1923(1 / 2) wide range of nucleases.

[0259] SEQ ID NO: 73 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QE adapter 1923(1 / 2) wide range of nucleases.

[0260] SEQ ID NO: 74 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QQ adapter 1923(1 / 2) wide range of nucleases.

[0261] SEQ ID NO: 75 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QQ adapter 1923(1 / 2) wide range of nucleases.

[0262] SEQ ID NO: 76 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QQ adapter 1923(1 / 2) wide range of nucleases.

[0263] SEQ ID NO: 77 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QQ adapter 1923(1 / 2) wide range of nucleases.

[0264] SEQ ID NO: 78 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QE adapter 1923(1 / 2) wide range of nucleases.

[0265] SEQ ID NO: 79 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QE adapter 1923(1 / 2) wide range of nucleases.

[0266] SEQ ID NO: 80 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QE adapter 1923(1 / 2) wide range of nucleases.

[0267] SEQ ID NO: 81 shows the nucleic acid sequence of the mRNA construct encoding the HBV 11-12L.1090QE adapter 1923(1 / 2) wide range of nucleases.

[0268] SEQ ID NO: 82 shows the nucleic acid sequence of the 5' ALB untranslated region (UTR).

[0269] SEQ ID NO: 83 shows the nucleic acid sequence of the 3' SNRPB UTR.

[0270] SEQ ID NO: 84 shows the nucleic acid sequence of the Kozak sequence. Detailed Implementation

[0271] 1.1 References and Definitions The patents and scientific literature mentioned herein establish knowledge available to those skilled in the art. Granted U.S. patents, granted applications, published foreign applications, and references, including those from the GenBank database sequence, cited herein are incorporated by reference to the extent that each is specifically and individually indicated for inclusion by reference.

[0272] This invention may be embodied in various forms and should not be construed as limited to the embodiments listed herein. Rather, these embodiments are provided to make this disclosure complete and full, and to fully convey the scope of this disclosure to those skilled in the art. For example, features shown with respect to one embodiment may be incorporated into other embodiments, and features shown with respect to a particular embodiment may be removed from that embodiment. Furthermore, many variations and additions to the embodiments set forth herein will be apparent to those skilled in the art without departing from this disclosure.

[0273] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. The terminology used herein to describe this disclosure is for the purpose of describing particular embodiments only and is not intended to limit this disclosure.

[0274] All publicly available publications, patent applications, patents, and other references mentioned in this article are incorporated herein by reference in their entirety.

[0275] As used herein, “a / an” or “the” can refer to one or more. For example, “a” cell can refer to a single cell or multiple cells.

[0276] As used herein, unless otherwise specifically indicated, the word “or” is used in the inclusive sense of “and / or” rather than in the exclusive sense of “any / or”.

[0277] As used herein, the terms “nuclease” and “endonuclease” are used interchangeably to refer to naturally occurring or engineered enzymes that cleave phosphodiester bonds within a polynucleotide chain.

[0278] As used herein, the terms “cleavage” or “cleavage” refer to the hydrolysis of phosphodiester bonds within the target sequence’s recognition sequence backbone, which results in a double-strand break within the target sequence, referred to herein as the “cleavage site”.

[0279] As used herein, the term "vast nuclease" refers to a nuclease that binds to double-stranded DNA at a recognition sequence of more than 12 base pairs. In some embodiments, the recognition sequence for the vast nuclease used in this disclosure is 22 base pairs. A vast nuclease may be a nuclease derived from I-CreI (SEQ ID NO:1) and may refer to an engineered variant of I-CreI modified relative to native I-CreI in, for example, DNA binding specificity, DNA cleavage activity, DNA binding affinity, or dimerizing properties. Methods for generating such modified I-CreI variants are known in the art (e.g., WO2007 / 047859, incorporated herein by reference in its entirety). As used herein, a vast nuclease binds to double-stranded DNA as a heterodimer. A vast nuclease may also be a "single-stranded vast nuclease" in which a pair of DNA-binding domains are linked into a single polypeptide using a peptide linker. The term "homing nuclease" is synonymous with the term "vast nuclease." When expressed in the target cells described herein, the broad range of nucleases disclosed herein are substantially nontoxic, allowing cells to be transfected and maintained at 37°C without observed adverse effects on cell viability or a significant reduction in broad range nuclease cleavage activity (when measured using the methods described herein).

[0280] As used herein, the term "single-stranded macronuclease" refers to a polypeptide containing a pair of nuclease subunits linked by a linker. A single-stranded macronuclease is organized as follows: N-terminal subunit – linker – C-terminal subunit. The two macronuclease subunits are typically different in amino acid sequence and bind to different DNA sequences. Therefore, single-stranded macronucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences. A single-stranded macronuclease may be referred to as a "single-stranded heterodimer" or "single-stranded heterodimeric macronuclease," although it is not actually a dimer. For clarity, unless otherwise stated, the term "macronuclease" may refer to either a dimer or a single-stranded macronuclease.

[0281] As used herein, the term "linker" refers to a foreign peptide sequence used to link two broadly defined nuclease subunits into a single polypeptide. Linkers may tend to form specific three-dimensional structures under physiological conditions, such as turns or coils. In some embodiments, the linker is 31 amino acids in length. In some embodiments, the linker may have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 15.

[0282] As used herein, with respect to proteins, the terms "recombinant" or "engineered" refer to proteins with altered amino acid sequences resulting from the application of genetic engineering techniques to the nucleic acids encoding the protein and the cells or organisms expressing the protein. With respect to nucleic acids, the terms "recombinant" or "engineered" refer to nucleic acid sequences with altered sequences resulting from the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning; transfection, transformation, and other gene transfer techniques; homologous recombination; site-directed mutagenesis; and gene fusion. According to this definition, proteins having the same amino acid sequence as naturally occurring proteins but produced through cloning and expression in a heterologous host are not considered recombinant or engineered.

[0283] As used herein, the term "wildtype" refers to the most common naturally occurring allele (i.e., polynucleotide sequence) in a population of alleles of the same type of gene, wherein the polypeptide encoded by the wild-type allele has its original function. The term "wildtype" also refers to a polypeptide encoded by a wild-type allele. Wild-type alleles (i.e., polynucleotides) and polypeptides are distinguishable from mutant or variant alleles and polypeptides that contain one or more mutations and / or substitutions relative to the wild-type sequence. Given that wild-type alleles or polypeptides can confer a normal phenotype to an organism, in some cases, mutant or variant alleles or polypeptides can confer an altered phenotype. Wild-type nucleases are distinguishable from recombinant or non-naturally occurring nucleases. The term "wildtype" can also refer to cells, organisms, and / or subjects possessing a wild-type allele of a specific gene, or cells, organisms, and / or subjects used for comparative purposes.

[0284] As used herein, the term "genetically modified" refers to a cell or organism in which the genomic DNA sequence of itself or its ancestor has been intentionally modified by recombination technology. As used herein, the term "genetically modified" includes the term "transgenic".

[0285] As used herein, the term “modification” in relation to recombinant proteins refers to any insertion, deletion, or substitution of amino acid residues in the recombinant sequence relative to a reference sequence (e.g., wild-type or natural sequence).

[0286] As used herein, the term "recognition sequence" or "recognition site" refers to the DNA sequence that is bound and cleaved by a nuclease. In the case of macronucleases, the recognition sequence comprises a pair of inverted 9-base-pair "half-sites" separated by 4 base pairs. In the case of single-stranded macronucleases, the N-terminal domain of the protein contacts the first half-site, and the C-terminal domain of the protein contacts the second half-site. Cleavage by macronucleases produces a 3' "overhang" of four base pairs. "Overhangs" or "sticky ends" are short single-stranded DNA fragments that can be produced by cleavage of double-stranded DNA sequences by endonucleases. In the case of macronucleases derived from I-CreI and single-stranded macronucleases, the overhang contains 22 base pairs of the recognition sequence (10⁻¹³).

[0287] As used herein, the terms “target site” or “target sequence” refer to a region of cellular chromosomal DNA that contains the recognition sequence of a nuclease.

[0288] As used herein, the term "DNA binding affinity" or "binding affinity" refers to the tendency of a broad range of nucleases to non-covalently associate with a reference DNA molecule (e.g., a recognition sequence or any sequence). Binding affinity is measured by the dissociation constant Kd. As used herein, a nuclease has an "altered" binding affinity if the percentage change in Kd of the nuclease relative to the reference recognition sequence is statistically significant.

[0289] As used herein, the term "specificity" refers to the ability of a nuclease to recognize and cleave a double-stranded DNA molecule only at a specific base pair sequence, or only at a specific set of recognition sequences. This set of recognition sequences shares certain conserved positions or sequence motifs, but may be degenerate at one or more positions. Highly specific nucleases are capable of cleaving only one or a very small number of recognition sequences. Specificity can be determined by any method known in the art, such as unbiased DSB identification via sequencing (GUIDE-seq), oligonucleotide capture assays, whole-genome sequencing, and long-range next-generation sequencing of recognition sequences. In some embodiments, specificity is measured using GUIDE-seq. As used herein, "specificity" is synonymous with a low incidence of cleavage of sequences other than the target sequence (non-target sequences) (i.e., off-target cleavage). Low rates of off-target cutting can include off-target sequence cutting rates of less than 25%, less than 20%, less than 18%, less than 15%, less than 12.5%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.75%, less than 0.5%, or less than 0.25%.

[0290] As used herein, a macronuclease is considered "altered" specific if, under physiological conditions, it binds to and cleaves a recognition sequence that is not bound to and cleaved by a reference macronuclease (e.g., wild-type), or if the cleavage rate of the recognition sequence increases or decreases by a biologically significant amount (e.g., at least 2×, or 2×–10×) relative to the reference macronuclease.

[0291] In some embodiments, the engineered broad-spectrum nuclease disclosed herein exhibits improved (i.e., enhanced) specificity for the target recognition sequence (i.e., HBV 11-12) containing SEQ ID NO: 3, compared to the HBV 11-12 L.1090QQ adapter 1 broad-spectrum nuclease (whose amino acid sequence is shown in SEQ ID NO: 13). Therefore, in some embodiments, the engineered broad-spectrum nuclease disclosed herein exhibits reduced off-target cleavage compared to the HBV 11-12 L.1090QQ adapter 1 broad-spectrum nuclease. Off-target cleavage of a wide range of nucleases can be measured using any method known in the art, including, for example, oligonucleotide capture assays described herein, T7 endonuclease (T7E) assays described herein, digital PCR described herein, targeted sequencing of specific off-target sites, exome sequencing, whole-genome sequencing, direct in situ break marker enrichment and next-generation sequencing (BLESS) on streptavidin, whole-genome GUIDE-seq, and linear amplification-mediated high-throughput whole-genome translocation sequencing (LAM-HTGTS) (see, for example, Zischewski et al. (2017), Biotechnology Advances 35(1):95-104, the full text of which is incorporated herein by reference).

[0292] As used herein, the term “cleavage efficiency” refers to the rate at which a macronuclease cleaves the recognition sequence in a double-stranded DNA molecule relative to the rate at which all cleavage events occur on the DNA molecule. “Cleavage efficiency” is synonymous with DNA editing efficiency or mid-target editing. The cleavage and / or indel formation efficiency of a macronuclease can be measured using any method known in the art, including T7E assay, digital PCR (ddPCR), mismatch detection assay, mismatch cleavage assay, high-resolution melting analysis (HRMA), heteroduplex mobility assay, sequencing, and fluorescent PCR capillary gel electrophoresis (see, for example, Zischewski et al. (2017) Biotechnology Advances 35(1):95-104, the full text of which is incorporated herein by reference). In some embodiments, cleavage efficiency is measured by ddPCR. In some embodiments, the disclosed macronuclease produces a cleavage efficiency of at least about 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% at the recognition sequence.

[0293] As used herein, "indel" refers to the insertion or deletion of a nucleus base in a nucleic acid (such as DNA). In some embodiments, it is desirable to generate one or more insertions or deletions (i.e., indels) in nucleic acids, such as in exogenous nucleic acids like viral DNA. Therefore, as used herein, "indel formation efficiency" refers to the rate at which a macronuclease generates one or more indels by cleaving a recognition sequence relative to the rate at which all cleavage events occur on a DNA molecule. In some embodiments, indel formation efficiency is measured by ddPCR. In some embodiments, the disclosed macronuclease generates at least about 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% indel formation efficiency at the recognition sequence. The disclosed wide range of nucleases can produce at least approximately 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% cleavage efficiency and / or indel formation efficiency at the recognition sequence.

[0294] As used herein, the term “homological recombination” or “HR” refers to the natural cellular process in which a homologous DNA sequence is used as a repair template to repair double-stranded DNA breaks (see, for example, Cahill et al., (2006) Front. Biosci. 11:1958-1976). The homologous DNA sequence can be an endogenous chromosomal sequence or an exogenous nucleic acid delivered to the cell.

[0295] As used herein, the term “non-homologous end joining” or “NHEJ” refers to the natural cellular process in which double-strand DNA breaks are repaired by directly joining two non-homologous DNA fragments (see, for example, Cahill et al., (2006) Front. Biosci. 11:1958–1976). DNA repair via NHEJ is error-prone and often results in non-template additions or deletions of the DNA sequence at the repair site. In some cases, cleavage at the target recognition sequence leads to NHEJ at the target recognition site. Nuclease-induced cleavage at the target site in the gene coding sequence followed by DNA repair via NHEJ can introduce mutations that disrupt gene function into the coding sequence, such as frameshift mutations. Therefore, engineered large-scale nucleases can be used to efficiently knock out genes in cellular populations.

[0296] As used herein, the term "homologous arm" or "sequence homologous to a flanking sequence of a macronuclease cleavage site" refers to the 5' and 3' flanking sequences of a nucleic acid molecule that facilitate insertion of the nucleic acid molecule into a cleavage site generated by a macronuclease. Generally, the length of a homologous arm can be at least 50 base pairs, preferably at least 100 base pairs, and at most 2000 base pairs or more, and can have at least 90%, preferably at least 95%, or higher sequence homology with its corresponding sequence in the genome. In some embodiments, the homologous arm is about 500 base pairs.

[0297] As used in this paper regarding both amino acid sequences and nucleic acid sequences, the terms "percentage identity," "sequence identity," "percentage similarity," and "sequence similarity" refer to a measure of the degree of similarity between two sequences based on a sequence alignment that maximizes the similarity between aligned amino acid residues or nucleotides. This similarity varies with the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs can be used to determine sequence similarity using standard parameters. As used herein, sequence similarity was measured using the BLASTp procedure for amino acid sequences and the BLASTn procedure for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ) and are described, for example, in: Altschul et al., (1990) J.Mol. Biol. 215:403-410; Gish and States (1993) Nature Genet. 3:266-272; Madden et al., (1996) Meth. Enzymol. 266:131-141; Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402; and Zhang et al., (2000) J. Comput. Biol. 7(1-2):203-14. As used in this paper, the percentage similarity of two amino acid sequences is based on scores from the following parameters of the BLASTp algorithm: word length = 3; gap opening penalty = -11; gap extension penalty = -1; and score matrix = BLOSUM62. As used in this paper, the percentage similarity of two nucleic acid sequences is based on scores from the following parameters of the BLASTn algorithm: word length = 11; gap opening penalty = -5; gap extension penalty = -2; match reward = 1; and mismatch penalty = -3.

[0298] As used herein with respect to modifications of two protein or amino acid sequences, the term "corresponds to" indicates that a specified modification in the first protein is a substitution of the same amino acid residue in a modification of the second protein, and that when the two proteins are subjected to standard sequence alignment (e.g., using the BLASTp procedure) and alignment for maximum sequence identity across the entire subunit or protein, the amino acid position of the modification in the first protein corresponds to or aligns with the amino acid position of the modification in the second protein. Therefore, if residues X and Y correspond to each other in sequence alignment, even though X and Y may be located at different positions relative to the N-terminus or C-terminus, a modification of residue "X" to amino acid "A" in the first protein will correspond to a modification of residue "Y" to amino acid "A" in the second protein.

[0299] As used herein, the terms “recognition half-site”, “recognition sequence half-site”, or simply “half-site”, refer to a nucleic acid sequence in a double-stranded DNA molecule that is recognized and bound by a monomer of a homodimer or heterodimer macronuclease, or by a subunit of a single-stranded macronuclease, or by a subunit of a single-stranded macronuclease.

[0300] As used herein, the term "hypervariate region" refers to a local sequence within a macronuclease monomer or subunit containing amino acids with relatively high variability. The hypervariate region may contain about 50-60 consecutive residues, about 53-57 consecutive residues, or preferably about 56 residues. In some embodiments, the residues of the hypervariate region may correspond to positions 24-79 or 204-259 of SEQ ID NO: 5 or 6. The hypervariate region may contain one or more residues that contact DNA bases in the recognition sequence and may be modified to alter the base preference of the monomer or subunit. When the macronuclease binds to the double-stranded DNA recognition sequence, the hypervariate region may also contain one or more residues that bind to the DNA backbone. These residues may be modified to alter the binding affinity of the macronuclease to the DNA backbone and the target recognition sequence. In various embodiments, the hypervariate region may contain 1-20 residues exhibiting variability and may be modified to affect base preference and / or DNA binding affinity. In a particular implementation, the hypervariable region contains approximately 15-20 residues that exhibit variability and can be modified to affect base preference and / or DNA binding affinity.

[0301] In some embodiments, the variable residues within the hypervariable region correspond to one or more of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 5 or 6. In some embodiments, the variable residues within the hypervariable region correspond to position 51 of SEQ ID NO: 5 or 6. In some embodiments, the variable residues within the hypervariable region correspond to one or more of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 51, 68, 70, 75, and 77 of SEQ ID NO: 5 or 6.

[0302] In some embodiments, the variable residues within the hypervariable region correspond to one or more of positions 204, 206, 208, 210, 212, 213, 218, 220, 222, 224, 226, 248, 250, 255, and 257 of SEQ ID NO: 5 or 6. In some embodiments, the variable residues within the hypervariable region further correspond to one or more of positions 237, 241, 251, 252, and 253 of SEQ ID NO: 5 or 6. In other embodiments, the variable residues within the hypervariable region correspond to one or more of positions 204, 206, 208, 210, 212, 213, 218, 220, 222, 224, 226, 237, 241, 248, 250, 251, 252, 253, 255, and 257 of SEQ ID NO: 5 or 6.

[0303] In this document, the terms "recombinant DNA construct," "recombinant construct," "expression cassette," "cassette," "expression construct," "chimeric construct," "construct," and "recombinant DNA fragment" are used interchangeably and refer to single-stranded or double-stranded polynucleotides. A recombinant construct comprises an artificial combination of nucleic acid fragments, including but not limited to regulatory and coding sequences not found together in nature. For example, a recombinant DNA construct may contain regulatory and coding sequences derived from different sources, or from the same source but arranged in a manner different from that found in nature. Such constructs can be used alone or in combination with a vector.

[0304] As used herein, "vector" or "recombinant DNA vector" can be a construct comprising a replication system and a sequence capable of transcribing and translating a polypeptide-coding sequence in a given host cell. If a vector is used, the choice of vector depends on methods well known to those skilled in the art for transforming host cells. Vectors may include, but are not limited to, plasmid vectors and recombinant AAV vectors, or any other vectors known in the art suitable for delivering genes to target cells. Genetic elements that must be present on a vector are well known to those skilled in the art for successful transformation, selection, and amplification of host cells containing any isolated nucleotide or nucleic acid sequences described herein. In some embodiments, "vector" also refers to a viral vector. Viral vectors may include, but are not limited to, retroviral vectors, lentiviral vectors, adenovirus vectors, and adeno-associated virus vectors (AAV).

[0305] As used herein, a “polycistronic” mRNA refers to a single messenger RNA containing two or more coding sequences (i.e., cistrons) and encoding more than one protein. Polycistronic mRNAs may contain any element known in the art that allows translation of two or more genes from the same mRNA molecule, including, but not limited to, IRES elements, T2A elements, P2A elements, E2A elements, and F2A elements.

[0306] As used herein, the term "operably linked" is intended to refer to a functional connection between two or more elements. For example, an operably linked nucleic acid sequence encoding the nuclease described herein and a regulatory sequence (e.g., a promoter) is a functional connection that allows the expression of the nucleic acid sequence encoding the nuclease. Operatically linked elements can be contiguous or non-contiguous. When used to refer to the connection of two protein-coding regions, "operably linked" is intended to mean that the coding regions are in the same reading frame.

[0307] As used herein, "uridine depletion" refers to the removal or replacement of uridine residues from an RNA molecule (e.g., mRNA) or the removal or replacement of thymine residues from a DNA molecule encoding the RNA molecule. Urate can be replaced by any other residue (such as adenosine or pseudouridine) in any region of the RNA molecule, but in some embodiments, uridine is removed and replaced in the non-coding regions of the RNA molecule. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the uridine in the RNA molecule is removed or replaced.

[0308] As used herein, “codon optimization” refers to coding sequences modified to improve gene expression and translation efficiency by adapting to codon bias in a host organism or tissue, thereby mitigating species- and tissue-specific limitations associated with codon use and transfer RNA (tRNA) abundance. Codon-optimized coding sequences can be designed for expression in mammalian cells and / or specific tissues. It has been shown that the codon use bias of genes specifically expressed in the human liver differs from that of the coding DNA sequences in the human genome (Dittmar et al. (2006)). PLoS Genet. 2:e221), in some implementations, the sequence encoding the engineered wide-range nuclease described herein is optimized for expression in the liver.

[0309] As used herein, “inactivation” of a gene refers to the introduction of a mutation into a gene such that the resulting inactivated gene no longer encodes an active and / or full-length protein, or the gene is eliminated (i.e., degraded). The HBV genome or fragments thereof can be eliminated by subsequent degradation of the genome or genome fragments through cleavage, thereby inactivating any HBV genes present in the HBV genome or fragments thereof.

[0310] As used herein, “control” or “control cell” refers to a cell that provides a baseline for measuring changes in the genotype or phenotype of a genetically modified cell. Control cells may include, for example: (a) wild-type cells, i.e., cells with the same genotype as the starting material used to induce the genetic alteration in the genetically modified cell; (b) cells with the same genotype as the genetically modified cell but transformed with a null construct (i.e., a construct that has no known effect on the target trait); or (c) cells that are genetically identical to the genetically modified cell but not exposed to conditions or stimuli that induce the expression of the altered genotype or phenotype, or to further genetic modifications. Control subjects may include, for example: wild-type subjects, i.e., subjects without HBV infection who have not been exposed to conditions or stimuli or further genetic modifications (e.g., administration of the engineered wide-range nucleases described herein). Alternatively, control subjects may include, for example: subjects with HBV infection who have not been exposed to conditions or stimuli that may alter the subject’s HBV infection status, or to further genetic modifications (e.g., administration of the engineered wide-range nucleases described herein).

[0311] As used herein, the terms "treatment" or "treated subject" refer to the application of the engineered large-scale nuclease of this disclosure, or nucleic acid encoding the engineered large-scale nuclease of this disclosure, to an HBV-infected subject for the purpose of slowing or stopping the HBV replication rate by cleaving the genome of at least one HBV particle. Such treatment reduces or prevents HBV transfection and replication in the subject, and provides partial or complete relief of one or more symptoms of HBV infection or HBV-related illness in the subject. Assessment of symptom relief of HBV infection or HBV-related illness may include by measuring alanine aminotransferase (ALT) levels or by measuring liver function measurements of serum conversion (i.e., the disappearance and / or reduction of circulating HBeAg and / or HBsAg levels). Furthermore, symptom relief or reduction of HBV infection or HBV-related illness may be determined by liver biopsy and measurement of tissue fibrosis levels using methods known in the art. The number of circulating viral particles may be determined, for example, by measuring HBV DNA levels using PCR or by detecting HBsAg levels in the blood. The term "treatment" or "treatment of a subject" may further refer to the administration of cells (e.g., hepatocytes) containing nucleic acids encoding engineered macronucleases, wherein the cells are delivered to a target tissue (e.g., the liver) and produce an amount of engineered macronuclease sufficient to treat HBV infection or HBV-related disease in the subject, thereby resulting in partial or complete relief of one or more symptoms of HBV infection or HBV-related disease. In some aspects, the engineered macronucleases of this disclosure or their encoded nucleic acids are administered during treatment in the form of the pharmaceutical compositions of the present invention.

[0312] The term "disease associated with hepatitis B virus infection" refers to any condition related to or caused by hepatitis B virus infection, such as chronic liver disease / dysregulation, inflammation, fibrotic disorders, and proliferative disorders, such as liver cancer. Chronic persistent HBV infection can cause fatigue, liver damage, cirrhosis, and hepatocellular carcinoma (primary liver cancer).

[0313] As used herein, the terms “proliferating” and “proliferation” refer to HBV virus that actively divides and / or infects human cells, or HBV covalently closed circular DNA (cccDNA). Therefore, reduced proliferation refers to any reduction in HBV proliferation compared to an appropriate control not administered the engineered wide-ranging nucleases described herein or nucleic acids encoding engineered wide-ranging nucleases, including reductions of at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Throughout this application, the term “proliferative disorder” refers to any disease / condition characterized by undesirable or abnormal proliferation of cells or tissues. As used herein, the term “proliferative disorder” also refers to a condition in which unregulated and / or abnormal growth of cells may lead to the occurrence of an undesirable symptom or disease, which may be cancerous or non-cancerous.

[0314] As used herein, the term "effective amount" or "therapeutic effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical outcome. Therapeutic effective amounts vary depending on the formulation or composition used, the disease and its severity, and the age, weight, physical condition, and responsiveness of the subject being treated. In specific embodiments, the effective amount of the engineered broad-spectrum nuclease or pharmaceutical composition described herein can reduce HBV levels or proliferation, or alleviate at least one symptom of HBV infection-related disease in subjects with HBV infection.

[0315] The term “gc / kg” or “gene copy number per kilogram” refers to the copy number of nucleic acid encoding the engineered macronuclease described herein per kilogram of the weight of the subject administering the nucleic acid encoding the engineered macronuclease.

[0316] As used herein, the term "lipid nanoparticles" refers to lipid compositions that typically have a spherical structure with an average diameter of 10-1000 nanometers. In some formulations, lipid nanoparticles may comprise at least one cationic lipid, at least one non-cationic lipid, and at least one conjugated lipid. Lipid nanoparticles known in the art suitable for encapsulating nucleic acids (such as mRNA) are intended for use.

[0317] As used herein, the description of the numerical range of a variable is intended to convey that this disclosure can be practiced with a variable equal to any value within that range. Thus, for a variable that is inherently discrete, the variable can be equal to any integer value within that numerical range, including the endpoints of the range. Similarly, for a variable that is inherently continuous, the variable can be equal to any real value within that numerical range, including the endpoints of the range. By way of example and not limitation, if a variable is inherently discrete, a variable described as having values ​​between 0 and 2 can take the values ​​0, 1, or 2, and if a variable is inherently continuous, it can take the values ​​0.0, 0.1, 0.01, 0.001, or any other real value ≥ 0 and ≤ 2.

[0318] 2.1 Principle of the Invention This disclosure is based in part on the discovery of a large-scale HBV 11-12 nuclease with improved properties compared to previously described HBV 11-12 nucleases, such as improved (i.e., enhanced) specificity, resulting in reduced off-target cleavage, and enhanced (i.e., enhanced) cleavage efficiency and indel formation at HBV 11-12 recognition sequences, particularly in cells containing integrated HBV genome copies.

[0319] This paper discloses the HBV 11-12 broad-spectrum nuclease recognition sequence (SEQ ID NO: 3) in the polymerase (P) gene encoding the viral DNA polymerase in the hepatitis B virus genome. The HBV 11-12 recognition sequence is conserved at least in the HBV genotype AG, which advantageously allows the engineered broad-spectrum nuclease disclosed herein to target HBV infection globally.

[0320] The HBV 11-12 macronucleases disclosed herein are designated as HBV 11-12L.1090QQ adapter 1923(1 / 2), as shown in SEQ ID NO: 5, and HBV 11-12L.1090QE adapter 1923(1 / 2), as shown in SEQ ID NO: 6. The designation "QQ" indicates the presence of amino acid residue Q at positions 80 and 260 of SEQ ID NO: 5 and positions 80 and 271 of SEQ ID NO: 13. The designation "QE" indicates the presence of amino acid residue Q at position 80 of SEQ ID NO: 6 and SEQ ID NO: 14, and the presence of amino acid residue E at positions 260 of SEQ ID NO: 6 and 271 of SEQ ID NO: 14. The sequence of linker 1923, which binds to the first and second subunits of these engineered macronucleases, is shown in SEQ ID NO: 15, and its length (i.e., 31 amino acid residues) is shorter than the linker 1 sequence (i.e., 42 amino acid residues) shown in SEQ ID NO: 16, which was previously identified in the HBV 11-12L.1090QQ linker 1 engineered macronuclease. The term "(1 / 2)" refers to the presence of amino acid modifications in the first and second subunits of the engineered macronuclease when the subunits bind via the linker 1923 sequence. In the HBV11-12L.1090 macronuclease containing the linker 1923 sequence described herein, the N-terminal subunit contains an A at position 96, an A at position 99, and a D at position 100; and the C-terminal subunit contains a Y at position 57 corresponding to SEQ ID NO: 1 (i.e., position 237 of SEQ ID NO: 5 or 6) and a T at position 61 corresponding to SEQ ID NO: 1 (i.e., position 241 of SEQ ID NO: 5 or 6). While not bound by any particular theory or mechanism of action, it is believed that linker 1923 interacts with these modified “(1 / 2)” residues within the I-CreI scaffold, thereby leading to the stabilization of the engineered macronuclease.

[0321] Cleavage at the HBV 11-12 recognition sequence allows for non-homologous end joining (NHEJ) at the cleavage site, and the expression of one or more viral proteins (e.g., viral DNA polymerase) can be disrupted due to NHEJs at the cleavage site that result in insertions, deletions, or frameshift mutations. Alternatively, cleavage of the HBV genome at the HBV 11-12 recognition sequence can promote the degradation of the HBV genome and / or HBV cccDNA. Disruption of viral protein expression can reduce or eliminate HBV infection and / or replication.

[0322] Furthermore, cleavage at the HBV 11-12 recognition sequence can also allow exogenous nucleic acid sequences to directly recombine into the HBV genome to disrupt the expression of one or more viral proteins. For example, a "suicide gene" can be introduced into the HBV genome via homologous recombination.

[0323] Therefore, this disclosure covers engineered macronucleases that recognize and cleave HBV 11-12 recognition sequences within the HBV genome. This disclosure also covers the use of such engineered macronucleases in pharmaceutical compositions and in methods for inactivating polymerase genes of the HBV genome or HBV genome fragments in eukaryotic cells, as well as methods for treating HBV infection or HBV infection-related diseases. Furthermore, this disclosure covers pharmaceutical compositions comprising engineered macronuclease proteins or nucleic acids encoding engineered macronucleases, and the use of such compositions for treating HBV infection and HBV infection-related diseases (e.g., hepatocellular carcinoma (HCC)).

[0324] 2.2 A wide range of nucleases that recognize and cleave the HBV 11-12 recognition sequence within the hepatitis B virus genome Identification Sequence It is known in the art that it is possible to use site-specific nucleases to induce DNA breaks in the viral genome, and that such DNA breaks can be repaired by mutagenic NHEJ or by homologous recombination with transgenic DNA sequences leading to permanent modifications of the genome, thereby preventing HBV viral particles from dividing / replicating or infecting human cells.

[0325] The engineered, wide-ranging nuclease disclosed herein is designed to bind to and cleave the HBV 11-12 recognition sequence (SEQ ID NO: 3). The HBV 11-12 recognition sequence is located within the ORF of the polymerase gene for multiple HBV genotypes (including at least genotypes A, B, C, D, E, F, and G).

[0326] Exemplary engineered large-scale nucleases It is known in the art that site-specific nucleases can be used to induce DNA breaks in the viral genome, and that such DNA breaks can lead to permanent modifications of the genome via NHEJ, rendering HBV viral particles unable to divide / replicate or infect human cells. Inducing DNA breaks in the viral genome can also lead to viral genome degradation, rendering it unable to divide, replicate, or become infectious.

[0327] Therefore, in some embodiments, this disclosure provides engineered nucleases, particularly engineered large-scale nucleases. In a particular embodiment, the large-scale nuclease is a single-stranded large-scale nuclease. The single-stranded large-scale nuclease comprises an N-terminal subunit and a C-terminal subunit bound by a linker peptide. Each of the two domains recognizes half of a recognition sequence (i.e., a recognition half-site), and the DNA cleavage site is located in the middle of the recognition sequence near the interface between the two subunits. The DNA strand break is offset by four base pairs, such that DNA cleavage by the large-scale nuclease produces a pair of four-base-pair 3' single-stranded overhangs.

[0328] In some embodiments, the engineered large-scale nuclease disclosed herein exhibits at least one optimized property compared to the previously described large-scale nuclease HBV 11-12L.1090QQ adapter 1. Such optimized properties include improved (i.e., enhanced) specificity, resulting in reduced off-target cleavage, and enhanced (i.e., increased) efficiency of cleavage and indel (i.e., insertion or deletion) formation at the HBV 11-12 recognition sequence, particularly in cells containing integrated HBV genome copies. Therefore, in certain embodiments, when delivered to a population of HBV-infected target cells, the engineered large-scale nuclease disclosed herein is able to produce a higher percentage of viral particles or cells with cleavage and / or indels within the HBV genome, whether integrated or unintegrated. In some of these embodiments, the population of HBV or target cells comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of HBV or target cells containing cleavages and / or indels in the HBV genome (whether integrated or unintegrated). Extensive nuclease-induced cleavages and / or indel formation can be measured using any method known in the art, including T7E assays, digital PCR, mismatch detection assays, mismatch cleavage assays, high-resolution melt analysis (HRMA), heteroduplex mobility assays, sequencing, and fluorescent PCR capillary gel electrophoresis (see, for example, Zischewski et al. (2017) Biotechnology Advances 35(1):95-104, the full text of which is incorporated herein by reference).

[0329] In some embodiments, the target cells are liver cells. In some embodiments, the liver cells are hepatocytes. In some embodiments, the target cells are primary human hepatocytes (PHH). In some embodiments, the target cells are non-human mammalian hepatocytes.

[0330] The engineered large-scale nuclease described herein comprises a first subunit and a second subunit, the first subunit comprising a first hypervariable (HVR1) region and the second subunit comprising a second hypervariable (HVR2) region. Furthermore, the first subunit binds a first recognition half-site (i.e., the HBV11 half-site) in the recognition sequence and the second subunit binds a second recognition half-site (i.e., the HBV12 half-site) in the recognition sequence. In an embodiment where the engineered large-scale nuclease is a single-stranded large-scale nuclease, the first and second subunits may be oriented such that the first subunit containing the HVR1 region and binding the first half-site is positioned as an N-terminal subunit, and the second subunit containing the HVR2 region and binding the second half-site is positioned as a C-terminal subunit. In an alternative embodiment, the first and second subunits may be oriented such that the first subunit containing the HVR1 region and binding the first half-site is positioned as a C-terminal subunit, and the second subunit containing the HVR2 region and binding the second half-site is positioned as an N-terminal subunit. Exemplary engineered wide-range nucleases that identify and cleave HBV 11-12 recognition sequences are provided in SEQ ID NO: 5 and 6, and are further described below.

[0331] HBV 11-12L.1090QQ connector 1923(1 / 2) (SEQ ID NO: 5) In some embodiments, the HVR1 region contains an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 204-259 of SEQ ID NO: 5.

[0332] In some embodiments, the HVR1 region contains an amino acid sequence that has at least 97% sequence identity with residues 204-259 of SEQ ID NO: 5.

[0333] In some embodiments, the HVR1 region contains one or more residues corresponding to residues 204, 206, 208, 210, 212, 213, 218, 220, 222, 224, 226, 248, 250, 255 and 257 of SEQ ID NO: 5.

[0334] In some implementations, the HVR1 region contains residues 204, 206, 208, 210, 212, 213, 218, 220, 222, 224, 226, 248, 250, 255 and 257 corresponding to SEQ ID NO: 5.

[0335] In some implementations, the HVR1 region contains residue 237 corresponding to SEQ ID NO: 5.

[0336] In some implementations, the HVR1 region contains residues corresponding to residue 241 of SEQ ID NO: 5.

[0337] In some implementations, the HVR1 region contains residue 251 corresponding to SEQ ID NO: 5.

[0338] In some implementations, the HVR1 region contains residue 252 corresponding to SEQ ID NO: 5.

[0339] In some implementations, the HVR1 region contains residue 253 corresponding to SEQ ID NO: 5.

[0340] In some embodiments, the HVR1 region contains Y, R, K, or D at residue 246 corresponding to SEQ ID NO: 5.

[0341] In some implementations, the HVR1 region contains residues 204-259 of SEQ ID NO: 5.

[0342] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 187-333 of SEQ ID NO: 5.

[0343] In some embodiments, the first subunit comprises an amino acid sequence having at least 99% sequence identity with residues 187-333 of SEQ ID NO: 5.

[0344] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 185-343 of SEQ ID NO: 5.

[0345] In some embodiments, the first subunit comprises an amino acid sequence having at least 99% sequence identity with residues 185-343 of SEQ ID NO: 5.

[0346] In some embodiments, the first subunit contains G, S, or A at residue 199 corresponding to SEQ ID NO: 5.

[0347] In some embodiments, the first subunit contains E, Q, or K at residue 260 corresponding to SEQ ID NO: 5.

[0348] In some implementations, the first subunit comprises residues 187-333 of SEQ ID NO: 5.

[0349] In some implementations, the first subunit comprises residues 185-343 of SEQ ID NO: 5.

[0350] In some embodiments, the HVR2 region contains an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 24-79 of SEQ ID NO: 5.

[0351] In some implementations, the HVR2 region contains one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75 and 77 of SEQ ID NO: 5.

[0352] In some implementations, the HVR2 region contains residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75 and 77 corresponding to SEQ ID NO: 5.

[0353] In some implementations, the HVR2 region contains residue 51 corresponding to SEQ ID NO: 5.

[0354] In some implementations, the HVR2 region contains Y, R, K, or D at residue 66 corresponding to SEQ ID NO: 5.

[0355] In some implementations, the HVR2 region contains residues 24-79 of SEQ ID NO: 5.

[0356] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 7-153 of SEQ ID NO: 5.

[0357] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 7-153 of SEQ ID NO: 5.

[0358] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 6-153 of SEQ ID NO: 5.

[0359] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 6-153 of SEQ ID NO: 5.

[0360] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 5-153 of SEQ ID NO: 5.

[0361] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 5-153 of SEQ ID NO: 5.

[0362] In some embodiments, the second subunit is an N-terminal subunit and contains an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 4-153 of SEQ ID NO: 5.

[0363] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 4-153 of SEQ ID NO: 5.

[0364] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 3-153 of SEQ ID NO: 5.

[0365] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 3-153 of SEQ ID NO: 5.

[0366] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 2-153 of SEQ ID NO: 5. In some embodiments, the second subunit comprises residues other than M at position 1 corresponding to SEQ ID NO: 5.

[0367] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 2-153 of SEQ ID NO: 5.

[0368] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 1-153 of SEQ ID NO: 5.

[0369] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 1-153 of SEQ ID NO: 5.

[0370] In some embodiments, the second subunit contains residue 19 corresponding to SEQ ID NO: 5.

[0371] In some embodiments, the second subunit contains residue 80 corresponding to residue SEQ ID NO: 5.

[0372] In some embodiments, the second subunit contains residue 96 corresponding to SEQ ID NO: 5.

[0373] In some embodiments, the second subunit contains residue 99 corresponding to SEQ ID NO: 5.

[0374] In some embodiments, the second subunit contains residue 100 corresponding to SEQ ID NO: 5.

[0375] In some embodiments, the second subunit contains G, S, or A at residue 19 corresponding to SEQ ID NO: 5.

[0376] In some embodiments, the second subunit contains E, Q, or K at residue 80 corresponding to SEQ ID NO: 5.

[0377] In some implementations, the second subunit comprises residues 7-153 of SEQ ID NO: 5.

[0378] In some implementations, the second subunit comprises residues 6-153 of SEQ ID NO: 5.

[0379] In some implementations, the second subunit comprises residues 5-153 of SEQ ID NO: 5.

[0380] In some implementations, the second subunit comprises residues 4-153 of SEQ ID NO: 5.

[0381] In some implementations, the second subunit comprises residues 3-153 of SEQ ID NO: 5.

[0382] In some embodiments, the second subunit comprises residues 2-153 of SEQ ID NO: 5. In some embodiments, the second subunit comprises residues other than M at position 1 corresponding to SEQ ID NO: 5.

[0383] In some embodiments, the second subunit comprises residues 1-153 of SEQ ID NO: 5.

[0384] In some implementations, the engineered large-scale nuclease is a single-stranded large-scale nuclease containing a linker, wherein the linker is covalently bonded to a first subunit and a second subunit.

[0385] In some embodiments, the adapter comprises the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16.

[0386] In some embodiments, the adapter comprises the amino acid sequence shown in SEQ ID NO: 15.

[0387] In some embodiments, the N-terminus of the connector is fused with a residue corresponding to residue 153 of SEQ ID NO: 5 (i.e., the D residue), and the C-terminus of the connector is fused with a residue corresponding to residue 185 of SEQ ID NO: 5 (i.e., the Y residue).

[0388] In some embodiments, the engineered wide-range nuclease comprises a first subunit containing an amino acid sequence having at least 99% sequence identity with residues 187-333 of SEQ ID NO: 5; a linker containing the amino acid sequence shown in SEQ ID NO: 15, wherein the linker covalently binds to the first and second subunits; and a second subunit containing an amino acid sequence having at least 99% sequence identity with residues 7-153 of SEQ ID NO: 5.

[0389] In some embodiments, the engineered wide-range nuclease comprises: a first subunit comprising an amino acid sequence having at least 99% sequence identity with residues 185-343 of SEQ ID NO: 5; a linker comprising the amino acid sequence shown in SEQ ID NO: 15, wherein the linker covalently binds to the first and second subunits; and a second subunit comprising an amino acid sequence having at least 99% sequence identity with residues 1-153 of SEQ ID NO: 5.

[0390] In some embodiments, the engineered wide range of nucleases comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 4-343 of SEQ ID NO: 5.

[0391] In some implementations, the engineered wide-range nuclease comprises an amino acid sequence having at least 92% sequence identity with residues 4-343 of SEQ ID NO: 5.

[0392] In some embodiments, the engineered wide range of nucleases comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 3-343 of SEQ ID NO: 5.

[0393] In some implementations, the engineered wide-range nuclease comprises an amino acid sequence having at least 92% sequence identity with residues 3-343 of SEQ ID NO: 5.

[0394] In some embodiments, the engineered broad-spectrum nuclease comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or higher sequence identity with residues 2-343 of SEQ ID NO: 5. In some embodiments, the second subunit comprises residues other than M at position 1 corresponding to SEQ ID NO: 5.

[0395] In some implementations, the engineered wide-range nuclease comprises an amino acid sequence having at least 92% sequence identity with residues 2-343 of SEQ ID NO: 5.

[0396] In some embodiments, the engineered broad-spectrum nuclease comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or higher sequence identity with SEQ ID NO: 5. In some embodiments, the engineered broad-spectrum nuclease comprises an amino acid sequence having at least 92% sequence identity with SEQ ID NO: 5.

[0397] In some implementations, the engineered wide-range nuclease comprises the amino acid sequence of residues 4-343 of SEQ ID NO: 5.

[0398] In some implementations, the engineered wide range of nucleases comprises the amino acid sequence of residues 3-343 of SEQ ID NO: 5.

[0399] In some embodiments, the engineered broad-spectrum nuclease comprises the amino acid sequence of residues 2-343 of SEQ ID NO: 5. In some embodiments, the second subunit contains residues other than M at position 1 corresponding to SEQ ID NO: 5.

[0400] In some implementations, the engineered wide-range nuclease contains the amino acid sequence of SEQ ID NO: 5.

[0401] In some implementations, the engineered wide-range nuclease is encoded by a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence of SEQ ID NO: 11.

[0402] In some implementations, the engineered wide-range nuclease is encoded by the nucleic acid sequence of SEQ ID NO: 11.

[0403] In some implementations, engineered large-scale nucleases contain nuclear localization signals.

[0404] In some implementations, the nuclear localization signal is located at the N-terminus of the engineered large-scale nuclease.

[0405] In some implementations, the nuclear localization signal is located at the C-terminus of engineered large-scale nucleases.

[0406] In some implementations, engineered wide-range nucleases include a first nuclear localization signal at the N-terminus and a second nuclear localization signal at the C-terminus.

[0407] In some embodiments, the nuclear localization signal comprises an amino acid sequence having at least 80% or at least 90% sequence identity with SEQ ID NO: 17. In some embodiments, the nuclear localization signal comprises SEQ ID NO: 17.

[0408] HBV 11-12L.1090QE connector 1923(1 / 2) (SEQ ID NO: 6) In some embodiments, the HVR1 region contains an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 204-259 of SEQ ID NO: 6.

[0409] In some embodiments, the HVR1 region contains an amino acid sequence that has at least 97% sequence identity with residues 204-259 of SEQ ID NO: 6.

[0410] In some embodiments, the HVR1 region contains one or more residues corresponding to residues 204, 206, 208, 210, 212, 213, 218, 220, 222, 224, 226, 248, 250, 255 and 257 of SEQ ID NO: 6.

[0411] In some implementations, the HVR1 region contains residues 204, 206, 208, 210, 212, 213, 218, 220, 222, 224, 226, 248, 250, 255 and 257 corresponding to SEQ ID NO: 6.

[0412] In some implementations, the HVR1 region contains residue 237 corresponding to SEQ ID NO: 6.

[0413] In some implementations, the HVR1 region contains residue 241 corresponding to SEQ ID NO: 6.

[0414] In some implementations, the HVR1 region contains residue 251 corresponding to SEQ ID NO: 6.

[0415] In some implementations, the HVR1 region contains residue 252 corresponding to SEQ ID NO: 6.

[0416] In some implementations, the HVR1 region contains residue 253 corresponding to SEQ ID NO: 6.

[0417] In some implementations, the HVR1 region contains Y, R, K, or D at residue 246 corresponding to SEQ ID NO: 6.

[0418] In some implementations, the HVR1 region contains residues 204-259 of SEQ ID NO: 6.

[0419] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 187-333 of SEQ ID NO: 6.

[0420] In some embodiments, the first subunit comprises an amino acid sequence having at least 99% sequence identity with residues 187-333 of SEQ ID NO: 6.

[0421] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 185-343 of SEQ ID NO: 6.

[0422] In some embodiments, the first subunit comprises an amino acid sequence having at least 99% sequence identity with residues 185-343 of SEQ ID NO: 6.

[0423] In some implementations, the first subunit contains residue 260 corresponding to SEQ ID NO: 6.

[0424] In some embodiments, the first subunit contains G, S, or A at residue 199 corresponding to SEQ ID NO: 6.

[0425] In some embodiments, the first subunit contains E, Q, or K at the residue corresponding to residue 260 of SEQ ID NO: 6.

[0426] In some embodiments, the first subunit comprises residues 187-333 of any of SEQ ID NO: 6.

[0427] In some implementations, the first subunit comprises residues 185-343 of any of SEQ ID NO: 6.

[0428] In some embodiments, the HVR2 region contains an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 24-79 of SEQ ID NO: 6.

[0429] In some implementations, the HVR2 region contains one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75 and 77 of SEQ ID NO: 6.

[0430] In some implementations, the HVR2 region contains residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75 and 77 corresponding to SEQ ID NO: 6.

[0431] In some implementations, the HVR2 region contains residue 51 corresponding to SEQ ID NO: 6.

[0432] In some implementations, the HVR2 region contains Y, R, K, or D at residue 66 corresponding to SEQ ID NO: 6.

[0433] In some implementations, the HVR2 region contains residues 24-79 of SEQ ID NO: 6.

[0434] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 7-153 of SEQ ID NO: 6.

[0435] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 7-153 of SEQ ID NO: 6.

[0436] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 6-153 of SEQ ID NO: 6.

[0437] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 6-153 of SEQ ID NO: 6.

[0438] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 5-153 of SEQ ID NO: 6.

[0439] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 5-153 of SEQ ID NO: 6.

[0440] In some embodiments, the second subunit is an N-terminal subunit and contains an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 4-153 of SEQ ID NO: 6.

[0441] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 4-153 of SEQ ID NO: 6.

[0442] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 3-153 of SEQ ID NO: 6.

[0443] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 3-153 of SEQ ID NO: 6.

[0444] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or higher sequence identity with residues 2-153 of SEQ ID NO: 6. In some embodiments, the second subunit comprises residues other than M at position 1 corresponding to SEQ ID NO: 6.

[0445] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 2-153 of SEQ ID NO: 6.

[0446] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 1-153 of SEQ ID NO: 6.

[0447] In some embodiments, the second subunit comprises an amino acid sequence having at least 99% sequence identity with residues 1-153 of SEQ ID NO: 6.

[0448] In some embodiments, the second subunit contains residue 19 corresponding to SEQ ID NO: 6.

[0449] In some embodiments, the second subunit contains residue 80 corresponding to SEQ ID NO: 6.

[0450] In some embodiments, the second subunit contains residue 96 corresponding to SEQ ID NO: 6.

[0451] In some implementations, the second subunit contains residue 99 corresponding to SEQ ID NO: 6.

[0452] In some embodiments, the second subunit contains residue 100 corresponding to SEQ ID NO: 6.

[0453] In some embodiments, the second subunit contains G, S, or A at residue 19 corresponding to SEQ ID NO: 6.

[0454] In some embodiments, the second subunit contains E, Q, or K at residue 80 corresponding to SEQ ID NO: 6.

[0455] In some implementations, the second subunit comprises residues 7-153 of SEQ ID NO: 6.

[0456] In some implementations, the second subunit comprises residues 6-153 of SEQ ID NO: 6.

[0457] In some implementations, the second subunit comprises residues 5-153 of SEQ ID NO: 6.

[0458] In some implementations, the second subunit comprises residues 4-153 of SEQ ID NO: 6.

[0459] In some implementations, the second subunit comprises residues 3-153 of SEQ ID NO: 6.

[0460] In some embodiments, the second subunit comprises residues 2-153 of SEQ ID NO: 6. In some embodiments, the second subunit comprises residues other than M at position 1 corresponding to SEQ ID NO: 5 or SEQ ID NO: 6.

[0461] In some implementations, the second subunit comprises residues 1-153 of SEQ ID NO: 6.

[0462] In some implementations, the engineered large-scale nuclease is a single-stranded large-scale nuclease containing a linker, wherein the linker is covalently bonded to a first subunit and a second subunit.

[0463] In some embodiments, the adapter comprises the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16.

[0464] In some embodiments, the adapter comprises the amino acid sequence shown in SEQ ID NO: 15.

[0465] In some embodiments, the N-terminus of the connector is fused with a residue corresponding to residue 153 of SEQ ID NO: 6 (i.e., the D residue), and the C-terminus of the connector is fused with a residue corresponding to residue 185 of SEQ ID NO: 6 (i.e., the Y residue).

[0466] In some embodiments, the engineered wide-range nuclease comprises a first subunit containing an amino acid sequence having at least 99% sequence identity with residues 187-333 of SEQ ID NO: 6; a linker containing the amino acid sequence shown in SEQ ID NO: 15, wherein the linker covalently binds to the first and second subunits; and a second subunit containing an amino acid sequence having at least 99% sequence identity with residues 7-153 of SEQ ID NO: 6.

[0467] In some embodiments, the engineered wide-range nuclease comprises a first subunit containing an amino acid sequence having at least 99% sequence identity with residues 185-343 of SEQ ID NO: 6; a linker containing the amino acid sequence shown in SEQ ID NO: 15, wherein the linker covalently binds the first subunit and the second subunit; and a second subunit containing an amino acid sequence having at least 99% sequence identity with residues 1-153 of SEQ ID NO: 6.

[0468] In some embodiments, the engineered wide range of nucleases comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 4-343 of SEQ ID NO: 6.

[0469] In some implementations, the engineered wide-range nuclease comprises an amino acid sequence having at least 92% sequence identity with residues 4-343 of SEQ ID NO: 6.

[0470] In some embodiments, the engineered wide range of nucleases comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 3-343 of SEQ ID NO: 6.

[0471] In some implementations, the engineered wide-range nuclease comprises an amino acid sequence that has at least 92% sequence identity with residues 3-343 of SEQ ID NO: 6.

[0472] In some embodiments, the engineered broad-spectrum nuclease comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with residues 2-343 of SEQ ID NO: 6. In some embodiments, the second subunit comprises residues other than M at position 1 corresponding to SEQ ID NO: 6.

[0473] In some implementations, the engineered wide-range nuclease comprises an amino acid sequence having at least 92% sequence identity with residues 2-343 of SEQ ID NO: 6.

[0474] In some implementations, the engineered wide range of nucleases comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 6.

[0475] In some implementations, the engineered wide-range nuclease comprises an amino acid sequence that has at least 92% sequence identity with SEQ ID NO: 6.

[0476] In some implementations, the engineered wide range of nucleases comprises the amino acid sequence of residues 4-343 of SEQ ID NO: 6.

[0477] In some implementations, the engineered wide-range nuclease comprises the amino acid sequence of residues 3-343 of SEQ ID NO: 6.

[0478] In some embodiments, the engineered wide-range nuclease comprises the amino acid sequence of residues 2-343 of SEQ ID NO: 6. In some embodiments, the second subunit contains residues other than M at position 1 corresponding to SEQ ID NO: 6.

[0479] In some implementations, the engineered wide-range nuclease contains the amino acid sequence of SEQ ID NO: 6.

[0480] In some implementations, the engineered wide-range nuclease is encoded by a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the nucleic acid sequence of SEQ ID NO: 12.

[0481] In some implementations, the engineered wide-range nuclease is encoded by the nucleic acid sequence of SEQ ID NO: 12.

[0482] In some implementations, engineered large-scale nucleases contain nuclear localization signals.

[0483] In some implementations, the nuclear localization signal is located at the N-terminus of the engineered large-scale nuclease.

[0484] In some implementations, the nuclear localization signal is located at the C-terminus of engineered large-scale nucleases.

[0485] In some implementations, engineered wide-range nucleases include a first nuclear localization signal at the N-terminus and a second nuclear localization signal at the C-terminus.

[0486] In some embodiments, the nuclear localization signal comprises an amino acid sequence having at least 80% or at least 90% sequence identity with SEQ ID NO: 17. In some embodiments, the nuclear localization signal comprises SEQ ID NO: 17.

[0487] In some embodiments of the engineered wide-range nucleases described herein, the first subunit (i.e., containing HVR1) may be positioned as a C-terminal subunit, and the second subunit (i.e., containing HVR2) may be positioned as an N-terminal subunit. In some embodiments of this configuration, such as those exemplified in SEQ ID NO: 5 and SEQ ID NO: 6, the first subunit (i.e., the C-terminal subunit) may lack residues 1-4 corresponding to wild-type I-CreI at its N-terminus, since the binding site of the polypeptide linker is located at the Y residue at position 5 corresponding to wild-type I-CreI. The first subunit may also contain residues 154-163 corresponding to wild-type I-CreI at its C-terminus. Furthermore, in some embodiments of this configuration, the second subunit (i.e., the N-terminal subunit) may lack residues 154-163 corresponding to wild-type I-CreI at its C-terminus, since the binding site of the polypeptide linker is located at the D residue at position 153 corresponding to wild-type I-CreI. The second subunit may also contain one or more residues (e.g., residues 1-6, 2-6, 3-6, 4-6, or 5-6) corresponding to one or more of the residues 1-6 of wild-type I-CreI at its N-terminus.

[0488] In other embodiments of the engineered wide-range nuclease described herein, the first subunit (i.e., containing HVR1) may be positioned as an N-terminal subunit, and the second subunit (i.e., containing HVR2) may be positioned as a C-terminal subunit. In some embodiments of this configuration, the first subunit (i.e., the N-terminal subunit) may lack residues corresponding to wild-type I-CreI 154-163 at its C-terminus, because the binding site of the polypeptide linker is located at the D residue corresponding to position 153 of wild-type I-CreI. The first subunit may also contain one or more residues corresponding to residues 1-6 of wild-type I-CreI (e.g., residues 1-6, 2-6, 3-6, 4-6, or 5-6) at its N-terminus. Moreover, in some embodiments of this configuration, the second subunit (i.e., the C-terminal subunit) may lack residues corresponding to residues 1-4 of wild-type I-CreI at its N-terminus, because the binding site of the polypeptide linker is located at the Y residue corresponding to position 5 of wild-type I-CreI. The second subunit may also contain residues 154-163 at its C-terminus that correspond to wild-type I-CreI.

[0489] In some embodiments, the disclosed engineered wide-range nuclease comprises (i) inactivated amino acids in the N-terminal subunit that reduce or eliminate cleavage activity; (ii) inactivated amino acids in the C-terminal subunit that reduce or eliminate cleavage activity; or (iii) inactivated amino acids in both the N-terminal and C-terminal subunits that reduce or eliminate cleavage activity.

[0490] As used herein, the inactivating amino acid that “reduces” the cleavage activity of an engineered wide-range nuclease only inactivates the subunit containing that amino acid, without affecting the ability of the other subunit to cleave its DNA strand. For example, if only one subunit contains the inactivating amino acid that reduces cleavage activity, the other subunit remains active, and the engineered wide-range nuclease becomes a cleaving enzyme that can still cleave one strand of double-stranded DNA. In other cases where two subunits contain the inactivating amino acid that reduces cleavage activity, both subunits are inactive, the engineered wide-range nuclease has no cleavage activity, and it cannot produce single-strand or double-strand breaks in DNA.

[0491] In contrast, the inactivating amino acid that "eliminates" the cleavage activity of engineered large-scale nucleases can be present in only one subunit, but inactivating both subunits of the engineered large-scale nuclease renders it without any cleavage activity and unable to produce single-strand or double-strand breaks in DNA.

[0492] In some embodiments, the inactivated amino acid is position A at position 20 or 200 corresponding to SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the inactivated amino acid is position E at position 47 or 227 corresponding to SEQ ID NO: 5 or SEQ ID NO: 6.

[0493] In some embodiments, the N-terminal subunit is contained at position E corresponding to position 47 of SEQ ID NO: 5 or SEQ ID NO: 6, and the C-terminal subunit is contained at position E corresponding to position 227 of SEQ ID NO: 5 or SEQ ID NO: 6, wherein the engineered wide-range nuclease does not contain cleavage activity (i.e., the activity is eliminated).

[0494] In some embodiments, the N-terminal subunit is contained at position A corresponding to position 20 of SEQ ID NO: 5 or SEQ ID NO: 6, and / or the C-terminal subunit is contained at position A corresponding to position 200 of SEQ ID NO: 5 or SEQ ID NO: 6, wherein the engineered wide-range nuclease does not contain cleavage activity (i.e., activity is eliminated).

[0495] In some embodiments, the N-terminal subunit contains an E at position 47 corresponding to SEQ ID NO: 5 or SEQ ID NO: 6, and the C-terminal subunit does not contain an inactivating amino acid, wherein the engineered broad-spectrum nuclease is a cleavage enzyme capable only of cleaving the antisense strand of the dsDNA target site.

[0496] In some embodiments, the C-terminal subunit contains an E at position 227 corresponding to SEQ ID NO: 5 or SEQ ID NO: 6, and the N-terminal subunit does not contain an inactivating amino acid, wherein the engineered broad-spectrum nuclease is a nickase capable of cleaving only the positive strand of the dsDNA target site.

[0497] In embodiments where the engineered macronuclease lacks cleavage activity (i.e., activity is eliminated) due to one or more inactivating amino acid modifications, such engineered macronucleases are capable of binding to double-stranded DNA containing the recognition sequence of SEQ ID NO: 3 (i.e., HBV 11-12) without cleaving the double-stranded DNA. In embodiments where the engineered macronuclease contains inactivating amino acid modifications such that only one subunit has cleavage activity and the engineered macronuclease is a nicking enzyme, such engineered macronucleases are capable of binding to double-stranded DNA containing the recognition sequence of SEQ ID NO: 3 (i.e., HBV 11-12) and cleaving either the sense or antisense strand of the DNA.

[0498] 2.3 Methods for the delivery and expression optimization of engineered, large-scale nucleases This document describes a method for inactivating a polymerase (pol) gene of the HBV genome or a fragment thereof, comprising introducing the engineered macronuclease described herein or a nucleic acid encoding the engineered macronuclease into a eukaryotic cell containing the HBV genome or a fragment thereof, wherein the engineered macronuclease produces a cleavage site at a recognition sequence comprising or consisting of SEQ ID NO: 3 (i.e., HBV 11-12), and the pol gene is inactivated by introducing an indel at the cleavage site or by eliminating the HBV genome or a fragment thereof. Similarly, methods are provided for reducing symptoms of HBV infection and / or HBV-related disease in subjects, reducing the amount of HBV, reducing the rate of HBV proliferation, and / or treating HBV infection and / or HBV-related disease in subjects, comprising administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the engineered macronuclease described herein or a nucleic acid encoding the engineered macronuclease or expressing the engineered macronuclease. In the methods described herein, the engineered wide-range nucleases of this disclosure can be delivered to target cells and / or expressed from DNA / RNA in target cells, which can provide engineered wide-range nucleases to the HBV genome.

[0499] Introducing engineered, wide-range nucleases into cells The engineered, wide-ranging nuclease proteins described herein, or the polynucleotides encoding them, can be delivered into cells via a variety of mechanisms known in the art to cleave genomic DNA or HBV genome fragments, including those detailed below.

[0500] The engineered wide-range nucleases described herein can be delivered to cells in protein form, or preferably, as polynucleotides containing a nucleic acid sequence encoding the engineered wide-range nuclease. Such polynucleotides can be, for example, DNA (e.g., circular or linearized plasmid DNA, PCR products, or viral genomes) or RNA (e.g., mRNA).

[0501] For implementation schemes where engineered large-scale nuclease coding sequences are delivered in DNA form, they should be operatively linked to a promoter to facilitate transcription of the large-scale nuclease gene. Suitable mammalian promoters include constitutive promoters, such as the early cytomegalovirus (CMV) promoter (Thomsen et al. (1984)). Proc Natl Acad Sci USA .8 1(3):659-63) or SV40 early promoter (Benoist and Chambon (1981), Nature . 290(5804):304-10), and inductive promoters, such as tetracycline-inductive promoters (Dingermann et al. (1992), Mol Cell Biol .12(9):4038-45). The engineered wide-range nucleases of this disclosure can also be operatively linked to synthetic promoters. Synthetic promoters may include, but are not limited to, the JeT promoter (WO 2002 / 012514). In specific embodiments, the nucleic acid sequence encoding the engineered wide-range nucleases described herein may be operatively linked to liver-specific promoters. Examples of liver-specific promoters include, but are not limited to, the human α-1 antitrypsin promoter, heterozygous liver-specific promoters (the liver locus control region (ApoE-HCR) from the ApoE gene and the liver-specific α1-antitrypsin promoter), the human thyroxine-binding globulin (TBG) promoter, and the apolipoprotein A-II promoter.

[0502] In some embodiments, where a single polynucleotide comprises two separate nucleic acid sequences each encoding an engineered large-scale nuclease (as described herein), the large-scale nuclease gene is operatively linked to two separate promoters. In alternative embodiments, the two large-scale nuclease genes are operatively linked to a single promoter, and in some instances this can be via an internal ribosome entry site (IRES) or a 2A peptide sequence (Szymczak and Vignali (2005)). Expert Opin Biol Ther (5:627-38) Separated. This 2A peptide sequence may include, for example, T2A, P2A, E2A or F2A sequences.

[0503] In certain embodiments, a polynucleotide comprising a nucleic acid sequence encoding at least one of the engineered broad-spectrum nucleases described herein is delivered on a recombinant DNA construct or expression cassette. For example, the recombinant DNA construct may comprise an expression cassette (i.e., a “cassette”) containing a promoter and a nucleic acid sequence encoding the engineered broad-spectrum nucleases described herein.

[0504] In other embodiments, the recombinant DNA construct comprises at least a first box and a second box, wherein the first box contains a promoter and a nucleic acid sequence encoding the engineered wide-range nuclease described herein, and wherein the second box contains a promoter and a nucleic acid sequence encoding a second engineered wide-range nuclease that binds to and cleaves a second recognition sequence present in the hepatitis B virus genome but different from SEQ ID NO: 3.

[0505] In other embodiments, the recombinant DNA construct comprises a cassette containing a promoter and a polycistronic nucleic acid sequence, wherein the promoter drives the expression of the polycistronic nucleic acid sequence to produce the polycistronic mRNA described herein in target cells.

[0506] In another specific embodiment, a single-stranded DNA template is used to introduce a polynucleotide containing a nucleic acid sequence encoding the engineered wide-ranging nucleases described herein into a cell. The single-stranded DNA may also contain 5' and / or 3' AAV inverted terminal repeats (ITRs) upstream and / or downstream of the sequence encoding the engineered nuclease. The single-stranded DNA may also contain 5' and / or 3' homologous arms upstream and / or downstream of the sequence encoding the engineered wide-ranging nuclease.

[0507] In another specific embodiment, a linearized DNA template is used to introduce a polynucleotide containing a nucleic acid sequence encoding a wide range of engineered nucleases described herein into a cell. Such a linearized DNA template can be generated by methods known in the art. For example, plasmid DNA encoding a nuclease can be digested with one or more restriction enzymes, such that the circular plasmid DNA is linearized before being introduced into the cell.

[0508] In some implementations, the mRNA encoding a large-scale engineered nuclease is delivered to the cell because this reduces the likelihood of the gene encoding the large-scale engineered nuclease being integrated into the cell genome.

[0509] Such mRNA can be produced using methods known in the art, such as in vitro transcription. In some embodiments, the mRNA is produced using 7-methyl-guanosine, an anti-reverse cap analog (ARCA) (US7,074,596), or CleanCap. ®Analogs such as Cap1 analogs (Trilink, San Diego, CA) are 5' capped, or enzymatically capped using vaccinia virus capping enzymes or analogs. In some embodiments, the mRNA may be polyadenylated. The mRNA may contain various 5' and 3' untranslated sequence elements to enhance the expression of the encoded engineered wide-ranging nucleases and / or the stability of the mRNA itself. Non-limiting examples of such elements include: the 5' albumin (ALB) untranslated region (UTR) as shown in SEQ ID NO: 82 (e.g., encoded by SEQ ID NO: 18), the Kozak sequence as shown in SEQ ID NO: 84 (e.g., encoded by SEQ ID NO: 21), the 3' small nucleoribonucleoprotein B (SNRPB) UTR as shown in SEQ ID NO: 83 (e.g., encoded by SEQ ID NO: 19), and the poly-A termination sequence as shown in SEQ ID NO: 20. These elements may include, for example, post-translational regulatory elements, such as marmot hepatitis virus post-translational regulatory elements. mRNA may contain nucleoside analogs or naturally occurring nucleosides, such as pseudouridine, 5-methylcytidine, N6-methyladenosine, 5-methyluridine, or 2-thiouridine. Other nucleoside analogs include, for example, those described in US8,278,036. Because uridine-rich RNA sequences may trigger an innate immune response, mRNA can undergo uridine depletion. Polynucleotides containing nucleic acid sequences encoding the engineered wide range of nucleases described herein can be codon-optimized for expression in mammalian cells, and more specifically, for expression in specific tissues such as the liver.

[0510] In certain embodiments, the mRNA encoding the engineered large-scale nucleases described herein may be a polycistronic mRNA encoding two or more large-scale nucleases (as described herein) that are simultaneously expressed in the cell. In some embodiments, the polycistronic mRNA may encode at least a first and a second engineered large-scale nuclease, wherein the first engineered large-scale nuclease is the engineered large-scale nuclease described herein, and wherein the second engineered large-scale nuclease binds to and cleaves a second recognition sequence in the HBV genome that is different from SEQ ID NO: 3, such that the HBV genome is cleaved at multiple sites. In some embodiments, the polycistronic mRNA may encode the engineered large-scale nucleases described herein and at least one additional protein that induces a therapeutically beneficial effect in the cell. The polycistronic mRNA may contain any element known in the art that allows translation of two or more genes from the same mRNA molecule, including but not limited to IRES elements, T2A elements, P2A elements, E2A elements, and F2A elements.

[0511] In some embodiments, the method includes delivering the engineered wide-range nuclease (or its encoded nucleic acid) described herein, along with a nucleic acid containing a polynucleotide sequence encoding a suicide gene and a sequence homologous to a flanking sequence of the wide-range nuclease cleavage site, wherein the engineered wide-range nuclease binds to and cleaves a recognition sequence containing SEQ ID NO: 3 or composed thereof within the hepatitis B virus genome, thereby cleaving the HBV genome, wherein the suicide gene is inserted into the cleaved HBV genome via homologous recombination.

[0512] Suicide genes are products that encode cell death, either on their own or in the presence of other compounds. A representative example of such a suicide gene is the gene encoding thymidine kinase for herpes simplex virus. Other examples include genes encoding thymidine kinase for varicella-zoster virus and bacterial genes encoding cytosine deaminases that convert 5-fluorocytosine into the highly toxic compound 5-fluorouracil. Suicide genes also include, as non-limiting examples, genes encoding caspase-9, caspase-8, or cytosine deaminase. In some instances, caspase-9 can be activated using a specific chemically induced dimerizing agent (CID). In some embodiments, the suicide gene is directly lethal to HBV or target cells (e.g., HCC cells). In some such embodiments, the directly lethal suicide gene encodes a toxic polypeptide or apoptotic protein. In some embodiments, the suicide gene is indirectly lethal to target cells and directs the subject's own immune system to kill the target cells. In some such embodiments, the indirect lethal suicide gene encodes a cell surface protein that is recognized as a foreign substance by the subject's immune system and targeted by humoral or cellular immune responses. In other such embodiments, the indirect lethal suicide gene encodes a polypeptide presented by an MHC class I molecule that is recognized as a foreign substance by the subject's immune system and targeted by a cytotoxic immune response.

[0513] Purified, wide-ranging nuclease proteins can be delivered into cells to cleave genomic DNA through a variety of mechanisms known in the art, including those detailed below.

[0514] Target tissues for delivering the engineered wide-range nucleases of this disclosure include, but are not limited to, liver cells, such as hepatocytes or preferably primary hepatocytes, more preferably human hepatocytes or primary human hepatocytes, HepG2.2.15 or HepG2-hNTCP cells. As discussed, the wide-range nucleases disclosed herein can be delivered as purified proteins or as RNA or DNA encoding the wide-range nuclease. In one embodiment, the wide-range nuclease protein or the mRNA or DNA vector encoding the wide-range nuclease is provided to target cells (e.g., cells in the liver) by direct injection into the target tissue. Alternatively, the wide-range nuclease protein, mRNA, DNA, or cells expressing the wide-range nuclease can be delivered systemically via the circulatory system.

[0515] In some embodiments, a broad range of nuclease proteins or DNA / mRNA encoding a broad range of nucleases are coupled to cell-penetrating peptides or targeting ligands to facilitate cellular uptake. Examples of cell-penetrating peptides known in the art include polyarginine (Jearawiriyapaisarn et al., (2008) Mol Ther. 16:1624-9), TAT peptides from HIV (Hudecz et al., (2005) Med. Res. Rev. 25:679-736), MPG (Simeoni et al., (2003) Nucleic Acids Res. 31:2717-2724), Pep-1 (Deshayes et al., (2004) Biochemistry 43:7698-7706), and HSV-1 VP-22 (Deshayes et al., (2005) Cell Mol Life Sci. 62:1839-49). In an alternative implementation, the macronuclease protein or the DNA / mRNA encoding the macronuclease is covalently or non-covalently coupled to an antibody that recognizes a specific cell surface receptor expressed on a target cell, thereby binding the macronuclease protein / DNA / mRNA to the target cell and being internalized by the target cell. Alternatively, the macronuclease protein / DNA / mRNA may be covalently or non-covalently coupled to a natural ligand (or a portion of a natural ligand) of such a cell surface receptor (McCall et al., (2014) Tissue Barriers. 2(4):e944449; Dinda et al., (2013) Curr. Pharm. Biotechnol. 14:1264-74; Kang et al., (2014) Curr. Pharm. Biotechnol. 15(3):220-30; Qian et al., (2014) Expert Opin. Drug Metab Toxicol. 10(11):1491-508).

[0516] In some embodiments, a wide range of nuclease proteins or DNA / mRNA encoding a wide range of nucleases are encapsulated within a biodegradable hydrogel for injection or implantation into desired areas of the liver (e.g., near sinusoidal endothelial cells or hematopoietic endothelial cells, or progenitor cells differentiating into the aforementioned cells). The hydrogel can provide a sustained and tunable release of the therapeutic payload to desired areas of the target tissue without requiring frequent injections, and stimulus-responsive materials (e.g., temperature and pH-responsive hydrogels) can be programmed to release the payload in response to environmental or externally applied cues (Kang Derwent et al., (2008) Trans Am. Ophthalmol. Soc. 106:206-214).

[0517] In some embodiments, a large-scale nuclease protein or DNA / mRNA encoding a large-scale nuclease is covalently or preferably non-covalently coupled to or encapsulated within a nanoparticle using methods known in the art (Sharma et al., (2014) Biomed. Res. Int. 2014). The nanoparticles are nanoscale delivery systems with a length scale <1 μm, preferably <100 nm. Such nanoparticles can be designed using a core composed of metals, lipids (called lipid nanoparticles), polymers, or biomacromolecules, and multiple copies of the large-scale nuclease protein, mRNA, or DNA can be attached to or encapsulated within the nanoparticle core. This increases the number of protein / mRNA / DNA copies delivered to each cell and thus increases the intracellular expression of each large-scale nuclease to maximize the likelihood of target recognition sequence cleavage. The surface of such nanoparticles can be further modified with polymers or lipids (e.g., chitosan, cationic polymers, or cationic lipids) to form core-shell nanoparticles that confer additional functions on their surface, thereby enhancing cellular delivery and payload uptake (Jian et al., (2012) Biomaterials. 33(30): 7621-30). Nanoparticles can also be advantageously coupled to targeting molecules to direct the nanoparticles to suitable cell types and / or increase the likelihood of cellular uptake. Examples of such targeting molecules include antibodies specific to cell surface receptors and natural ligands (or portions of natural ligands) of cell surface receptors.

[0518] In some implementations, a wide range of nuclease proteins or DNA / mRNA encoding a wide range of nucleases are encapsulated in liposomes or cationic lipid complexes are used (see, for example, LIPOFECTAMINE™, Life Technologies Corp., Carlsbad, CA; Zuris et al., (2015) Nat. Biotechnol. 33:73-80; Mishra et al., (2011) J. Drug Deliv. 2011:863734). Liposomes and cationic liposome formulations can protect payloads from degradation, enhance accumulation and retention at target sites, and promote cellular uptake and delivery efficiency by fusing with and / or disrupting the cell membrane of target cells.

[0519] In some implementations, a wide range of nuclease proteins or DNA / mRNA encoding a wide range of nucleases are encapsulated within a polymer scaffold (e.g., PLGA) or in combination with cationic polymers (e.g., PEI, PLL) (Tamboli et al., (2011) Ther Deliv. 2(4):523-536). Polymer carriers can be engineered to provide tunable drug release rates by controlling polymer erosion and drug diffusion, and high drug encapsulation efficiency can provide protection of the therapeutic payload until intracellular delivery to the desired target cell population.

[0520] In some embodiments, a large-scale nuclease protein or DNA / mRNA encoding an engineered large-scale nuclease is combined with an amphiphilic molecule that self-assembles into micelles (Tong et al., (2007) J. Gene Med. 9(11):956-66). Polymer micelles may include a micelle shell formed of a hydrophilic polymer (e.g., polyethylene glycol) that can prevent aggregation, mask charge interactions, and reduce nonspecific interactions.

[0521] In some embodiments, a wide range of nuclease proteins or DNA / mRNA encoding a wide range of nucleases are formulated into emulsions or nanoemulsions (i.e., having an average particle size of <1 nm) for application and / or delivery to target cells. The term "emulsion" refers to (but is not limited to) any oil-in-water, water-in-oil, water-in-oil-in-water, or water-in-oil-in-oil dispersion or droplet, including lipid structures that can be formed as a result of hydrophobic forces when an immiscible phase is mixed with an aqueous phase, said hydrophobic forces driving nonpolar residues (e.g., long hydrocarbon chains) away from water and polar head groups toward water. These other lipid structures include, but are not limited to, monolayer, thin-layer, and multilayer lipid vesicles, micelles, and lamellar phases. Emulsions consist of an aqueous phase and a lipophilic phase (typically containing oils and organic solvents). Emulsions also frequently contain one or more surfactants. Nanoemulsion formulations are well known, for example, as described in U.S. Patent Nos. 6,015,832, 6,506,803, 6,635,676, 6,559,189 and 7,767,216, each of which is incorporated herein by reference in its entirety.

[0522] In some implementations, a wide range of nuclease proteins or DNA / mRNA encoding a wide range of nucleases are covalently or non-covalently associated with multifunctional polymer conjugates, DNA dendrimers, and polymeric dendrimers (Mastorakos et al., (2015) Nanoscale. 7(9):3845-56; Cheng et al., (2008) J. Pharm Sci. 97(1):123-43). The generation of dendrimers allows for control over effective load capacity and size, and can provide high drug loading capacity. Furthermore, the display of multiple surface groups can be utilized to improve stability, reduce nonspecific interactions, and enhance cell-specific targeting and drug release.

[0523] In some implementations, a recombinant virus (i.e., a recombinant viral vector) is used to introduce a polynucleotide containing a nucleic acid sequence encoding the engineered broad-spectrum nucleases described herein into cells. Such recombinant viruses are known in the art and include recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, and recombinant adeno-associated viruses (AAVs) (reviewed in Vannucci et al., (2013)). New Microbiol.36:1-22). Recombinant AAV may have any serotype that allows for viral transduction into target cell types and expression of a wide range of nuclease genes in the target cells. For example, in some embodiments, recombinant AAV has serotypes (i.e., capsids) of AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV12. In some embodiments, the viral vector is injected directly into the target tissue (e.g., liver tissue). In alternative embodiments, the viral vector is delivered systemically via the circulatory system. It is known in the art that different AAVs tend to localize to different tissues (Wang et al., Expert Opin Drug Deliv 11(3). 2014). In liver target tissues, for example, efficient transduction of hepatocytes has been demonstrated using AAV serotypes 2, 8, and 9 (Sands(2011) Methods Mol. Biol. 807:141-157). In some embodiments, the AAV serotype is AAV2. In some embodiments, the AAV serotype is AAV8. In some embodiments, the AAV serotype is AAV9. AAVs can also be self-complementary, such that they do not require second-strand DNA synthesis in the host cell (McCarty et al., (2001) Gene Ther. 8:1248-54).

[0524] The polynucleotides delivered via recombinant AAV may include left (5') and right (3') inverted terminal repeat sequences as part of the viral genome. In some embodiments, the recombinant virus is injected directly into the target tissue. In alternative embodiments, the recombinant virus is delivered systemically via the circulatory system.

[0525] In one embodiment, the recombinant virus used for the delivery of the macronuclease gene is a self-limiting recombinant virus. Because the viral genome contains recognition sequences for engineered macronucleases, self-limiting viruses can have a limited duration of existence in cells or organisms. Therefore, self-limiting recombinant viruses can be engineered to provide coding sequences for a promoter, the engineered macronuclease described herein, and a macronuclease recognition site within the ITR. The self-limiting recombinant virus delivers the macronuclease gene to cells, tissues, or organisms, causing the macronuclease to be expressed and capable of cleaving the cell's genome at an endogenous recognition sequence within the genome. The delivered macronuclease will also find its target site in the self-limiting recombinant virus genome and cleave the recombinant viral genome at that target site. Once cleaved, the 5' and 3' ends of the viral genome are exposed and degraded by exonucleases, thereby killing the virus and stopping the production of the macronuclease.

[0526] If a polynucleotide containing a nucleic acid sequence encoding an engineered macronuclease as described herein is delivered to a cell via a recombinant virus (e.g., AAV), the nucleic acid sequence encoding the engineered macronuclease can be operatively linked to a promoter. In some embodiments, this can be a viral promoter, such as an endogenous promoter from a recombinant virus (e.g., the LTR of a lentivirus) or a well-known early promoter of cytomegalovirus or SV40 virus. In certain embodiments, the nucleic acid sequence encoding the engineered macronuclease is operatively linked to a promoter that preferentially drives gene expression in target cells (e.g., hepatocytes). Examples of liver-specific promoters include, but are not limited to, the human α-1 antitrypsin promoter, heterozygous liver-specific promoters (the liver locus control region (ApoE-HCR) and the liver-specific α1-antitrypsin promoter from the ApoE gene), the human thyroxine-binding globulin (TBG) promoter, and the apolipoprotein A-II promoter.

[0527] In certain embodiments, the viral vector comprises a cassette containing a promoter and a nucleic acid sequence encoding the engineered broad-spectrum nuclease described herein. The viral vector may also comprise two or more cassettes, wherein at least a first cassette contains a promoter and a nucleic acid sequence encoding the engineered broad-spectrum nuclease described herein, and wherein at least a second cassette contains a promoter and a nucleic acid sequence encoding an engineered broad-spectrum nuclease specific to different HBV recognition sequences other than the HBV 11-12 recognition sequence. In some embodiments, the viral vector comprises a cassette containing a promoter and a polycistronic nucleic acid sequence, wherein the promoter drives the expression of the polycistronic nucleic acid sequence to produce polycistronic mRNA in target cells, such as polycistronic mRNA encoding the engineered broad-spectrum nuclease described herein.

[0528] In some embodiments, the engineered wide range of nucleases described herein, or polynucleotides (e.g., mRNA) containing nucleic acid sequences encoding the engineered wide range of nucleases described herein, are encapsulated in lipid nanoparticles and introduced into cells.

[0529] Some lipid nanoparticles considered for use comprise at least one cationic lipid, at least one non-cationic lipid, and at least one conjugated lipid. In more specific examples, lipid nanoparticles may comprise about 50 mol% to about 85 mol% of cationic lipids, about 13 mol% to about 49.5 mol% of non-cationic lipids, and about 0.5 mol% to about 10 mol% of lipid conjugates, and are produced in a non-layered (i.e., non-bilayer) morphology. In other specific examples, lipid nanoparticles may comprise about 40 mol% to about 85 mol% of cationic lipids, about 13 mol% to about 49.5 mol% of non-cationic lipids, and about 0.5 mol% to about 10 mol% of lipid conjugates, and are produced in a non-layered (i.e., non-bilayer) morphology.

[0530] Cationic lipids may include, for example, one or more of the following: palmitoyl-oleoyl-norarginine (PONA), MPDACA, GUADACA, ((6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate) (MC3), LenMC3, CP-LenMC3, γ-LenMC3, CP-γ-LenMC3, MC3MC, MC2MC, MC3 ether, MC4 ether, MC3 amide, Pan-MC3, Pan-MC4, and Pan MC5, 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-Dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane 2,2-Dilinyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-Dilinyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-Dilinyl-4-N-methylpiperazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2 -Dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleoylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleenylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane hydrochloride (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane hydrochloride (DLin-TAP).Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleenylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleenyloxy-N,N-dimethyl Aminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-diolenoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-diolenoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) N-(1,2-Dimyristoyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-diolenyloxy-N-[2-(spermine-formamido)ethyl]-N,N-dimethyl-1-propanetrimonium trifluoroacetate (DOSPA), di-octadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2- [5'-(cholest-5-en-3-β-oxy)-3'-oxaproloxy]-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleenylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), or mixtures thereof. Cationic lipids can also be DLinDMA, DLin-K-C2-DMA (“XTC2”), MC3, LenMC3, CP-LenMC3, γ-LenMC3, CP-γ-LenMC3, MC3MC, MC2MC, MC3 ether, MC4 ether, MC3 amide, Pan-MC3, Pan-MC4, PanMC5, or mixtures thereof.

[0531] In various embodiments, cationic lipids may comprise about 50 mol% to about 90 mol%, about 50 mol% to about 85 mol%, about 50 mol% to about 80 mol%, about 50 mol% to about 75 mol%, about 50 mol% to about 70 mol%, about 50 mol% to about 65 mol%, or about 50 mol% to about 60 mol% of the total lipids present in the particles.

[0532] In other embodiments, cationic lipids may comprise about 40 mol% to about 90 mol%, about 40 mol% to about 85 mol%, about 40 mol% to about 80 mol%, about 40 mol% to about 75 mol%, about 40 mol% to about 70 mol%, about 40 mol% to about 65 mol%, or about 40 mol% to about 60 mol% of the total lipids present in the particles.

[0533] Noncationic lipids may comprise, for example, one or more anionic lipids and / or neutral lipids. In a particular embodiment, the noncationic lipid comprises one of the following neutral lipid components: (1) cholesterol or a derivative thereof; (2) phospholipids; or (3) a mixture of phospholipids and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholesterolanol, cholesterol ketone, cholesterol ketone, coprosterol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, and mixtures thereof. Phospholipids can be neutral lipids, including but not limited to dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, ditransoleoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lecithinylcholine (EPC), and mixtures thereof. In some embodiments, the phospholipid is DPPC, DSPC, or a mixture thereof.

[0534] In some embodiments, non-cationic lipids (e.g., one or more phospholipids and / or cholesterol) may comprise about 10 mol% to about 60 mol%, about 15 mol% to about 60 mol%, about 20 mol% to about 60 mol%, about 25 mol% to about 60 mol%, about 30 mol% to about 60 mol%, about 10 mol% to about 55 mol%, about 15 mol% to about 55 mol%, about 20 mol% to about 55 mol%, about 25 mol% to about 55 mol%, about 30 mol% to about 55 mol%, about 13 mol% to about 50 mol%, about 15 mol% to about 50 mol%, or about 20 mol% to about 50 mol%. When the non-cationic lipids are a mixture of phospholipids and cholesterol or cholesterol derivatives, the mixture may comprise up to about 40, 50, or 60 mol% of the total lipids present in the particles.

[0535] Conjugated lipids that inhibit particle aggregation may comprise one or more of the following: polyethylene glycol (PEG)-lipid conjugates, polyamide (ATTA)-lipid conjugates, cationic polymer-lipid conjugates (CPL), or mixtures thereof. In one specific embodiment, the nucleic acid-lipid particles comprise a PEG-lipid conjugate or an ATTA-lipid conjugate. In some embodiments, the PEG-lipid conjugate or ATTA-lipid conjugate is used in conjunction with CPL. Conjugated lipids that inhibit particle aggregation may comprise PEG-lipids, including, for example, PEG-diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. PEG-DAA conjugates may be PEG-dilauroyloxypropyl (C12), PEG-dimyristoyloxypropyl (C14), PEG-dispalmitoyloxypropyl (C16), PEG-distearateyloxypropyl (C18), or mixtures thereof.

[0536] Other suitable PEG-lipid conjugates include, but are not limited to, mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglycerol (PEG-C-DOMG). The synthesis of PEG-C-DOMG is described in PCT application PCT / US08 / 88676. Other suitable PEG-lipid conjugates also include, but are not limited to, 1-[8'-(1,2-dimyristoyl-3-propoxy)-carbamoyl-3',6'-dioxaoctyl]carbamoyl-ω-methyl-poly(ethylene glycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in US Patent No. 7,404,969.

[0537] In some cases, the conjugated lipids that inhibit particle aggregation (e.g., PEG-lipid conjugates) may comprise about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol%, about 1 mol% to about 2 mol%, about 0.6 mol% to about 1.9 mol%, about 0.7 mol% to about 1.8 mol%, about 0.8 mol% to about 1.7 mol%, about 1 mol% to about 1.8 mol%, about 1.2 mol% to about 1.8 mol%, about 1.2 mol% to about 1.7 mol%, about 1.3 mol% to about 1.6 mol%, about 1.4 mol% to about 1.5 mol%, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol% (or any fraction thereof or range thereof) of the total lipids present in the particles. Typically, in such cases, the PEG moiety has an average molecular weight of about 2,000 Daltons. In other cases, the conjugated lipids that inhibit particle aggregation (e.g., PEG-lipid conjugates) may comprise about 5.0 mol% to about 10 mol%, about 5 mol% to about 9 mol%, about 5 mol% to about 8 mol%, about 6 mol% to about 9 mol%, about 6 mol% to about 8 mol%, or about 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, or 10 mol% (or any fraction thereof or range thereof) of the total lipids present in the particles. Typically, in such cases, the PEG moiety has an average molecular weight of about 750 Daltons.

[0538] In other embodiments, the composition may comprise amphoteric liposomes containing at least one positively charged carrier and at least one negatively charged carrier, the negatively charged carrier being different from the positively charged carrier, and the isoelectric point of the liposomes being 4-8. This is achieved due to the fact that the liposomes are prepared with a pH-dependent, variable charge.

[0539] For example, when the amount of cationic charge carriers in a membrane-forming or membrane-based system exceeds that of anionic charge carriers at low pH and this ratio reverses at higher pH, a liposome structure with the desired properties is formed. This is always the case when the pKa value of the ionizable component is between 4 and 9. As the pH of the medium decreases, all cationic charge carriers become more charged and all anionic charge carriers lose their charge.

[0540] Cationic compounds that can be used in amphoteric liposomes include those previously described above. Without limitation, strong cationic compounds may include, for example: DC-Chol 3-β-[N-(N',N'-dimethylmethane)carbamoyl]cholesterol, TC-Chol 3-β-[N-(N',N',N'-trimethylaminoethane)carbamoyl]cholesterol, BGSC biguanide-spermine-cholesterol, BGTC biguanide-tren-cholesterol, DOTAP (1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride, DOSPER (1,3-dioleoyloxy-2-(6-carboxy-spermine)-propionamide), DOTMA (1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride) (Lipofectin). ® ), DORIE (1,2-dioleoyloxypropyl)-3-dimethylhydroxyethylammonium bromide, DOSC (1,2-dioleoyl-3-succinoyl-sn-glycerolcholine ester), DOGSDSO (1,2-dioleoyl-sn-glycerol-3-succinoyl-2-hydroxyethyl ornithine disulfide), DDAB dimethyl di-octadecyl ammonium bromide, DOGS ((C18)2GlySper3+) N,N-di-octadecylamido-diol-spermine (Transfectam) ® (C18)2Gly+ N,N-di-octadecylamino-glycine, CTAB cetyltrimethylammonium bromide, CpyC cetylpyridine chloride, DOOPC 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine or other O-alkyl-phosphatidylcholine or ethanolamine, amides and phosphatidylethanolamine derived from lysine, arginine or ornithine.

[0541] Examples of weakly cationic compounds include, but are not limited to: His-Chol (histyl-cholesterol hemisuccinate), Mo-Chol (morpholino-N-ethylamino-cholesterol hemisuccinate), or histyl-PE.

[0542] Examples of neutral compounds include, but are not limited to: cholesterol, ceramide, phosphatidylcholine, phosphatidylethanolamine, tetraether lipids, or diacylglycerols.

[0543] Anionic compounds that can be used in amphoteric liposomes include the noncationic compounds described above. Without limitation, examples of weak anionic compounds may include CHEMS (cholesterol hemisuccinate), alkyl carboxylic acids having 8 to 25 carbon atoms, or diacylglycerol hemisuccinates. Other weak anionic compounds may include amides of aspartic acid or glutamic acid and PE, as well as PS and amides of glycine, alanine, glutamine, asparagine, serine, cysteine, threonine, tyrosine, glutamic acid, aspartic acid, or other amino acids or aminodicarboxylic acids. Following the same principle, esters of hydroxycarboxylic acids or hydroxydicarboxylic acids and PS are also weak anionic compounds.

[0544] In some embodiments, the amphoteric liposomes may comprise conjugated lipids, such as those described above. Specific examples of useful conjugated lipids include, but are not limited to, PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacoxypropane-3-amines. In particular, PEG-modified diacylglycerols and dialkylglycerols.

[0545] In some embodiments, neutral lipids may comprise about 10 mol% to about 60 mol%, about 15 mol% to about 60 mol%, about 20 mol% to about 60 mol%, about 25 mol% to about 60 mol%, about 30 mol% to about 60 mol%, about 10 mol% to about 55 mol%, about 15 mol% to about 55 mol%, about 20 mol% to about 55 mol%, about 25 mol% to about 55 mol%, about 30 mol% to about 55 mol%, about 13 mol% to about 50 mol%, about 15 mol% to about 50 mol%, or about 20 mol% to about 50 mol% of the total lipids present in the particles.

[0546] In some cases, the conjugated lipids that inhibit particle aggregation (e.g., PEG-lipid conjugates) may comprise about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol%, about 1 mol% to about 2 mol%, about 0.6 mol% to about 1.9 mol%, about 0.7 mol% to about 1.8 mol%, about 0.8 mol% to about 1.7 mol%, about 1 mol% to about 1.8 mol%, about 1.2 mol% to about 1.8 mol%, about 1.2 mol% to about 1.7 mol%, about 1.3 mol% to about 1.6 mol%, about 1.4 mol% to about 1.5 mol%, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol% (or any fraction thereof or range thereof) of the total lipids present in the particles. Typically, in such cases, the PEG moiety has an average molecular weight of about 2,000 Daltons. In other cases, the conjugated lipids that inhibit particle aggregation (e.g., PEG-lipid conjugates) may comprise about 5.0 mol% to about 10 mol%, about 5 mol% to about 9 mol%, about 5 mol% to about 8 mol%, about 6 mol% to about 9 mol%, about 6 mol% to about 8 mol%, or about 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, or 10 mol% (or any fraction thereof or range thereof) of the total lipids present in the particles. Typically, in such cases, the PEG moiety has an average molecular weight of about 750 Daltons.

[0547] Considering the total amount of neutral and conjugated lipids, the remainder of the amphoteric liposomes can contain a mixture of cationic and anionic compounds formulated in various ratios. The ratio of cationic to anionic lipids can be selected to achieve desired nucleic acid encapsulation properties, zeta potential, pKa, or other physicochemical properties that depend at least in part on the presence of charged lipid components.

[0548] In some implementations, lipid nanoparticles have a composition that specifically enhances delivery and uptake within the liver, particularly within hepatocytes.

[0549] Methods and compositions for delivering the wide range of nucleases described herein to the liver of an HBV-infected subject are provided. In one embodiment, native hepatocytes taken from a mammal can be transduced using a vector encoding an engineered wide range of nucleases. Alternatively, native hepatocytes from an HBV-infected subject can be transduced ex vivo using an adenovirus vector encoding an engineered wide range of nucleases (i.e., an AAV vector) and / or a molecule that stimulates liver regeneration (such as a hepatotoxin). Preferably, the hepatotoxin is uPA and is modified to inhibit its secretion from hepatocytes once expressed by the viral vector. In another embodiment, the vector encodes tPA, which can stimulate de novo hepatocyte regeneration. The transduced hepatocytes taken from the mammal can then be reinfused into the mammal, where conditions favorable for the expression of the engineered wide range of nucleases are provided. Typically, the transduced hepatocytes can be reinfused into the patient via infusion through the splenic or portal vascular system, and administration can be a single dose or multiple doses over a period of 1 to 5 days or more.

[0550] application In the in vivo aspect of the methods described herein, retroviruses, pseudoviruses, or adeno-associated viruses (i.e., AAVs) encoding engineered large-scale nucleases are constructed and administered to subjects. Administration of the vector encoding the engineered large-scale nuclease can be performed simultaneously with administration of an adenovirus vector encoding a hepatotoxic toxin or an adenovirus vector encoding tPA (which stimulates hepatocyte regeneration without acting as a hepatotoxic toxin).

[0551] In various embodiments of the methods and compositions described herein, engineered large-scale nucleases, those encoding polynucleotides, recombinant viruses containing such polynucleotides, or lipid nanoparticles containing such polynucleotides, as described herein, can be administered via any suitable route of administration known in the art. Such routes of administration may include, for example, intravenous, intramuscular, intraperitoneal, subcutaneous, intrahepatic, transmucosal, transcutaneous, intraarterial, and sublingual administration. In some embodiments, engineered large-scale nuclease proteins, those encoding polynucleotides, recombinant viruses containing such polynucleotides, or lipid nanoparticles containing such polynucleotides are supplied to target cells (e.g., hepatocytes) by direct injection into target tissue (e.g., liver tissue). Other suitable routes of administration may be readily determined by the treating physician as needed.

[0552] In some embodiments, a therapeutically effective amount of the engineered macronuclease described herein or its encoding polynucleotide is administered to a subject in need for the treatment of HBV infection or HBV infection-related disease. Such administration may continue until sAg is no longer detectable in the subject's serum or plasma. In some embodiments, after one or more administrations of one or more engineered macronucleases described herein, polynucleotides encoding such engineered macronucleases, lipid nanoparticles containing one or more polynucleotides encoding such engineered macronucleases, or recombinant viruses, optionally in conjunction with one or more other therapeutic agents described herein, the subject does not exhibit symptoms of HBV infection-related disease in the absence of antiviral treatment. In some embodiments, after one or more administrations of one or more engineered macronucleases described herein, polynucleotides encoding such engineered macronucleases, or carriers containing one or more polynucleotides encoding such engineered macronucleases, optionally in conjunction with one or more other therapeutic agents described herein, sAg is no longer detectable in the subject's serum or plasma in the absence of antiviral treatment.

[0553] In some embodiments, a pharmaceutical composition comprising a polynucleotide containing a nucleic acid sequence encoding an engineered broad-spectrum nuclease as described herein is administered to a subject, wherein the nucleic acid sequence is in the form of approximately 1 x 10^6 nucleotides. 10 gc / kg to approximately 1x10 14 gc / kg (e.g., about 1 x 10) 10 gc / kg, approximately 1x10 11 gc / kg, approximately 1x10 12 gc / kg, approximately 1x10 13 gc / kg or approximately 1x10 14 Administered at a dose of gc / kg. In some embodiments, the subject is given a pharmaceutical composition comprising a multinucleotide containing a nucleic acid sequence encoding an engineered broad-spectrum nuclease as described herein, wherein the nucleic acid sequence is expressed in approximately 1 x 10-1 gc / kg. 10 gc / kg, approximately 1x10 11 gc / kg, approximately 1x10 12 gc / kg, approximately 1x10 13 gc / kg or approximately 1x10 14 Administered at a dose of gc / kg. In some embodiments, the subject is given a pharmaceutical composition comprising a multinucleotide containing a nucleic acid sequence encoding an engineered broad-spectrum nuclease as described herein, wherein the nucleic acid sequence is in the form of approximately 1 x 10^6 g / kg. 10 gc / kg to approximately 1x10 11 gc / kg, approximately 1x10 11 gc / kg to approximately 1x10 12 gc / kg, approximately 1x1012 gc / kg to approximately 1x10 13 gc / kg or approximately 1x10 13 gc / kg to approximately 1x10 14 Dosage administration in gc / kg. It should be understood that these dosages may involve the administration of a single polynucleotide comprising a single nucleic acid sequence encoding a single engineered macronuclease as described herein, or alternatively, may involve a single polynucleotide comprising a first nucleic acid sequence encoding the first engineered macronuclease as described herein and a second nucleic acid sequence encoding the second engineered macronuclease as described herein, or a polynucleotide of an engineered macronuclease that is combined with and, in some embodiments, cleaves the recognition sequences within the HBV genome other than HBV 11-12, wherein each of the two encoding nucleic acid sequences is administered at the indicated dosage.

[0554] In some embodiments, a lipid nanoparticle formulation comprising mRNA containing a nucleic acid sequence encoding an engineered wide range of nucleases described herein is administered to a subject. In some such embodiments, the dose of mRNA is from about 0.1 mg / kg to about 3 mg / kg. In some embodiments, a lipid nanoparticle formulation comprising mRNA containing a nucleic acid sequence encoding an engineered wide range of nucleases described herein is administered to a subject, wherein the dose of mRNA is from about 0.1 mg / kg, about 0.25 mg / kg, about 0.5 mg / kg, about 0.75 mg / kg, about 1.0 mg / kg, about 1.5 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, or about 3.0 mg / kg. In some embodiments, a lipid nanoparticle formulation comprising mRNA containing a nucleic acid sequence encoding the engineered wide range of nucleases described herein is administered to a subject, wherein the dose of mRNA is about 0.1 mg / kg to about 0.25 mg / kg, about 0.25 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 0.75 mg / kg, about 0.75 mg / kg to about 1.0 mg / kg, about 1.0 mg / kg to about 1.5 mg / kg, about 1.5 mg / kg to about 2.0 mg / kg, about 2.0 mg / kg to about 2.5 mg / kg, or about 2.5 mg / kg to about 3.0 mg / kg.

[0555] When appropriate, the dosage or frequency of administration of engineered large-scale nucleases or their encoded polynucleotides may be adjusted during treatment based on the attending physician's judgment. Among other factors, the appropriate dosage depends on the specific characteristics of any AAV selected (e.g., serotype, etc.), any lipid nanoparticles selected, the route of administration, the subject being treated (i.e., the subject's age, weight, sex, and general condition), and the method of administration. Therefore, the appropriate dosage may vary from patient to patient. Those skilled in the art or the attending physician can readily determine the appropriate effective amount. Dosage treatment may be a single-dose regimen or, if multiple administrations are required, a multi-dose regimen. Furthermore, appropriate multiple doses may be administered to the subject. Those skilled in the art can readily determine the appropriate number of doses. Alternative routes of administration or balancing therapeutic effects with any side effects may need to be considered when adjusting the dosage.

[0556] 2.4 Pharmaceutical Composition In some embodiments, this disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an engineered macronuclease as described herein, or a pharmaceutically acceptable carrier and a polynucleotide as described herein encoding a nucleic acid sequence of an engineered macronuclease as described herein. Such polynucleotides may be, for example, mRNA or DNA as described herein. In some such examples, the polynucleotide in the pharmaceutical composition may be contained in lipid nanoparticles or may be contained in a recombinant virus (e.g., recombinant AAV). The pharmaceutical compositions of this disclosure may be used to treat subjects suffering from HBV, reduce HBV levels or proliferation, reduce at least one symptom of HBV infection-related disease, or treat HBV infection-related disease.

[0557] Pharmaceutical compositions can be designed or selected based on the genotype of the target HBV strain. As detailed herein, the broad-spectrum nucleases described herein are engineered to recognize and cleave recognition sequences in specific HBV genotypes. The HBV 11-12 broad-spectrum nucleases disclosed herein (e.g., SEQ ID NO: 5 and 6) recognize and cleave HBV 11-12 recognition sequences found in the genomes of at least HBV genotypes A, B, C, D, E, F, and G. In some embodiments, the pharmaceutical compositions described herein can be administered to subjects having any HBV genotype containing the recognition sequence shown in SEQ ID NO: 3.

[0558] Such pharmaceutical compositions can be prepared using known techniques. See, for example, Remington, The Science and Practice of Pharmacy (21). st(See Philadelphia, Lippincott, Williams & Wilkins, 2005). In preparing pharmaceutical formulations according to this disclosure, the engineered wide-ranging nucleases described herein, those encoding polynucleotides, or cells expressing them are typically mixed with a pharmaceutically acceptable carrier, and the resulting composition is administered to a subject. The carrier must be acceptable in terms of compatibility with any other components of the formulation and must not be harmful to the subject. The carrier may be solid or liquid, or both, and may be formulated with the compound into a unit-dose formulation.

[0559] In some embodiments, the pharmaceutical compositions disclosed herein may also comprise one or more additional pharmaceutical agents or biomolecules that can be used to treat a disease in the subject. Similarly, the additional pharmaceutical agents and / or biomolecules may be administered as separate compositions.

[0560] Given that the engineered large-scale nucleases disclosed herein can have improved (i.e., enhanced) specificity leading to reduced off-target cleavage, and enhanced (i.e., enhanced) efficiency of cleavage and indel formation, particularly in cells containing an integrated copy of the HBV genome, in some embodiments, the pharmaceutical compositions disclosed herein containing optimized engineered large-scale nucleases, their encoding nucleic acid sequences, or cells expressing them also have improved (i.e., enhanced) efficacy in treating HBV infection, reducing HBV levels or proliferation, reducing at least one symptom of HBV infection-related disease, or treating HBV infection-related disease in subjects, compared to pharmaceutical compositions containing HBV 11-12L.1090QQ adapter 1 large-scale nucleases.

[0561] In certain embodiments, the pharmaceutical compositions disclosed herein may include a combination of engineered macronucleases (or nucleic acids encoding engineered macronucleases or cells expressing engineered macronucleases) as described herein. In other embodiments, the pharmaceutical composition may include at least two engineered macronucleases (or nucleic acids encoding engineered macronucleases or cells expressing engineered macronucleases), wherein at least the first engineered macronuclease is a macronuclease as described herein that binds to and cleaves the HBV 11-12 recognition sequence, and wherein at least the second engineered macronuclease binds to and cleaves a second recognition sequence in the HBV genome other than the HBV 11-12 recognition sequence, such that a single pharmaceutical composition is broadly applicable to the treatment of a wide variety of HBV genotypes and / or genotype isolates in a subject. Similarly, in other embodiments, the pharmaceutical composition may include polycistronic mRNA (or a recombinant DNA construct or viral vector having a cassette that produces polycistronic mRNA upon expression) encoding multiple engineered macronucleases as described herein. In other embodiments, the pharmaceutical composition may include a polycistronic mRNA encoding at least two engineered macronucleases (or a recombinant DNA construct or viral vector having a cassette that produces polycistronic mRNA upon expression), wherein at least the first engineered macronuclease is a macronuclease described herein that binds to and cleaves the HBV 11-12 recognition sequence, and wherein at least the second engineered macronuclease binds to and cleaves a second recognition sequence in the HBV genome other than the HBV 11-12 recognition sequence. Such pharmaceutical compositions are also broadly applicable for treating a wide variety of HBV genotypes and / or genotype isolates in subjects. In either case, such pharmaceutical compositions may be used as a single treatment when a particular HBV genotype or isolate is known or unknown in the subject.

[0562] For example, pharmaceutical compositions comprising multiple different engineered macronucleases (including at least one engineered macronuclease described herein) or comprising nucleic acid molecules encoding multiple different engineered macronucleases (including at least one engineered macronuclease described herein) can be administered to patients infected with multiple HBV genotypes or infected with unknown HBV genotypes. Therefore, in situations where resources do not allow for accurate HBV genotyping and where rapid and extensive treatment regimens are required, providing pharmaceutical compositions with multiple different engineered macronucleases or comprising nucleic acid molecules encoding multiple different engineered macronucleases offers a flexible option for the treatment and control of HBV infection.

[0563] The pharmaceutical compositions described herein may comprise a therapeutically effective amount of any engineered broad-spectrum nuclease described herein or any polynucleotide described herein encoding any engineered broad-spectrum nuclease described herein. For example, in some embodiments, the pharmaceutical composition may comprise any dose (e.g., gc / kg of the encoding nucleic acid sequence or mg / kg of mRNA) of the polynucleotide described herein.

[0564] In some embodiments, the pharmaceutical composition may also contain one or more additional agents that can be used to treat HBV infection or HBV-related diseases in the subject.

[0565] This disclosure also provides the engineered macronuclease described herein (or cells encoding nucleic acids or expressing macronucleases) for use as a medicament. This disclosure further provides the use of the engineered macronuclease described herein (or cells encoding nucleic acids or expressing macronucleases) in the preparation of medicaments for treating HBV infection, reducing HBV levels or proliferation, reducing symptoms of HBV-related diseases, or treating HBV-related diseases.

[0566] 2.5 Engineered Wide Range of Nuclease Variants Embodiments of this disclosure include the engineered wide-ranging nucleases described herein and their variants. Further embodiments of this disclosure include polynucleotides comprising nucleic acid sequences encoding the wide-ranging nucleases described herein, and variants of such polynucleotides.

[0567] As used herein, “variant” is intended to refer to substantially similar sequences. A “variant” polypeptide is intended to refer to a polypeptide derived from a “natural” polypeptide by deleting or adding one or more amino acids at one or more internal sites of the natural protein and / or by substituting one or more amino acids at one or more sites of the natural polypeptide. As used herein, a “natural” polynucleotide or polypeptide contains a variant derived from its parental sequence. The variant polypeptides covered by the embodiments are biologically active. That is, they retain the desired biological activity of the natural protein; namely, the ability to recognize and cleave the HBV 11-12 recognition sequence (SEQ ID NO: 3) within the hepatitis B virus genome, and in some embodiments, exhibit at least one improved property compared to previously described engineered HBV macronucleases (e.g., HBV 11-12 L.1090QQ linker 1 macronuclease), such as improved (i.e., increased) specificity and enhanced (i.e., increased) cleavage and indel formation efficiency. Such variants can be produced, for example, by artificial manipulation. The bioactive variants of the natural polypeptides (e.g., SEQ ID NO: 5 and 6) of the embodiments, or the bioactive variants of the recognition half-site binding subunits described herein, will have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the amino acid sequence of the natural polypeptide, natural subunit, natural HVR1 region, and / or natural HVR2 region, as determined by the sequence alignment procedures and parameters described elsewhere herein. The bioactive variants of the peptide or subunit of the embodiment may differ from the peptide or subunit by as few as about 1-40 amino acid residues, as few as about 1-20, as few as about 1-10, as few as about 5, as few as 4, 3, 2 or even 1 amino acid residue.

[0568] The peptides of the implementation scheme can be modified in various ways, including amino acid substitution, deletion, truncation, and insertion. Methods for these manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by mutations in DNA. Methods for mutagenesis and polynucleotide alteration are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al., (1987) Methods Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds., (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York), and the references cited therein. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), which is incorporated herein by reference. Conservative substitutions (such as exchanging one amino acid with another amino acid with similar properties) are likely optimal.

[0569] In some embodiments, the engineered large-scale nucleases described herein may include variants of the HVR1 and HVR2 regions described herein. The parental HVR region may contain, for example, residues 24-79 or 204-259 of the exemplary engineered large-scale nuclease. Thus, the variant HVR region may contain an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the amino acid sequence corresponding to residues 24-79 or 204-259 of the engineered large-scale nuclease exemplified herein, such that the variant HVR region maintains the biological activity of the engineered large-scale nuclease (i.e., binding and cleaving of the recognition sequence). Furthermore, in some embodiments of this disclosure, the variant HVR1 region or variant HVR2 region may contain residues corresponding to amino acid residues found at specific positions in the parental HVR. In this context, "corresponding to" means that the amino acid residue in the variant HVR is the same amino acid residue (i.e., a single identical residue) that exists at the same relative position (i.e., relative to the rest of the amino acids in the parental sequence) in the parental HVR sequence. For example, if the parental HVR sequence contains a serine residue at position 26, then the variant HVR that "contains a residue corresponding to residue 26" also contains a serine residue at a position relative to (i.e., corresponding to) parental position 26.

[0570] In a particular embodiment, the engineered wide-range nuclease described herein comprises HVR1, which has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the amino acid sequence corresponding to residues 204-259 of SEQ ID NO: 5 or 6.

[0571] In some embodiments, the engineered wide-range nuclease described herein comprises an HVR2 region having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 5 or 6.

[0572] Numerous amino acid modifications to the DNA recognition domain of wild-type I-CreI macronucleases have previously been identified (e.g., US 8,021,867), which, individually or in combination, produce engineered macronucleases with specificity at a single base altered within a half-site of the DNA recognition sequence, resulting in rationally designed macronucleases with half-site specificity distinct from that of the wild-type enzyme. Table 1 provides potential substitutions that can be performed in engineered macronuclease monomers or subunits to enhance specificity based on the bases present at each half-site position (-1 to -9) of the recognition half-site.

[0573] Table 1.

[0574] Bold items are wild-type contact residues and do not constitute "modifications" as used herein. An asterisk indicates that the residue is in contact with a base on the antisense strand.

[0575] Certain modifications can be made to engineered wide-range nuclease monomers or subunits to modulate DNA binding affinity and / or activity. For example, the engineered wide-range nuclease monomers or subunits described herein may contain G, S, or A at residues corresponding to position 19 of I-CreI or position 199 of SEQ ID NO: 5 or 6 (WO 2009 / 001159), Y, R, K, or D at residues corresponding to position 66 of I-CreI or position 246 of SEQ ID NO: 5 or 6, and / or E, Q, or K at residues corresponding to position 80 of I-CreI or position 260 of SEQ ID NO: 5 or 6 (US Patent No. 8,021,867).

[0576] For polynucleotides, a “variant” includes the deletion and / or addition of one or more nucleotides at one or more sites within a native polynucleotide. Those skilled in the art will recognize that variants of the nucleic acids of the embodiments are constructed such that an open reading frame is maintained. For polynucleotides, conserved variants include those sequences that encode the amino acid sequence of one of the polypeptides of the embodiments due to the degeneracy of the genetic code. Variant polynucleotides include synthetically derived polynucleotides, for example, those produced by site-directed mutagenesis but still encoding a recombinant nuclease of the embodiments. Generally, variants of a particular polynucleotide of the embodiments have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher sequence identity with that particular polynucleotide, as determined by the sequence alignment procedures and parameters described elsewhere herein. Variants of a specific polynucleotide (i.e., the reference polynucleotide) in an implementation scheme can also be assessed by comparing the percentage of sequence identity between a polypeptide encoded by the variant polynucleotide and a polypeptide encoded by the reference polynucleotide.

[0577] Deletion, insertion, and substitution of the protein sequences included herein are not expected to result in fundamental changes to the peptide characteristics. However, when the exact effect of a substitution, deletion, or insertion is difficult to predict before it is performed, those skilled in the art will understand that the effect will be evaluated by screening for the expected activity of the peptide. For example, screening for variants of engineered broad-spectrum nucleases that preferentially bind to and cleave the HBV 11-12 recognition sequence within the hepatitis B virus genome.

[0578] 2.6 Combination Therapy for HBV In some embodiments, a method for treating HBV infection or HBV-related disease in humans who are infected or at risk of infection is provided, comprising administering to a human a therapeutically effective amount of the engineered macronuclease described herein or its encoded nucleic acid, in combination with a therapeutically effective amount of one or more additional therapeutic agents (e.g., an agent in combination with one, two, three, four, one or two, one to three, or one to four additional therapeutic agents, or administered co-administered). In one embodiment, a method for treating HBV infection or HBV-related disease in humans who are infected or at risk of infection is provided, comprising administering to a human a therapeutically effective amount of the engineered macronuclease described herein or its encoded nucleic acid, in combination with a therapeutically effective amount of one or more (e.g., one, two, three, four, one or two, one to three, or one to four) additional therapeutic agents.

[0579] In some embodiments, this disclosure provides a method of treating HBV infection or HBV-related disease, comprising administering to a patient in need a therapeutically effective amount of the engineered wide-ranging nuclease described herein or its encoded nucleic acid, in combination with a therapeutically effective amount of one or more (e.g., one, two, three, four, one or two, one to three, or one to four) additional therapeutic agents suitable for treating HBV infection or HBV-related disease.

[0580] In some embodiments, the engineered large-scale nuclease described herein or its encoding nucleic acid is combined with one, two, three, four, or more additional therapeutic agents. In some embodiments, the engineered large-scale nuclease described herein or its encoding nucleic acid is combined with two additional therapeutic agents. In other embodiments, the engineered large-scale nuclease described herein or its encoding nucleic acid is combined with three additional therapeutic agents. In a further embodiment, the engineered large-scale nuclease described herein or its encoding nucleic acid is combined with four additional therapeutic agents. The one, two, three, four, or more additional therapeutic agents may be different therapeutic agents selected from the same class of therapeutic agents, and / or they may be selected from different classes of therapeutic agents.

[0581] Administration of HBV combination therapy In some embodiments, when the engineered wide-ranging nucleases described herein or their encoded nucleic acids are combined with one or more additional therapeutic agents as described above, the components of the composition are administered in a simultaneous or sequential manner. When administered sequentially, the composition may be administered in two or more doses.

[0582] The co-administration of the engineered large-scale nuclease or its encoded nucleic acid described herein with one or more other therapeutic agents generally refers to the simultaneous or sequential administration of the engineered large-scale nuclease or its encoded nucleic acid as described herein with one or more other therapeutic agents, such that a therapeutically effective amount of each agent is present in the patient.

[0583] Co-administration includes administering a unit dose of the compound described herein before or after administering a unit dose of one or more other therapeutic agents. The engineered large-scale nuclease or its encoded nucleic acid described herein may be administered within seconds, minutes, or hours after administering one or more other therapeutic agents. For example, in some embodiments, a unit dose of the engineered large-scale nuclease or its encoded nucleic acid described herein is administered first, followed by a unit dose of one or more other therapeutic agents within seconds or minutes. Alternatively, in other embodiments, a unit dose of one or more other therapeutic agents is administered first, followed by a unit dose of the engineered large-scale nuclease or its encoded nucleic acid described herein within seconds or minutes. In some embodiments, a unit dose of the engineered large-scale nuclease or its encoded nucleic acid is administered first, followed by a unit dose of one or more other therapeutic agents after a period of several hours (e.g., 1-12 hours). In other embodiments, a unit dose of one or more other therapeutic agents is administered first, followed by a unit dose of the engineered large-scale nuclease or its encoded nucleic acid after a period of several hours (e.g., 1-12 hours).

[0584] In some implementations, the engineered wide-range nucleases described herein or their encoded nucleic acids are combined with one or more additional therapeutic agents in a single dosage form for simultaneous administration to a patient.

[0585] HBV combination therapy The engineered wide-range nucleases or their encoded nucleic acids described herein may be combined or co-administered with one, two, three, four or more other therapeutic agents selected from the following: HBV combination drugs, HBV vaccines, HBV DNA polymerase inhibitors, immunomodulators, Toll-like receptor (TLR) modulators, interferon α receptor ligands, hyaluronidase inhibitors, HBV antigen inhibitors (e.g., HBV core antigen (HBcAg) inhibitors, HBV surface antigen (HBsAg) inhibitors, HBx inhibitors, HBV E antigen inhibitors), anti-HBV antigen antibodies, HBV-targeting inhibitory nucleic acids (e.g., antisense oligonucleotides, short interfering RNA (siRNA), DNA-directed RNA interference (ddRNAi)), HBsAg secretion or assembly inhibitors, HBV virus entry inhibitors, immune checkpoint inhibitors, cytotoxic T lymphocyte-associated protein 4 (CTLA4) inhibitors, cyclin inhibitors, endonuclease modulators, ribonucleotide reductase inhibitors, and covalently closed circular DNA. (cccDNA) inhibitors, farnesoid X receptor (FXR) agonists, STING agonists, anti-HBV antibodies, CCR2 chemokine antagonists, thymosin agonists, cytokines, nucleoprotein regulators, retinoic acid-induced gene 1 stimulators, NOD2 stimulators, phosphatidylinositol 3-kinase (PI3K) inhibitors, indoleamine-2,3-dioxygenase (IDO) pathway inhibitors, PD-1 inhibitors, PD-L1 inhibitors, recombinant thymosin α-1, Bruton's tyrosine kinase (BTK) inhibitors, KDM inhibitors, HBV replication inhibitors, arginase inhibitors, gene therapy and cell therapy, gene editors, cell therapy, TCR-T cell therapy, and other HBV drugs.

[0586] In some embodiments, the engineered wide range of nucleases or their encoded nucleic acids described herein may be used or combined with one or more of the following: chemotherapeutic agents, immunomodulators, immunotherapeutic agents, therapeutic antibodies, therapeutic vaccines, bispecific antibodies and “antibody-like” therapeutic proteins (such as DARPins®, anti-pMHC TCR-like antibodies, DARTs®, Duobodies®, Bits®, XmAbs®, TandAbs®, Fab derivatives), antibody-drug conjugates (ADCs), gene modifiers or gene editors targeting HBV (e.g., CRISPR-Cas (e.g., Cas9, Cas12, Cascade, Cas13), zinc finger nucleases, homing nucleases (e.g., engineered I-CreI variant wide range of nucleases), synthetic nucleases, TALENs, cell therapies (e.g., T cells, NK cells, macrophages with chimeric antigen receptors (CARs)) and TCR-T (engineered T cell receptors) or any combination thereof.

[0587] In some embodiments, the engineered broad-spectrum nuclease described herein is combined with one or more additional engineered nucleases that bind to and cleave a second recognition sequence in the HBV genome that is different from the HBV 11-12 recognition sequence. For example, one, two, three, or more additional engineered nucleases (e.g., one, two, three, or more engineered broad-spectrum nucleases) (that bind to and cleave recognition sequences other than HBV 11-12) may be combined with the engineered broad-spectrum nuclease disclosed herein (e.g., SEQ ID NO: 5 or 6). One or more additional engineered nucleases may bind to and cleave recognition sequences found at any location within the HBV genome, including one or more HBV gene coding or non-coding sequences. For example, one or more additional engineered nucleases can bind to and cleave recognition sequences within the HBV genome sequence (including, but not limited to, gene S encoding the major hepatitis B surface antigen (HBsAg) protein; the sequence upstream of gene S encoding the pre-S domain; gene C encoding the hepatitis B core antigen (HBcAg); the P region encoding the viral reverse transcriptase; gene X encoding the HBx viral protein; and the pre-core region encoding the HBeAg gene). In some embodiments, one or more additional engineered nucleases bind to and cleave the recognition sequences described in PCT / US2017 / 056638. In some embodiments, one or more additional nucleases are engineered I-CreI-derived broad-spectrum nucleases, such as those described in PCT / US2017 / 056638 or variants of the engineered broad-spectrum nucleases described therein, wherein one or more additional engineered broad-spectrum nucleases bind to and cleave recognition sequences other than HBV 11-12 (SEQ ID NO: 3).

[0588] In some embodiments, the engineered wide-range nucleases described herein or their encoding nucleic acids are optionally combined with or co-administered with one, two, three, four or more other therapeutic agents, such as 3-dioxygenase (IDO) inhibitors, apolipoprotein A1 modulators, arginase inhibitors, B lymphocyte and T lymphocyte attenuator inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, CCR2 chemokine antagonists, CD137 inhibitors, CD160 inhibitors, CD305 inhibitors, CD4 agonists and modulators, compounds targeting hepatitis B core antigen (HBcAg), core protein allosteric modulators, covalently closed circular DNA (cccDNA) inhibitors, cyclophilin inhibitors, cytotoxic T lymphocyte-associated protein 4 (CTLA4) inhibitors, DNA polymerase inhibitors, endonuclease modulators, epigenetic modifiers, farnesol X receptor (FXR) agonists, HBV DNA polymerase inhibitors, HBV replication inhibitors, and HBV RNase inhibitors, HBV virus entry inhibitors, HBx inhibitors, hepatitis B large envelope protein modulators, hepatitis B large envelope protein stimulators, hepatitis B structural protein modulators, hepatitis B surface antigen (HBsAg) inhibitors, hepatitis B surface antigen (HBsAg) secretion or assembly inhibitors, hepatitis B virus e antigen inhibitors, hepatitis B virus replication inhibitors, hepatitis virus structural protein inhibitors, HIV-1 reverse transcriptase inhibitors, hyaluronidase inhibitors, apoptosis protein family protein inhibitors (IAPs) inhibitors, IL-2 agonists, IL-7 agonists, immunomodulators, indoleamine-2 inhibitors, ribonucleotide reductase inhibitors, interleukin-2 ligands, ipi4 inhibitors, lysine demethylase inhibitors, histone demethylase inhibitors, KDM1 inhibitors, KDM5 inhibitors, cytotoxic cell lectin-like receptor G subfamily member 1 inhibitors, lymphocyte activation gene 3 inhibitors, lymphotoxic agents. β-receptor activators, Axl modulators, B7-H3 modulators, B7-H4 modulators, CD160 modulators, CD161 modulators, CD27 modulators, CD47 modulators, CD70 modulators, GITR modulators, HEVEM modulators, ICOS modulators, Mer modulators, NKG2A modulators, NKG2D modulators, OX40 modulators, SIRPalpha modulators, TIGIT modulators, Tim-4 modulators, Tyro modulators, Na+-taurocholic acid cotransporter (NTCP) inhibitors, natural killer cell receptor 2B4 inhibitors, NOD2 gene stimulators, nucleoprotein inhibitors, nucleoprotein modulators, OX-40 receptor agonists, PD-1 inhibitors, PD-L1 inhibitors, peptidylprolyl isomerase inhibitors, phosphatidylinositol-3 kinase (PI3K) inhibitors, retinoic acid-induced gene 1 stimulators, reverse transcriptase inhibitors, ribonuclease inhibitors, RNADNA polymerase inhibitors, SLC10A1 gene inhibitors, SMAC mimics, Src tyrosine kinase inhibitors, interferon gene stimulators (STING) agonists, NOD1 stimulators, T cell surface glycoprotein CD28 inhibitors, T cell surface glycoprotein CD8 regulators, thymosin agonists, thymosin α1 ligands, Tim-3 inhibitors, TLR-3 agonists, TLR-7 agonists, TLR-9 agonists, TLR9 gene stimulators, Toll-like receptor (TLR) regulators, viral ribonucleotide reductase inhibitors, and combinations thereof.

[0589] HBV inhibitory antiviral drugs In various embodiments, the engineered large-scale nuclease described herein, or the nucleic acid it encodes, is combined with or co-administered with one or more antiviral agents. In some embodiments, one or more antiviral agents are selected from lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT), tenofovir disoproxil fumarate (TDF), tenofovir disoproxil fumarate, emtricitabine (TRUVADA®), tenofovir alafenamide (TAF or VEMLIDY®), and ledipasvir and sofosbuvir (HARVONI®). In some embodiments, the engineered large-scale nuclease, or the nucleic acid it encodes, is combined with a long-acting form of an anti-HBV drug. Illustrative long-acting forms of anti-HBV drugs that can be combined include entecavir (subcutaneous reservoir type), long-acting tenofovir (TFD and TAF) implants (devices), or subcutaneous reservoir types. Case studies of long-acting entecavir are described in Henry et al., Eur J Pharm Sci. 2019 Aug 1;136:104958.

[0590] Other HBV medications Examples of other medications used to treat HBV infection or HBV-related conditions include alpha-hydroxytophenone, amadoxovir, duroquinol, beta-hydroxycytosine nucleoside, ARB-199, CCC-0975, ccc-R08, evitabine, ezetimibe, cyclosporine A, gentiopicroside, HH-003, hapratide, JNJ-56136379, nitrozonide, birenapa, NJK14047, NOV-205 (molixan, BAM-205), oligotide, mivodiamide, feron, GST-HG-131, levamisole, cassuccinate, alloferon, WS-007, Y-101 (tifentazone), rSIFN-co, PEG-IIFNm, KW-3, BP-Inter-014, oleanolic acid, HepB-nRNA, cTP-5 (rTP-5), HSK-II-2, HEISCO-106-1, HEISCO-106, Hepbarna, IPB-006IA, Hepuyinfen, DasKloster 0014-01, ISA-204, Jiangantai (Ganxikang), MIV-210, OB-AI-004, PF-06, berberine, DasKloster-0039, hepulantai, IMB-2613, TCM-800B, reduced glutathione, RO-6864018, RG-7834, QL-007, sofosbuvir, ledipasvir, UB-551, and ZH-2N, as well as US20150210682 (Roche), US 2016 / 0122344 (Roche), WO2015173164, WO2016023877, US2015252057A (Roche), WO16128335A1 (Roche), and WO16120186A1 Compounds disclosed in (Roche), US2016237090A (Roche), WO16107833A1 (Roche), WO16107832A1 (Roche), US2016176899A (Roche), WO16102438A1 (Roche), WO16012470A1 (Roche), US2016220586A (Roche) and US2015031687A (Roche).

[0591] HBV vaccine HBV vaccines include both preventative and therapeutic vaccines. Examples of HBV preventative vaccines include Vaxelis, Hexaxim, Heplisav, Mosquirix, DTwP-HBV vaccine, Bio-Hep-B, D / T / P / HBV / M (LBVP-0101; LBVW-0101), DTwP-Hepb-Hib-IPV vaccine, Heberpenta L, DTwP-HepB-Hib, V-419, CVI-HBV-001, Tetrabhay, Hepatitis B preventative vaccine (Advax Super D), Hepatrol-07, GSK-223192A, ENGERIX B®, recombinant hepatitis B vaccine (intramuscular, Kangtai Biological Products), and recombinant hepatitis B vaccine (Hansenula polymorpha yeast, intramuscular, Hualan Biological). Engineering), Recombinant Hepatitis B Surface Antigen Vaccine, Bimmugen, CARG-101, Eufravac, Eutravac, Anrix-DTaP-IPV-Hep B, HBAI-20, Infanrix-DTaP-IPV-Hep B-Hib, Pentabio Vaksin DTP-HB-Hib, Comvac 4, Twinrix, Euvax-B, Tritanrix HB, InfanrixHep B, Comvax, DTP-Hib-HBV Vaccine, DTP-HBV Vaccine, Yi Tai, Heberbiovac HB, Trivac HB, GerVax, DTwP-Hep B-Hib Vaccine, Bilive, Hepavax-Gene, SUPERVAX, Comvac5, Shanvac-B, Hepbulin, Recombivax HB, Revac B mcf, Revac B+, Fendrix, DTwP-HepB-Hib, DNA-001, Shan5, Shan6, rhHBsAG vaccine, HBI pentavalent vaccine, LBVD, Infanrix HeXa, YS-HBV-001 and DTaP-rHB-Hib vaccine.

[0592] Examples of therapeutic HBV vaccines include HBsAG-HBIG complex, ARB-1598, Bio-Hep-B, NASVAC, abi-HB (intravenous), ABX-203, Tetrabhay, GX-110E, GS-4774, peptide vaccine (epsilonPA-44), Hepatrol-07, NASVAC (NASTERAP), IMP-321, BEVAC, Revac B mcf, Revac B+, MGN-1333, KW-2, CVI-HBV-002, AltraHepB, VGX-6200, FP-02, FP-02.2 (HepTcell), NU-500, HBVax, im / TriGrid / antigen vaccine, Mega-CD40L adjuvanted vaccine, HepB-v, RG7944 (INO-1800), and therapeutic vaccines based on recombinant VLPs (HBV infection, VLP). Biotech), AdTG-17909, AdTG-17910, AdTG-18202, ChronVac-B, TG-1050, VVX-001, GSK-3528869A (ChAd155-hli-HBV + MVA-HBV + Hbc-HBs / AS01B-4), VBI-2601, VTP-300 (ChAdOx1-SIi-HBV-CPmut-TPA-Ssh primary immunization and MVA-SIi-HBV-CPmut-TPA-Ssh booster), MVA-BN, and Lm HBV. HBV isovirus vaccines are described, for example, in WO2017076988 and WO2017198726.

[0593] HBV DNA polymerase inhibitors Examples of HBV DNA polymerase inhibitors include adefovir (HEPSERA®), emtricitabine (EMTRIVA®), tenofovir disoproxil fumarate (VIREAD®), tenofovir alafenamide, tenofovir, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, tenofovir disoproxil fumarate, tenofovir disoproxil fumarate, tenofovir octadecyloxyethyl ester, CMX-157, tenofovir alafenamide, bexifovir, entecavir (BARACLUDE®), and entecavir maleate. Ticovir, telbivudine (TYZEKA®), filocilovir, pradefovir, clavidine, ribavirin, lamivudine (EPIVIR-HBV®), phosphatidylcholine, faxil, fusolin, metacavir, SNC-019754, FMCA, AGX-1009, AR-II-04-26, HIP-1302, tenofovir disoproxil fumarate aspartate, tenofovir disoproxil fumarate orotate, and HS-10234.

[0594] Immunomodulators Examples of immunomodulators include rintatolimod, imidol hydrochloride, ingaron, dermaVir, hydroxychloroquine sulfate (hydroxychloroquine), proleukin, hydroxyurea, mycophenolic acid (MPA) and its ester derivative mycophenolic ester (MMF), JNJ-440, WF-10, AB-452, ribavirin, IL-12, INO-9112, polymeric polyethyleneimine (PEI), Gepon, VGV-1, MOR-22, CRV-431, JNJ-0535, TG-1050, ABI-H2158, BMS-936559, GS-9688, RO-7011785, RG-7854, RO-6871765, AIC-649, and IR-103.

[0595] Toll-like receptor (TLR) agonists In various embodiments, the engineered wide range of nucleases or their encoding nucleic acids described herein are combined with agonists of Toll-like receptors (TLRs), such as agonists of TLR1 (NCBI Gene ID: 7096), TLR2 (NCBI Gene ID: 7097), TLR3 (NCBI Gene ID: 7098), TLR4 (NCBI Gene ID: 7099), TLR5 (NCBI Gene ID: 7100), TLR6 (NCBI Gene ID: 10333), TLR7 (NCBI Gene ID: 51284), TLR8 (NCBI Gene ID: 51311), TLR9 (NCBI Gene ID: 54106) and / or TLR10 (NCBI Gene ID: 81793), TLR11, TLR12 and TLR13. Examples of TLR3 modifiers include rintatolimod, poly-ICLC, RIBOXXON®, Apoxxim, RIBOXXIM®, IPH-33, MCT-465, MCT-475, and ND-1.1.

[0596] Examples of TLR7 agonists that can be co-administered include, but are not limited to, AL-034, DSP-0509, GS-9620 (Vesamod), LHC-165, TMX-101 (Imiquimod), GSK-2245035, Remiquimod, DSR-6434, DSP-3025, IMO-4200, MCT-465, MEDI-9197, 3M-051, SB-9922, 3M-052, Limtop, TMX-30X, TMX-202, RG-7863, RG-7854, RG-7795, and US20100143301 (Gilead Sciences), US20110098248 (Gilead Sciences), US20090047249 (Gilead Sciences), and US20140045849. (Janssen), US20140073642 (Janssen), WO2014 / 056953 (Janssen), WO2014 / 076221 (Janssen), WO2014 / 128189 (Janssen), US20140350031 (Janssen), WO2014 / 023813 (Janssen), US20080234251 (Array Biopharma), US20080306050 (Array Biopharma), US20100029585 (Ventirx Pharma), US20110092485 (Ventirx Pharma), US20110118235 (VentirxPharma), US20120082658 (Ventirx Pharma), US20120219615 (Ventirx Compounds disclosed in US20140066432 (Ventirx Pharma), US20140088085 (Ventirx Pharma), US20140275167 (Novira Therapeutics), and US20130251673 (Novira Therapeutics).

[0597] The TLR7 / TLR8 agonists that can be used together are NKTR-262, telratolimod, and BDB-001.

[0598] Examples of TLR8 agonists that can be co-administered include, but are not limited to, E-6887, IMO-4200, IMO-8400, IMO-9200, MCT-465, MEDI-9197, motolimod, remiquimod, selgantolimod (GS-9688), VTX-1463, VTX-763, 3M-051, 3M-052, ZG-170607, and US20140045849 (Janssen), US20140073642 (Janssen), WO2014 / 056953 (Janssen), WO2014 / 076221 (Janssen), WO2014 / 128189 (Janssen), US20140350031 (Janssen), and WO2014 / 023813. (Janssen), US20080234251 (Array Biopharma), US20080306050 (Array Biopharma), US20100029585 (Ventirx Pharma), US20110092485 (Ventirx Pharma), US20110118235 (Ventirx Pharma), US20120082658 (Ventirx Pharma), US20120219615 (Ventirx Pharma), US20140066432 (Ventirx Pharma), US20140088085 (Ventirx Pharma), US20140275167 (Novira Therapeutics) and US20130251673 (Novira Therapeutics), US Patent No. 9670205 (Gilead Sciences), The compounds disclosed in US20160289229 (Gilead Sciences, Inc.), WO2017 / 048727 (Gilead Sciences, Inc.), US20180065938 (Gilead Sciences, Inc.) and US20180086755 (Gilead Sciences, Inc.).

[0599] Examples of TLR9 agonists that can be used in combination include, but are not limited to, AST-008, cobitolimod, CMP-001, IMO-2055, IMO-2125, IMO-3100, IMO-8400, IR-103, IMO-9200, agatolimod, DIMS-9054, DV-1079, DV-1179, AZD-1419, lefitolimod (MGN-1703), CYT-003, CYT-003-QbG10, tilsotolimod, and PUL-042.

[0600] Examples of TLR7, TLR8, and TLR9 regulators include WO2017047769 (Teika Seiyaku), WO2015014815 (Janssen), WO2018045150 (Gilead Sciences, Inc.), WO2018045144 (Gilead Sciences, Inc.), WO2015162075 (Roche), WO2017034986 (University of Kansas), WO2018095426 (Jiangsu Hengrui Medicine Co Ltd), WO2016091698 (Roche), WO2016075661 (GlaxoSmithKline Biologicals), WO2016180743 (Roche), WO2018089695 (Dynavax Technologies), WO2016055553 (Roche), WO2015168279 (Novartis), WO2016107536 (Medshine Discovery), WO2018086593 (Livo (Shanghai) Pharmaceutical), WO2017106607 (Merck), WO2017061532 (Sumitomo Dainippon Pharma), WO2016023511 (Chia Tai Tianqing Pharmaceutical), WO2017076346 (Chia Tai Tianqing Pharmaceutical), WO2017046112 (Roche), WO2018078149 (Roche), WO2017040233 (3M Co), WO2016141092 (Gilead Sciences), WO2018049089 (Bristol Myers Squibb), WO2015057655 (Eisai Co Ltd), WO2017001307 (Roche), WO2018005586 (Bristol Myers Squibb), WO201704023 (3M Co), WO2017163264 (Council of Scientific and Industrial Research (India)), WO2018046460(GlaxoSmithKlineBiologicals), WO2018047081 (Novartis), WO2016142250 (Roche), WO2015168269 (Novartis), WO201804163 (Roche), WO2018038877 (3M Co), WO2015057659 (Eisai CoLtd), WO2017202704 (Roche), WO2018026620 (BristolMyers Squibb), WO2016029077 (Janus Biotherapeutics), WO201803143 (Merck), WO2016096778 (Roche), WO2017190669 (Shanghai De Novo Pharmatech), US09884866 (University of Compounds disclosed in WO2017219931 (Sichuan Kelun Biotech Biopharmaceutical), WO2018002319 (Janssen Sciences), WO2017216054 (Roche), WO2017202703 (Roche), WO2017184735 (IFM Therapeutics), WO2017184746 (IFM Therapeutics), WO2015088045 (Takeda Pharmaceutical), WO2017038909 (Takeda Pharmaceutical), WO2015095780 (University of Kansas), and WO2015023958 (University of Kansas).

[0601] In some implementations, the engineered wide-range nucleases described herein or their encoded nucleic acids are co-administered with TLR7, TLR8, or TLR9 agonists.

[0602] Interferon α receptor ligand Examples of interferon α receptor ligands include interferon α-2b (INTRON A®), pegylated interferon α-2a (PEGASYS®), pegylated interferon α-1b, interferon α-1b (HAPGEN®), Veldona, Infradure, Roferon-A, YPEG-interferon α-2a (YPEG-rhIFNalpha-2a), P-1101, Algeron, Alfarona, Ingaron (interferon γ), rSIFN-co (recombinant high-efficiency complex interferon), Ypeginterferon alfa-2b (YPEG-rhIFNalpha-2b), MOR-22, pegylated interferon α-2b (PEG-INTRON®), Bioferon, Novaferon, Inmutag (Inferon), MULTIFERON®, interferon α-n1 (HUMOFERON®), interferon β-1a (AVONEX®), Shaferon, and interferon α-2b. (Axxo), Alfaferone, Interferon α-2b (BioGeneric Pharma), Interferon-α 2 (CJ), Laferonum, VIPEG, BLAUFERON-A, BLAUFERON-B, Intermax Alpha, Realdiron, Lanstion, Pegaferon, PDferon-B, PDferon-B, Interferon α-2b (IFN, Laboratorios Bioprofarma), alfainterferon 2b, Kalferon, Pegnano, Feronsure, PegiHep, Interferon α-2b (Zydus-Cadila), Interferon α-2a, Optipeg A, Realfa 2B, Reliferon, Interferon α-2b (Amega), Interferon α-2b (Virchow), ropeginterferon alfa-2b, rHSA-IFN α-2a (recombinant human serum albumin interferon α-2a fusion protein), PEG-IFN-α, rHSA-IFN α2b, recombinant human interferon α-(1b, 2a,2b) Pegylated interferon α-2b (Amega), pegylated interferon α-2a, Reaferon-EC, Proquiferon, Uniferon, Urifron, interferon α-2b (Changchun Institute of Biological Products), Anterferon, Shanferon, Layfferon, Shang Sheng Lei Tai, INTEFEN, SINOGEN, Fukangtai, Pegstat, rHSA-IFN α-2b, SFR-9216, and Interapo (Interapa).

[0603] Hyaluronidase inhibitor Examples of hyaluronidase inhibitors include astodrimer.

[0604] Hepatitis B surface antigen (HBsAg) inhibitors Examples of HBsAg inhibitors include AK-074, HBF-0259, PBHBV-001, PBHBV-2-15, PBHBV-2-1, REP-9AC, REP-9C, REP-9, REP-2139, REP-2139-Ca, REP-2055, REP-2163, REP-2165, REP-2053, REP-2031, REP-006, and REP-9AC′.

[0605] Examples of HBsAg secretion inhibitors include BM601, GST-HG-131, and AB-452.

[0606] Cytotoxic T-lymphocyte-associated protein 4 (ipi4) inhibitors Examples of cytotoxic T-lymphocyte-associated protein 4 (ipi4) inhibitors include AGEN-2041, AGEN-1884, ipilimumab, beracip, PSI-001, PRS-010, Probody monoclonal antibody, tremelimumab, and JHL-1155.

[0607] Cyclophilic inhibitors Examples of cyclophilin inhibitors include CPI-431-32, EDP-494, OCB-030, SCY-635, NVP-015, NVP-018, NVP-019, STG-175, and compounds disclosed in US8513184 (Gilead Sciences), US20140030221 (Gilead Sciences), US20130344030 (Gilead Sciences), and US20130344029 (Gilead Sciences).

[0608] HBV virus enters inhibitor Examples of HBV virus inhibitors include Myrcludex B.

[0609] Antisense oligonucleotides targeting viral mRNA Examples of antisense oligonucleotides targeting viral mRNA include ISIS-HBVRx, IONIS-HBVRx, IONIS-HBV-LRx, IONIS-GSK6-LRx, GSK-3389404, and RG-6004.

[0610] Short interfering RNA (siRNA) and ddRNAi Examples of siRNAs include TKM-HBV (TKM-HepB), ALN-HBV, SR-008, HepB-nRNA, ARC-520, ARC-521, ARB-1740, ARB-1467, AB-729, DCR-HBVS, RG-6084 (PD-L1), RG-6217, ALN-HBV-02, JNJ-3989 (ARO-HBV), STSG-0002, ALG-010133, ALG-ASO, LUNAR-HBV, and DCR-HBVS (DCR-S219).

[0611] Examples of DNA-guided RNA interference (ddRNAi) include BB-HB-331.

[0612] Nucleotide endonuclease regulators Examples of endonuclease regulators include PGN-514.

[0613] Ribonucleotide reductase inhibitors Examples of ribonucleotide reductase inhibitors include Trimidox.

[0614] Non-nucleoside reverse transcriptase inhibitors Examples of non-nucleoside reverse transcriptase inhibitors (NNRTIs) include compounds disclosed in WO2018118826 (Merck), WO2018080903 (Merck), WO2018119013 (Merck), WO2017100108 (Idenix), WO2017027434 (Merck), WO2017007701 (Merck), and WO2008005555 (Gilead).

[0615] HBV replication inhibitors Examples of hepatitis B virus replication inhibitors include GP-31502, isotiafludine, IQP-HBV, RM-5038, and Xingantie.

[0616] Covalently closed circular DNA (cccDNA) inhibitors Examples of cccDNA inhibitors include BSBI-25, ccc-R08, and CHR-101.

[0617] Farnesol X receptor agonists Examples of farnesoid X receptor agonists include, for example, EYP-001, GS-9674, EDP-305, MET-409, Tropifexor, AKN-083, RDX-023, BWD-100, LMB-763, INV-3, NTX-023-1, EP-024297, and GS-8670.

[0618] Anti-HBV antibody In various embodiments, the engineered wide-range nucleases or their encoded nucleic acids described herein are combined with or co-administered with one or more antibodies that specifically bind to HBV antigens, including HBV peptides (pMHC) presented in major histocompatibility molecules (MHC). Examples of HBV antibodies targeting hepatitis B surface antigen that can be combined or co-administered include lenvervimab (GC-1102), XTL-17, XTL-19, KN-003, IV Hepabulin SN, and fully human monoclonal antibody therapy (hepatitis B virus infection, Humabs BioMed). Antibodies targeting HBV X protein (HBx) that can be combined or co-administered are described, for example, by Kornyeyev et al. J Virol. July 30, 2019; 93(16). pii: e00248-19.

[0619] Examples of HBV antibodies (including monoclonal and polyclonal antibodies) that can be used in combination or in combination include Zutectra, Shang Sheng Gan Di, Uman Big (Hepatitis B Hyperimmune), Omri-Hep-B, Nabi-HB, Hepatect CP, HepaGam B, igantibe, Niuliva, CT-P24, Hepatitis B Immunoglobulin (intravenous, pH 4, HBV infection, Shanghai RAAS Blood Products), and Fovepta (BT-088).

[0620] Examples of fully human HBV monoclonal antibodies that can be used in combination or in combination include HBC-34.

[0621] Antibodies against HBV viral peptide / major histocompatibility complex (MHC) class I complexes (pMHC) that can be combined or co-administered are described, for example, by Sastry et al. J Virol. March 2011; 85(5):1935-42 and WO2011062562.

[0622] CCR2 chemokine antagonists Examples of CCR2 chemokine antagonists include propargyl germanium.

[0623] thymopeptide agonists Examples of thymosin agonists include thymosin alpha and recombinant thymosin α1 (GeneScience).

[0624] Cytokines Examples of cytokines include recombinant IL-7, CYT-107, interleukin-2 (IL-2, Immunex), recombinant human interleukin-2 (Shenzhen Neptunus), IL-15, IL-21, IL-24, and celmoleukin.

[0625] Interleukin agonists In some embodiments, the engineered wide range of nucleases described herein, or their encoding nucleic acids, are combined with interleukin agonists, such as IL-2, IL-7, IL-15, IL-10, and IL-12 agonists. Examples of IL-2 agonists include adelrucin (aldeleukin, IL-2); PEGylated IL-2 (e.g., NKTR-214), modified variants of IL-2 (e.g., THOR-707), bempegaldesleukin, AIC-284, ALKS-4230, CUI-101, and Neo-2 / 15. Examples of IL-15 agonists include ALT-803, NKTR-255, hetIL-15, interleukin-15 / Fc fusion protein, AM-0015, NIZ-985, SO-C101, IL-15 Synthorin (PEGylated IL-15), P-22339, and IL-15-PD-1 fusion protein N-809. Examples of IL-7 include CYT-107.

[0626] Nucleoprotein regulators Nucleoprotein modulators can be HBV core or capsid protein inhibitors. Examples of nucleoprotein modulators include GS-4882, AB-423, AT-130, ALG-001075, ALG-001024, ALG-000184, EDP-514, GLS4, NVR-1221, NVR-3778, AL-3778, and BAY. 41-4109, Mofexedin Mesylate, ARB-168786, ARB-880, ARB-1820, GST-HG-141, JNJ-379, JNJ-632, RG-7907, GST-HG-141, HEC-72702, KL-060332, AB-506, ABI-H0731, ABI-H3733, JNJ-440, ABI-H2158, CB-HBV-001, and DVR-23.

[0627] Examples of capsid inhibitors include US20140275167 (Novira Therapeutics), US20130251673 (Novira Therapeutics), US20140343032 (Roche), WO2014037480 (Roche), US20130267517 (Roche), WO2014131847 (Janssen), WO2014033176 (Janssen), WO2014033170 (Janssen), WO2014033167 (Janssen), WO2015 / 059212 (Janssen), WO2015118057 (Janssen), WO2015011281 (Janssen), WO2014184365 (Janssen), WO2014184350 (Janssen), WO2014161888 (Janssen), WO2013096744 (Novira), US20150225355 (Novira), US20140178337 (Novira), US20150315159 (Novira), US20150197533 (Novira), US20150274652 (Novira), US20150259324 (Novira), US20150132258 (Novira), US9181288 (Novira), WO2014184350 (Janssen), WO2013144129 (Roche), WO2017198744 (Roche), US 20170334882 (Novira), US 20170334898 (Roche), WO2017202798 (Roche), WO2017214395 (Enanta), WO2018001944 (Roche), WO2018001952 (Roche), WO2018005881 (Novira), WO2018005883 (Novira), WO2018011100 (Roche), WO2018011160 (Roche), WO2018011162 (Roche), WO2018011163 (Roche), WO2018036941 (Roche), WO2018043747 (Kyoto Univ), US20180065929 (Janssen), WO2016168619 (IndianaCompounds disclosed in WO2016195982 (The Penn State Foundation), WO2017001655 (Janssen), WO2017048950 (Assembly Biosciences), WO2017048954 (Assembly Biosciences), WO2017048962 (Assembly Biosciences), US20170121328 (Novira), and US20170121329 (Novira).

[0628] Examples of transcription inhibitors include WO2017013046 (Roche), WO2017016960 (Roche), WO2017017042 (Roche), WO2017017043 (Roche), WO2017061466 (Toyoma chemicals), WO2016177655 (Roche), WO2016161268 (Enanta), WO2017001853 (Redex Pharma), WO2017211791 (Roche), WO2017216685 (Novartis), WO2017216686 (Novartis), WO2018019297 (Ginkgo Pharma), WO2018022282 (Newave Pharma), US20180030053 (Novartis), and WO2018045911 (Zhejiang). Compounds disclosed in Pharma.

[0629] Innate immune activators In some embodiments, the engineered wide-ranging nucleases described herein, or their encoded nucleic acids, are administered in combination with or co-administered with one or more innate immune activators. In various embodiments, one or more innate immune activators comprise an agonist selected from fms-associated tyrosine kinase 3 (FLT3), interferon gene stimulator (STING) receptors, DExD / H-box helicase 58 (DDX58; also known as RIG-I), and receptors containing a nucleotide-binding oligomerization domain 2 (NOD2). In some embodiments, the method requires the co-administration of GS-3583 and / or GS-9992.

[0630] STING agonists, RIG-I and NOD2 modulators In some embodiments, the engineered wide-ranging nucleases described herein, or their encoding nucleic acids, are combined or co-administered with interferon-reactive cGAMP-interacting protein 1 agonists (STING or STING1; NCBI Gene ID: 340061). In some embodiments, the STING / STING1 agonist or activator is selected from ADU-S100 (MIW-815), SB-11285, MK-1454, SR-8291, AdVCA0848, STINGVAX, GSK-532, SYN-STING, MSA-1, SR-8291, 5,6-dimethylxanthone-4-acetic acid (DMXAA), cyclic GAMP (cGAMP), and cyclic di-AMP. Examples of STING agonists that can be administered in combination or together include WO 2018065360 (Biolog Life Science InstituteForschungslabor und Biochemica-Vertrieb GmbH, Germany), WO 2018009466 (AduroBiotech), WO 2017186711 (InvivoGen), WO 2017161349 (Immune Sensor), WO2017106740 (Aduro Biotech), US 20170158724 (Glaxo Smithkline), WO 2017075477 (Aduro Biotech), US 20170044206 (Merck), WO 2014179760 (University of California), WO2018098203 (Janssen), and WO2018118665. Compounds disclosed in (Merck), WO2018118664 (Merck), WO2018100558 (Takeda), WO2018067423 (Merck), and WO2018060323 (Boehringer).

[0631] In some embodiments, the engineered wide-range nucleases described herein, or their encoding nucleic acid sequences, are administered in combination with or in combination with DExD / H-box helicase 58 (DDX58; also known as retinoic acid-inducible gene 1 (RIG-I), RIG1, RIGI, RLR-1, SGMRT2; NCBIGene ID: 23586). Exemplary RIG-I agonists that can be administered in combination or in combination include inarigivir soproxil (SB-9200, GS-9992), SB-40, SB-44, ORI-7246, ORI-9350, ORI-7537, ORI-9020, ORI-9198, ORI-7170, and RGT-100.

[0632] In some implementations, the engineered wide range of nucleases described herein or their encoding nucleic acid sequences are combined or co-administered with nucleotide-binding oligomerization domain 2 (NOD2; NCBI Gene ID: 64127) agonists, such as inarigivir soproxil (SB-9200, GS-9992) and IR-103.

[0633] Phosphatidylinositol 3-kinase (PI3K) inhibitors Examples of PI3K inhibitors include idelalisib, ACP-319, AZD-8186, AZD-8835, buparlisib, CDZ-173, CLR-457, pictilisib, neratinib, regoracitinib, regoracitinib sodium, EN-3342, TGR-1202, apeliximab, duverithione, IPI-549, UCB-5857, taselisib, XL-765, gedatolisib, ME-401, VS-5584, copanlisib, CAI orotate, perifoxine, RG-7666, and GSK-26367. 71. DS-7423, Panulisib, GSK-2269557, GSK-2126458, CUDC-907, PQR-309, INCB-40093, Pilaralisib, BAY-1082439, Proquatinib Mesylate, SAR-245409, AMG-319, RP-6530, ZSTK-474, MLN-1117, SF-1126, RV-1729, Sonolisib, LY-3023414, SAR-260301, TAK-117, HMPL-689, Tenalisib, Voxtalisib, and CLR-1401.

[0634] Immune checkpoint modulators In various embodiments, the engineered large-scale nucleases or their encoding nucleic acid sequences described herein are combined with blockers or inhibitors of one or more inhibitory immune checkpoint proteins or receptors and / or with one or more stimulators, activators, or agonists of one or more stimulating immune checkpoint proteins or receptors. Blockage or inhibition of inhibitory immune checkpoints can positively modulate the activation of T cells or NK cells and prevent immune escape by infected cells. Activation or stimulation of stimulating immune checkpoints can enhance the effects of immune checkpoint inhibitors in the treatment of infectious diseases. In various embodiments, immune checkpoint proteins or receptors regulate T cell responses (e.g., Xu et al., J Exp Clin Cancer Res (2018) 37:110 (Review). In various implementations, immune checkpoint proteins or receptors regulate NK cell responses (e.g., Davis et al., Semin Immunol (2017) 31:64-75 and Chiossone et al., Nat Rev Immunol (2018) 18(11):671-688 (Review).

[0635] Examples of immune checkpoint proteins or receptors include, but are not limited to: CD27 (NCBI Gene ID: 939); CD70 (NCBI Gene ID: 970); CD40 (NCBI Gene ID: 958); CD40LG (NCBI Gene ID: 959); CD47 (NCBI Gene ID: 961); CD48 (SLAMF2; NCBI Gene ID: 962); TMIGD2 (CD28H; NCBI Gene ID: 126259) containing transmembrane and immunoglobulin domains; CD84 (LY9B, SLAMF5; NCBI Gene ID: 8832); CD96 (NCBI Gene ID: 10225); CD160 (NCBI Gene ID: 11126); MS4A1 (CD20; NCBI Gene ID: 931); CD244 (SLAMF4; NCBI Gene ID: 51744); CD276 (B7H3; NCBI Gene ID: 931); CD244 (SLAMF4; NCBI Gene ID: 51744); CD276 (B7H3; NCBI Gene ID: 931). ID: 80381); T cell activation inhibitory factor 1 containing V-set domain (VTCN1, B7H4; NCBI Gene ID: 79679); V-set immunomodulatory receptor (VSIR, B7H5, VISTA; NCBI Gene ID: 64115); Immunoglobulin superfamily member 11 (IGSF11, VSIG3; NCBI Gene ID: 152404); Natural killer cell cytotoxic receptor 3 ligand 1 (NCR3LG1, B7H6; NCBI Gene ID: 374383); HERV-H LTR-associated 2 (HHLA2, B7H7; NCBI Gene ID: 11148); Inducible T cell costimulator (ICOS, CD278; NCBI Gene ID: 29851); Inducible T cell costimulator ligand (ICOSLG, B7H2; NCBI Gene ID: 23308); TNF receptor superfamily member 4 (TNFRSF4, OX40; NCBI Gene ID: 7293); TNF superfamily member 4 (TNFSF4, OX40L; NCBI Gene ID: 7292); TNFRSF8 (CD30; NCBI Gene ID: 943); TNFSF8 (CD30L; NCBI Gene ID: 944); TNFRSF10A (CD261, DR4, TRAILR1; NCBI Gene ID: 8797); TNFRSF9 (CD137; NCBI Gene ID: 3604); TNFSF9 (CD137L;NCBI Gene ID: 8744); TNFRSF10B (CD262, DR5, TRAILR2; NCBI Gene ID: 8795); TNFRSF10 (TRAIL; NCBI Gene ID: 8743); TNFRSF14 (HVEM, CD270; NCBI Gene ID: 8764); TNFSF14 (HVEML; NCBI Gene ID: 8740); CD272 (B and T lymphocyte associated (BTLA); NCBI Gene ID: 151888); TNFRSF17 (BCMA, CD269; NCBI Gene ID: 608); TNFSF13B (BAFF; NCBI Gene ID: 10673); TNFRSF18 (GITR; NCBI Gene ID: 8784); TNFSF18 (GITRL; NCBI Gene ID: 8995); MHC I polypeptide related sequence A (MICA; NCBI Gene ID: 100507436); MHC I peptide-related sequence B (MICB; NCBI GeneID: 4277); CD274 (CD274, PDL1, PD-L1; NCBI Gene ID: 29126); programmed cell death protein 1 (PDCD1, PD1, PD-1; NCBI Gene ID: 5133); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152; NCBI Gene ID: 1493); CD80 (B7-1; NCBI Gene ID: 941); CD28 (NCBI Gene ID: 940); connexin cell adhesion molecule 2 (NECTIN2, CD112; NCBI Gene ID: 5819); CD226 (DNAM-1; NCBI Gene ID: 10666); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155; NCBI Gene ID: 100507436); NCBI Gene ID: 4277; CD274 (CD274, PDL1, PD-L1; NCBI Gene ID: 29126); programmed cell death protein 1 (PDCD1, PD-1; NCBI Gene ID: 5133); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152; NCBI Gene ID: 1493); CD80 (B7-1; NCBI Gene ID: 941); CD28 (NCBI Gene ID: 940); connexin cell adhesion molecule 2 (NECTIN2, CD112; NCBI Gene ID: 5819); CD226 (DNAM-1; NCBI Gene ID: 10666); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155; NCBI Gene ID: 100507436); poliovirus receptor ( Gene ID: 5817); containing PVR-associated immunoglobulin domains (PVRIG, CD112R; NCBI Gene ID: 79037); T-cell immune receptors with Ig and ITIM domains (TIGIT; NCBI Gene ID: 201633); containing T-cell immunoglobulin and mucin domains 4 (TIMD4; TIM4; NCBI Gene ID: 91937); hepatitis A virus cell receptor 2 (HAVCR2, TIMD3, TIM3; NCBI Gene ID: 84868); galactohemagglutinin 9 (LGALS9;NCBI Gene ID: 3965); Lymphocyte activation gene 3 (LAG3, CD223; NCBI Gene ID: 3902); Signal transduction lymphocyte activation molecule family member 1 (SLAMF1, SLAM, CD150; NCBI Gene ID: 6504); Lymphocyte antigen 9 (LY9, CD229, SLAMF3; NCBI Gene ID: 4063); SLAM family member 6 (SLAMF6, CD352; NCBI Gene ID: 114836); SLAM family member 7 (SLAMF7, CD319; NCBI Gene ID: 57823); UL16 binding protein 1 (ULBP1; NCBI Gene ID: 80329); UL16 binding protein 2 (ULBP2; NCBI Gene ID: 80328); UL16 binding protein 3 (ULBP3; NCBI Gene ID: 79465); Retinyl early transcript 1E (RAET1E; ULBP4; NCBI Gene ID: 135250); Early retinoic acid transcript 1G (RAET1G; ULBP5; NCBI Gene ID: 353091); Early retinoic acid transcript 1L (RAET1L; ULBP6; NCBI Gene ID: 154064); Cytokine lectin-like receptor C1 (KLRC1, NKG2A, CD159A; NCBI Gene ID: 3821); Cytokine lectin-like receptor K1 (KLRK1, NKG2D, CD314; NCBI Gene ID: 22914); Cytokine lectin-like receptor C2 (KLRC2, CD159c, NKG2C; NCBI Gene ID: 3822); Cytokine lectin-like receptor C3 (KLRC3, NKG2E; NCBI Gene ID: 3822); NCBI Gene ID: 3823); Cytokine lectin-like receptor C4 (KLRC4, NKG2F; NCBI Gene ID: 8302); Cytokine immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1; NCBI Gene ID: 3802); Cytokine immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2; NCBI Gene ID: 3803); Cytokine immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3; NCBI Gene ID: 3804); Cytokine immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1, KIR, CD158E1);NCBI Gene ID: 3811 (e.g., lirelurumab (IPH2102 / BMS-986015), IPH-4102); and cytotoxic lectin-like receptor D1 (KLRD1; NCBI Gene ID: 3824).

[0636] In various implementations, the engineered wide range of nucleases described herein or their encoding nucleic acid sequences are used in combination with one or more blockers or inhibitors of one or more T-cell inhibitory immune checkpoint proteins or receptors. Illustrative T-cell suppressor immune checkpoint proteins or receptors include, but are not limited to, CD274 (CD274, PDL1, PD-L1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T-lymphocyte-associated protein 4 (CTLA4, CD152); CD276 (B7H3); V-set domain-containing T-cell activation inhibitor 1 (VTCN1, B7H4); V-set immunomodulatory receptors (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA)); and PVR-associated immunoglobulin domain-containing (PVRIG, CD112R); T-cell immune receptor with Ig and ITIM domains (TIGIT); lymphocyte activation 3 (LAG3, CD223); hepatitis A virus cell receptor 2 (HAVCR2, TIMD3, TIM3); galactoglobulin 9 (LGALS9); cytotoxic cell immunoglobulin-like receptor with three Ig domains and a long cytoplasmic tail 1 (KIR, CD158E1); cytotoxic cell immunoglobulin-like receptor with two Ig domains and a long cytoplasmic tail 1 (KIR2DL1); cytotoxic cell immunoglobulin-like receptor with two Ig domains and a long cytoplasmic tail 2 (KIR2DL2); cytotoxic cell immunoglobulin-like receptor with two Ig domains and a long cytoplasmic tail 3 (KIR2DL3); and cytotoxic cell immunoglobulin-like receptor with three Ig domains and a long cytoplasmic tail 1 (KIR3DL1). In various implementations, the engineered wide range of nucleases described herein or their encoding nucleic acid sequences are combined with one or more agonists or activators of one or more T-cell stimulating immune checkpoint proteins or receptors.Illustrative T-cell stimulating immune checkpoint proteins or receptors include, but are not limited to, CD27, CD70; CD40, CD40LG; inducible T-cell costimulatory molecule (ICOS, CD278); inducible T-cell costimulatory molecule ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; connexin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); CD244 (2B4, SLAMF4); and poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155). See, for example, Xu et al. J Exp Clin Cancer Res. (2018) 37:110.

[0637] In various embodiments, the engineered large-scale nucleases described herein or their encoding nucleic acid sequences are combined with one or more blockers or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors. Illustrative NK cell inhibitory immune checkpoint proteins or receptors include, but are not limited to, cytotoxic cell immunoglobulin-like receptors, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); cytotoxic cell immunoglobulin-like receptors, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); cytotoxic cell immunoglobulin-like receptors, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); cytotoxic cell immunoglobulin-like receptors, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); cytotoxic cell immunoglobulin-like receptors, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); cytotoxic cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); and cytotoxic cell lectin-like receptor D1 (KLRD1, CD94). In various embodiments, the engineered wide range of nucleases described herein, or their encoding nucleic acid sequences, are combined with one or more agonists or activators of one or more NK cell-stimulating immune checkpoint proteins or receptors. Illustrative NK cell-stimulating immune checkpoint proteins or receptors include, but are not limited to, CD16, CD226 (DNAM-1); CD244 (2B4, SLAMF4); cytotoxic lectin-like receptor K1 (KLRK1, NKG2D, CD314); and SLAM family member 7 (SLAMF7). See, for example, Davis et al. Semin Immunol.(2017) 31:64-75; Fang et al., Semin Immunol. (2017) 31:37-54; and Chiossone et al., Nat Rev Immunol. (2018) 18(11):671-688.

[0638] In some embodiments, one or more immune checkpoint inhibitors include protein-based (e.g., antibody or fragment thereof, or antibody mimic) inhibitors of PD-L1 (CD274), PD-1 (PDCD1), or CTLA4. In some embodiments, one or more immune checkpoint inhibitors include small organic molecule inhibitors of PD-L1 (CD274), PD-1 (PDCD1), or CTLA4. In some embodiments, the small molecule inhibitor of CD274 or PDCD1 is selected from GS-4224, GS-4416, INCB086550, and MAX10181. In some embodiments, the small molecule inhibitor of CTLA4 includes BPI-002.

[0639] Examples of CTLA4 inhibitors that can be co-administered include, but are not limited to, ipilimumab, trimemumab, BMS-986218, AGEN1181, AGEN1884, BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, JHL-1155, KN-044, CG-0161, ATOR-1144, PBI-5D3H5, BPI-002, and multispecific inhibitors FPT-155 (CTLA4 / PD-L1 / CD28), PF-06936308 (PD-1 / CTLA4), MGD-019 (PD-1 / CTLA4), and KN-046. (PD-1 / CTLA4), MEDI-5752 (CTLA4 / PD-1), XmAb-20717 (PD-1 / CTLA4) and AK-104 (CTLA4 / PD-1).

[0640] Examples of co-administerable PD-L1 (CD274) or PD-1 (PDCD1) inhibitors include, but are not limited to, pembrolizumab, nivolumab, cimipril, pildilizumab, AMP-224, MEDI0680 (AMP-514), spartazumab, atezolizumab, avelumab, durvalumab, ALN-PDL, BMS-936559, CK-301, PF-06801591, BGB-108, BGB-A317 (tislelizumab), GLS-010 (WBP-3055), and AK-103. (HX-008), GB-226, AK-105, CS-1003, HLX-10, MGA-012, BI-754091, PDR-001, AGEN-2034, JS-001 (Toripalimab), JNJ-63723283, Jeromezab (CBT-501), LZM-009, BCD-100, LY-3300054, SHR-1201, SHR-1210 (Camrelizumab), Sym-021, ABBV-181, PD1-PIK, BAT-1306, RO-6084 (PD-L1 antisense oligonucleotide), STI-1110, GX-P2, RG-7446, mDX-400, (MSB0010718C), CX-072, CBT-502, TSR-042 (dotalimab), MSB-2311, JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155), MEDI-0680, envorimab (KN-035), KD-033, KY-1003, IBI-308 (sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001), BCD-135, FAZ-053, TQB-2450, MDX1105-01, MSB-0010718C, GS-4224, GS-4416, INCB086550, MAX10181, and multispecific inhibitors FPT-155 (CTLA4 / PD-L1 / CD28), PF-06936308 (PD-1 / CTLA4), MGD-013 (PD-1 / LAG-3), FS-118 (LAG-3 / PD-L1), MGD-019 (PD-1 / CTLA4), KN-046 (PD-1 / CTLA4), MEDI-5752 (CTLA4 / PD-1), RO-7121661 (PD-1 / TIM-3), XmAb-20717 (PD-1 / CTLA4), AK-104 (CTLA4 / PD-1), M7824(PD-L1 / TGFβ-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM3 / PDL1), GNS-1480 (epidermal growth factor receptor antagonist; programmed cell death ligand 1 inhibitor), M-7824 (PD-L1 / TGF-β bifunctional fusion protein) and INBRX-105 (4-1BB / PDL1).

[0641] Examples of PD-1 inhibitors include WO2017112730 (Incyte Corp), WO2017087777 (IncyteCorp), WO2017017624, WO2014151634 (BristolMyers Squibb Co), WO201317322(BristolMyers Squibb Co), WO2018119286 (Incyte Corp), WO2018119266 (IncyteCorp), WO2018119263 (Incyte Corp), WO2018119236 (Incyte Corp), WO2018119221(Incyte Corp), WO2018118848 (BristolMyers Squibb Co), WO20161266460(BristolMyers Squibb Co), WO2017087678 (BristolMyers Squibb Co), WO2016149351(BristolMyers Squibb Co), WO2015033299 (Aurigene Discovery Technologies Ltd), WO2015179615 (Eisai Co Ltd; Eisai Research Institute), WO2017066227(BristolMyers Squibb Co), WO2016142886 (Aurigene Discovery Technologies Ltd), WO2016142852 (Aurigene Discovery Technologies Ltd), WO2016142835 (AurigeneDiscovery Technologies Ltd; Individual), WO2016142833 (Aurigene DiscoveryTechnologies Ltd), WO2018085750 (BristolMyers Squibb Co), WO2015033303(Aurigene Discovery Technologies Ltd), WO2017205464 (Incyte Corp), WO2016019232(3M Co; Individual;Texas A&M University System)、WO2015160641 (BristolMyersSquibb Co)、WO2017079669 (Incyte Corp)、WO2015033301 (Aurigene DiscoveryTechnologies Ltd)、WO2015034820 (BristolMyers Squibb Co)、WO2018073754(Aurigene Discovery Technologies Ltd)、WO2016077518 (BristolMyers Squibb Co)、WO2016057624 (BristolMyers Squibb Co)、WO2018044783 (Incyte Corp)、WO2016100608(BristolMyers Squibb Co)、WO2016100285 (BristolMyers Squibb Co)、WO2016039749(BristolMyers Squibb Co)、WO2015019284 (Cambridge Enterprise Ltd)、WO2016142894(Aurigene Discovery Technologies Ltd)、WO2015134605 (BristolMyers Squibb Co)、WO2018051255 (Aurigene Discovery Technologies Ltd)、WO2018051254 (AurigeneDiscovery Technologies Ltd)、WO2017222976 (Incyte Corp)、WO2017070089 (IncyteCorp)、WO2018044963 (BristolMyers Squibb Co)、WO2013144704 (Aurigene DiscoveryTechnologies Ltd)、WO2018013789 (Incyte Corp)、WO2017176608 (BristolMyersSquibb Co)、WO2018009505 (BristolMyers Squibb Co)、WO2011161699 (AurigeneDiscovery Technologies Ltd)、WO2015119944 (Incyte Corp;Merck Sharp&DohmeCorp), WO2017192961 (Incyte Corp), WO2017106634 (Incyte Corp), WO2013132317 (Aurigene Discovery Technologies Ltd), WO2012168944 (Aurigene Discovery Technologies Ltd), WO2015036927 (Aurigene Discovery Technologies Ltd) Ltd), WO2015044900 (Aurigene Discovery Technologies Ltd), WO2018026971 (Arising International). ;

[0642] In various implementations, the engineered wide range of nucleases described herein or their encoding nucleic acid sequences are combined with anti-TIGIT antibodies, such as BMS-986207, RG-6058, and AGEN-1307.

[0643] TNF receptor superfamily (TNFRSF) agonists or activators In various implementations, the engineered wide-range nucleases described herein or their encoding nucleic acid sequences are combined with agonists of one or more members of the TNF receptor superfamily (TNFRSF), such as TNFRSF1A (NCBI Gene ID: 7132), TNFRSF1B (NCBI Gene ID: 7133), TNFRSF4 (OX40, CD134; NCBI Gene ID: 7293), TNFRSF5 (CD40; NCBI Gene ID: 958), TNFRSF6 (FAS, NCBI Gene ID: 355), TNFRSF7 (CD27, NCBI Gene ID: 939), TNFRSF8 (CD30, NCBI Gene ID: 943), TNFRSF9 (4-1BB, CD137, NCBI Gene ID: 3604), TNFRSF10A (CD261, DR4, TRAILR1, NCBI Gene ID: 8797), TNFRSF10B (CD262 ...TNFRSF10B, TNFRSF10B, TNFRSF10B, TNFRSF10B, TNFRSF10B, TNFRSF10B, TNFRSF10B, TNFRSF10B, TNFRSF10B, TNFRSF10B, TNFRSF10B, TNFRSF10B, TNFRSF10B, TNFRSF10B, TNFRSF10B, TNFRSF10 DR5, TRAILR2, NCBI Gene ID: 8795), TNFRSF10C (CD263, TRAILR3, NCBI Gene ID: 8794), TNFRSF10D (CD264, TRAILR4, NCBI Gene ID: 8793), TNFRSF11A (CD265, RANK, NCBI Gene ID: 8792), TNFRSF11B (NCBI Gene ID: 8793) ID: 4982), TNFRSF12A (CD266, NCBI Gene ID: 51330), TNFRSF13B (CD267, NCBI Gene ID: 23495), TNFRSF13C (CD268, NCBI Gene ID: 115650), TNFRSF16 (NGFR, CD271, NCBIGene ID: 4804), TNFRSF17 (BCMA, CD269, NCBI Gene A single or more of the following agonists: TNFRSF18 (GITR, CD357, NCBI Gene ID: 8784), TNFRSF19 (NCBI Gene ID: 55504), TNFRSF21 (CD358, DR6, NCBI Gene ID: 27242), and TNFRSF25 (DR3, NCBI Gene ID: 8718).

[0644] Examples of co-administerable anti-TNFRSF4 (OX40) antibodies include, but are not limited to, MEDI6469, MEDI6383, MEDI0562 (talixizumab), MOXR0916, PF-04518600, RG-7888, GSK-3174998, INCAGN1949, BMS-986178, GBR-8383, ABBV-368, IBI-101, and those described in WO2016179517, WO2017096179, WO2017096182, WO2017096281, and WO2018089628.

[0645] Examples of anti-TNFRSF5 (CD40) antibodies that can be co-administered include, but are not limited to, RG7876, SEA-CD40, APX-005M and ABBV-428.

[0646] In some implementations, the anti-TNFRSF7 (CD27) antibody varigramab (CDX-1127) is co-administered.

[0647] Examples of anti-TNFRSF9 (4-1BB, CD137) antibodies that can be co-administered include, but are not limited to, urinumab, urinumab (PF-05082566), AGEN2373, and ADG-106.

[0648] Examples of co-administerable anti-TNFRSF18 (GITR) antibodies include, but are not limited to: MEDI1873, FPA-154, INCAGN-1876, TRX-518, BMS-986156, MK-1248, GWN-323, and those described in WO2017096179, WO2017096276, WO2017096189, and WO2018089628. In some embodiments, antibodies or fragments thereof that co-target TNFRSF4 (OX40) and TNFRSF18 (GITR) are co-administered. Such antibodies are described, for example, in WO2017096179 and WO2018089628.

[0649] Indoleamine-pyrrole-2,3-dioxygenase (IDO1) inhibitors In various implementation schemes, the engineered wide range of nucleases described herein or their encoding nucleic acid sequences are combined with inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1; NCBI Gene ID: 3620). Examples of IDO1 inhibitors include, but are not limited to, BLV-0801, epacadostat, resminostat, F-001287, GBV-1012, GBV-1028, GDC-0919, indoximod, NKTR-218, NLG-919-based vaccines, PF-06840003, pyranoquinone derivatives (SN-35837), SBLK-200802, BMS-986205 and shIDO-ST, EOS-200271, KHK-2455, LY-3381916, and compounds disclosed in US20100015178 (Incyte), US2016137652 (Flexus Biosciences, Inc.), WO2014073738 (Flexus Biosciences, Inc.), and WO2015188085 (Flexus Biosciences, Inc.).

[0650] LAG-3 and TIM-3 inhibitors In some implementations, the engineered wide range of nucleases described herein or their encoding nucleic acid sequences are combined with anti-TIM-3 antibodies, such as TSR-022, LY-3321367, MBG-453, and INCAGN-2390.

[0651] In some implementations, the engineered wide range of nucleases described herein or their encoding nucleic acid sequences are combined with anti-LAG-3 (lymphocyte activation) antibodies, such as renalalimab (ONO-4482), LAG-525, MK-4280, REGN-3767, and INCAGN2385.

[0652] Inhibitors of the apoptosis protein family (IAPs) Examples of IAP inhibitors include APG-1387.

[0653] Recombinant thymosin α-1 Examples of recombinant thymosin α-1 include NL-004 and PEGylated thymosin α-1.

[0654] Bruton's tyrosine kinase (BTK) inhibitors Examples of BTK inhibitors include ABBV-105, acalatinib (ACP-196), ARQ-531, BMS-986142, dasatinib, ibrutinib, GDC-0853, PRN-1008, SNS-062, ONO-4059, BGB-3111, ML-319, MSC-2364447, RDX-022, X-022, AC-058, RG-7845, sprutinib, TAS-5315, TP-0158, TP-4207, HM-71224, KBP-7536, M-2951, TAK-020, AC-0025, and US20140330015 (Ono Pharmaceutical), US20130079327 (Ono Pharmaceutical). The compounds disclosed in US20130217880 (Ono Pharmaceutical) and US20130217880 (Ono Pharmaceutical).

[0655] KDM inhibitors Examples of KDM5 inhibitors include compounds disclosed in WO2016057924 (Genentech / Constellation Pharmaceuticals), US20140275092 (Genentech / Constellation Pharmaceuticals), US20140371195 (Epitherapeutics), US20140371214 (Epitherapeutics), US20160102096 (Epitherapeutics), US20140194469 (Quanticel), US20140171432, US20140213591 (Quanticel), US20160039808 (Quanticel), US20140275084 (Quanticel), and WO2014164708 (Quanticel).

[0656] Examples of KDM1 inhibitors include compounds disclosed in US9186337B2 (Oryzon Genomics), GSK-2879552, RG-6016, and ORY-2001.

[0657] Arginase inhibitors Examples of arginase inhibitors include CB-1158, C-201, and remizole.

[0658] Bispecific and trispecific natural killer (NK) cell connectors In various embodiments, the engineered wide-range nucleases described herein or their encoding nucleic acid sequences are combined with bispecific NK cell connectors (BiKE) or trispecific NK cell connectors (TriKE) (e.g., without Fc) or bispecific antibodies against NK cell activation receptors (e.g., with Fc), such as CD16A, C-type lectin receptors (CD94 / NKG2C, NKG2D, NKG2E / H, and NKG2F), natural cytotoxic receptors (NKp30, NKp44, and NKp46), cytotoxic cell C-type lectin-like receptors (NKp65, NKp80), Fc receptor FcγR (which mediates antibody-dependent cytotoxicity), SLAM family receptors (e.g., 2B4, SLAM6, and SLAM7), cytotoxic cell immunoglobulin-like receptors (KIR) (KIR-2DS and KIR-3DS), DNAM-1, and CD137 (4-1BB). Where suitable, anti-CD16 binding bispecific molecules may or may not have Fc. Illustrative bispecific NK cell connectors that can be co-administered target CD16 and one or more HBV-associated antigens described herein. BiKEs and TriKEs are described, for example, by Felices et al. Methods Mol Biol (2016) 1441:333-346; Fang et al., Semin Immunol (2017) 31:37-54.

[0659] Gene therapy and cell therapy In some implementations, the engineered large-scale nucleases or their encoding nucleic acid sequences described herein are combined with gene or cell therapy protocols. Gene therapy and cell therapy include, but are not limited to, gene modification to silence genes; genetic methods that directly kill infected cells; infusions designed to replace a large portion of a patient's autoimmune system to enhance the immune response to infected cells, or to activate the patient's autoimmune system to kill infected cells or to find and kill infected cells; and / or genetic methods that alter cell activity to further modify the endogenous immune response to infection.

[0660] CAR-T cell therapy CAR-T cell therapy comprises a population of immune effector cells engineered to express a chimeric antigen receptor (CAR), wherein the CAR contains an HBV antigen-binding domain. In some embodiments, the antigen-binding domain is the domain described herein. In some embodiments, the antigen-binding domain is not the domain described herein. In some embodiments, the antigen is HBsAg (i.e., HBsAg-CART). The immune effector cells are T cells or NK cells. In some embodiments, the T cells are CD4+ T cells, CD8+ T cells, NK cells, or a combination thereof. The cells may be autologous or allogeneic. Examples of HBV-targeted CARs are described in Cytotherapy. 2018 May; 20(5):697-705 doi: 10.1016 / j.jcyt.2018.02.

[0661] TCR-T cell therapy TCR-T cell therapy involves T cells expressing HBV-specific T cell receptors. TCR-T cells are engineered to target HBV-derived peptides presented on the surface of virus-infected cells. An example of a TCR targeting HBV is described in Wisskirchen, K. et al. J Clin Invest 2019;129(7):2932-2945.

[0662] TCR-T cell therapy involves T cells that express HBV surface antigen (HBsAg)-specific TCRs.

[0663] TCR-T cell therapy includes TCR-T therapies targeting HBV, such as LTCR-H2-1.

[0664] In another specific embodiment, the engineered wide-range nucleases or their encoded nucleic acids and HBV DNA polymerase inhibitors described herein are selected from immunomodulators, TLR modulators, HBsAg inhibitors, HBsAg secretion or assembly inhibitors, HBV therapeutic vaccines, HBV antibodies (including HBV antibodies targeting hepatitis B virus surface antigen, as well as bispecific antibodies and "antibody-like" therapeutic proteins (such as DARTs®, DUOBODIES®, BITES®, XmAbs®, TandAbs®, Fab derivatives, or TCR-like antibodies)), cyclin inhibitors, and retinoic acid. One or two other therapeutic agents selected from gene-inducing stimulants, RIG-I-like receptor stimulants, PD-1 inhibitors, PD-L1 inhibitors, arginase inhibitors, PI3K inhibitors, IDO inhibitors, and NOD2 stimulants, and a combination of one or two other therapeutic agents selected from HBV virus entry inhibitors, NTCP inhibitors, HBx inhibitors, cccDNA inhibitors, HBV antibodies targeting hepatitis B virus surface antigen, siRNA, miRNA gene therapy agents, sshRNA, KDM5 inhibitors, and nucleoprotein modulators (HBV core or capsid protein modulators).

[0665] In another specific embodiment, the engineered wide range of nucleases described herein or their encoded nucleic acids are combined with at least one additional second therapeutic agent selected from the following: HBV DNA polymerase inhibitors, immunomodulators, TLR modulators, HBsAg inhibitors, HBV therapeutic vaccines, HBV antibodies (including HBV antibodies targeting hepatitis B surface antigen, as well as bispecific antibodies and "antibody-like" therapeutic proteins (such as DARPins®, anti-pMHC TCR-like antibodies, DARTs®, DUOBODIES®, BITES®, XmAbs®, TandAbs®, Fab derivatives, or TCR-like antibodies)), cyclophilic inhibitors, retinoic acid-induced gene stimulators 1, RIG-I-like receptor stimulators, PD-1 inhibitors, PD-L1 inhibitors, arginase inhibitors, PI3K inhibitors, IDO inhibitors, and NOD2 stimulators.

[0666] In another specific embodiment, the engineered large-scale nuclease described herein or its encoded nucleic acid is combined with at least one additional second therapeutic agent selected from the following: HBV DNA polymerase inhibitor, HBV virus entry inhibitor, NTCP inhibitor, HBx inhibitor, cccDNA inhibitor, HBV antibody targeting hepatitis B virus surface antigen, siRNA, miRNA gene therapy agent, sshRNA, KDM5 inhibitor, and nucleoprotein modulator (HBV core or capsid protein inhibitor).

[0667] In certain embodiments, the engineered wide-range nucleases described herein or their encoded nucleic acids are similar to those described in U.S. Publication No. 2010 / 0143301 (Gilead Sciences), U.S. Publication No. 2011 / 0098248 (Gilead Sciences), U.S. Publication No. 2009 / 0047249 (Gilead Sciences), U.S. Patent No. 8722054 (Gilead Sciences), U.S. Publication No. 2014 / 0045849 (Janssen), U.S. Publication No. 2014 / 0073642 (Janssen), WO2014 / 056953 (Janssen), WO2014 / 076221 (Janssen), WO2014 / 128189 (Janssen), U.S. Publication No. 2014 / 0350031 (Janssen), WO2014 / 023813 (Janssen), and U.S. Publication No. 2008 / 0234251 (Array Biopharma), US Public No. 2008 / 0306050 (Array Biopharma), US Public No. 2010 / 0029585 (Ventirx Pharma), US Public No. 2011 / 0092485 (Ventirx Pharma), US2011 / 0118235 (Ventirx Pharma), US Public No. 2012 / 0082658 (Ventirx Pharma), US Public No. 2012 / 0219615 (Ventirx Pharma), US Public No. 2014 / 0066432 (Ventirx Pharma), US Public No. 2014 / 0088085 (Ventirx Pharma), US Public No. 2014 / 0275167 (Novira Therapeutics), US Public No. 2013 / 0251673 (NoviraTherapeutics), US Patent No. 8513184 (Gilead Sciences), US Publication No. 2014 / 0030221 (Gilead Sciences), US Publication No. 2013 / 0344030 (Gilead Sciences), US Publication No. 2013 / 0344029 (Gilead Sciences), US Publication No.20140275167 (Novira Therapeutics), US20130251673 (Novira Therapeutics), US Publication No. 2014 / 0343032 (Roche), WO2014037480 (Roche), US Publication No. 2013 / 0267517 (Roche), WO2014131847 (Janssen), WO2014033176 (Janssen), WO2014033170 (Janssen), WO2014033167 (Janssen), WO2015 / 059212 (Janssen), WO2015118057 (Janssen), WO2015011281 (Janssen), WO2014184365 (Janssen), WO2014184350 (Janssen), WO2014161888 (Janssen), WO2013096744 (Novira), US20150225355 (Novira), US20140178337 (Novira), US20150315159 (Novira), US20150197533 (Novira), US20150274652 (Novira), US20150259324 (Novira), US20150132258 (Novira), US9181288 (Novira), WO2014184350 (Janssen), WO2013144129 (Roche), US20100015178 (Incyte), US2016137652 (Flexus Biosciences, Inc.), WO2014073738 (Flexus Biosciences, Inc.), WO2015188085 (Flexus Biosciences, Inc.), US Publication No. 2014 / 0330015 (Ono Pharmaceutical), US Publication No. 2013 / 0079327 (Ono Pharmaceutical), US Publication No.2013 / 0217880 (Onopharmaceutical), WO2016057924 (Genentech / Constellation Pharmaceuticals), US20140275092 (Genentech / Constellation Pharmaceuticals), US20140371195 (Epitherapeutics) and US20140371214 (Epitherapeutics), US20160102096 (Epitherapeutics), US20140194469 (Quanticel), US20140171432, US20140213591 (Quanticel), US20160039808 (Quanticel), US20140275084 (Quanticel), WO2014164708 (Quanticel), US9186337B2 (Oryzon Compounds disclosed in *Genomics*, as well as other drugs for the treatment of HBV, and combinations thereof.

[0668] In one embodiment, a kit is provided comprising the engineered wide-ranging nuclease described herein or its encoded nucleic acid in combination with one or more (e.g., one, two, three, four, one or two, one to three, or one to four) additional therapeutic agents.

[0669] Example The embodiments of this disclosure are further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will recognize or be able to identify many equivalents of the specific substances and procedures described herein using only conventional experiments. These equivalents are intended to be included within the scope of the claims following the examples below.

[0670] Example 1 Evaluation of novel peptide linkers and subunit substitution combinations in a wide range of HBV 11-12 nucleases 1. Method This study aimed to evaluate variants of the HBV 11-12L.1090 macronuclease (SEQ ID NO: 13) that bind to and cleave the HBV 11-12 recognition sequence (SEQ ID NO: 3) and contain two modified I-CreI subunits linked together by a polypeptide linker called linker 1 (SEQ ID NO: 16). The variants included in this study comprise novel linkers selected from linker 1923 (SEQ ID NO: 15), linker 1766 (SEQ ID NO: 42), linker 1771 (SEQ ID NO: 43), linker 1808 (SEQ ID NO: 44), and linker 1814 (SEQ ID NO: 45). Each of these novel linkers was developed for potential use in I-CreI-derived single-stranded macronucleases such as HBV 11-12L.1090. Similar to linker 1 in the parental HBV 11-12L.1090 wide-range nuclease, the N-terminus of each novel linker is linked to the N-terminal subunit at the D residue corresponding to position 153 of wild-type I-CreI, and the C-terminus of each novel linker is linked to the C-terminal subunit at the Y residue corresponding to position 5 of wild-type I-CreI. Some variants containing these novel linkers also include different combinations of amino acid modifications in the N-terminal and C-terminal subunits, which are incorporated in principle to function synergistically with the novel linker structure. These amino acid modifications are grouped as shown in Table 2 below: Table 2. Subunit Modification Grouping

[0671] The HBV 11-12L.1090 nuclease variants prepared for this experiment are shown in Table 3 below: Table 3.

[0672] The activities of the HBV 11-12 L.1090 macronuclease and its variants were evaluated using the previously described CHO cell reporter assay (see WO 2012 / 167192, the entire contents of which are incorporated herein by reference). For the analysis, CHO cell reporter cell lines carrying a non-functional green fluorescent protein (GFP) gene expression cassette integrated into the cell genome were prepared. In each cell line, the GFP gene was interrupted by a pair of recognition sequences, such that intracellular cleavage of either recognition sequence by the macronuclease stimulated homologous recombination, resulting in a functional GFP gene. In the CHO reporter cell lines developed in this study, one of the recognition sequences inserted into the GFP gene was the human HBV 11-12 recognition sequence. The second recognition sequence inserted into the GFP gene was the CHO-23 / 24 recognition sequence, which was recognized and cleaved by the control macronuclease, referred to as "CHO-23 / 24".

[0673] CHO reporter cells were transfected with mRNA encoding a single clone selected from two large-scale nuclease libraries containing two adapter libraries. In one well of a 96-well plate, CHO reporter cells were also transfected with mRNA encoding the CHO-23 / 24 large-scale nuclease. In each assay, 5e4 CHO reporter cells were transfected with 90 ng mRNA in a 96-well plate using Lipofectamine® MessengerMax (ThermoFisher) according to the manufacturer's instructions. Two days post-transfection, transfected CHO cells were evaluated by flow cytometry to determine the percentage of GFP-positive cells compared to an untransfected negative control. Data obtained at each time point were normalized against the %GFP-positive cells observed using the CHO-23 / 24 large-scale nuclease to determine an "activity score," and the normalized data from the earliest time point was subtracted from the data from the latest time point to determine a "toxicity score." The activity score and toxicity score were then summed to determine the “activity index,” which was subsequently normalized for the activity index of a wide range of CHO-23 / 24 nucleases to compare data between cell lines (“normalized activity index”).

[0674] 2. Results The activity results for each wide range of nuclease variants (with various combinations of different linkers and subunit modifications) are as follows: Figures 4A-4E As shown. Due to the high activity l...

Claims

1. An engineered wide-range nuclease that binds to and cleaves a recognition sequence containing SEQ ID NO: 3 within the hepatitis B virus (HBV) genome, wherein the engineered wide-range nuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and wherein the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region.

2. The engineered wide-range nuclease according to claim 1, wherein the HVR1 region comprises one or more residues corresponding to residues 204, 206, 208, 210, 212, 213, 218, 220, 222, 224, 226, 248, 250, 255 and 257 of SEQ ID NO: 5 or SEQ ID NO:

6.

3. The engineered wide-range nuclease according to claim 1 or claim 2, wherein the HVR1 region comprises residue 237 corresponding to residue 237 of SEQ ID NO: 5 or SEQ ID NO:

6.

4. The engineered wide-range nuclease according to any one of claims 1-3, wherein the HVR1 region comprises residue 241 corresponding to residue 241 of SEQ ID NO: 5 or SEQ ID NO:

6.

5. The engineered wide-range nuclease according to any one of claims 1-4, wherein the HVR1 region comprises residue 251 corresponding to residue 251 of SEQ ID NO: 5 or SEQ ID NO:

6.

6. The engineered wide-range nuclease according to any one of claims 1-5, wherein the HVR1 region comprises residue 252 corresponding to residue 252 of SEQ ID NO: 5 or SEQ ID NO:

6.

7. The engineered wide-range nuclease according to any one of claims 1-6, wherein the HVR1 region comprises residues corresponding to residue 253 in SEQ ID NO: 5 or SEQ ID NO:

6.

8. The engineered wide-range nuclease according to any one of claims 1-7, wherein the HVR1 region comprises residues 204-259 of SEQ ID NO: 5 or SEQ ID NO:

6.

9. The engineered wide-range nuclease according to any one of claims 1-8, wherein the first subunit comprises an amino acid sequence having at least 80% sequence identity with residues 187-333 of SEQ ID NO: 5 or SEQ ID NO:

6.

10. The engineered wide-range nuclease according to any one of claims 1-9, wherein the first subunit comprises a residue corresponding to residue 260 of SEQ ID NO:

6.

11. The engineered wide-range nuclease according to any one of claims 1-10, wherein the first subunit comprises residues 187-333 of any one of SEQ ID NO: 5 or SEQ ID NO:

6.

12. The engineered wide-range nuclease according to any one of claims 1-11, wherein the HVR2 region comprises one or more residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75 and 77 corresponding to SEQ ID NO: 5 or SEQ ID NO:

6.

13. The engineered wide-range nuclease according to any one of claims 1-12, wherein the HVR2 region comprises residue 51 corresponding to residue 51 of SEQ ID NO: 5 or SEQ ID NO:

6.

14. The engineered wide-range nuclease according to any one of claims 1-13, wherein the HVR2 region comprises residues 24-79 of SEQ ID NO: 5 or SEQ ID NO:

6.

15. The engineered wide-range nuclease according to any one of claims 1-14, wherein the second subunit comprises an amino acid sequence having at least 80% sequence identity with residues 7-153 of SEQ ID NO: 5 or SEQ ID NO:

6.

16. The engineered wide-range nuclease according to any one of claims 1-15, wherein the second subunit comprises residue 19 corresponding to residue 19 of SEQ ID NO: 5 or SEQ ID NO:

6.

17. The engineered wide-range nuclease according to any one of claims 1-16, wherein the second subunit comprises residue 80 corresponding to residue 80 of SEQ ID NO: 5 or SEQ ID NO:

6.

18. The engineered wide-range nuclease according to any one of claims 1-17, wherein the second subunit comprises residue 96 corresponding to residue 96 of SEQ ID NO: 5 or SEQ ID NO:

6.

19. The engineered wide-range nuclease according to any one of claims 1-18, wherein the second subunit comprises residue 99 corresponding to residue 99 of SEQ ID NO: 5 or SEQ ID NO:

6.

20. The engineered wide-range nuclease according to any one of claims 1-19, wherein the second subunit comprises residue 100 corresponding to residue SEQ ID NO: 5 or SEQ ID NO:

6.

21. The engineered wide-range nuclease according to any one of claims 1-20, wherein the second subunit comprises residues 7-153 of SEQ ID NO: 5 or SEQ ID NO:

6.

22. The engineered wide-range nuclease according to any one of claims 1-21, wherein the engineered wide-range nuclease is a single-stranded wide-range nuclease comprising a linker, and wherein the linker covalently binds the first subunit and the second subunit.

23. The engineered wide-range nuclease of claim 22, wherein the adapter comprises the amino acid sequence shown in SEQ ID NO:

15.

24. The engineered wide-range nuclease according to any one of claims 1-23, wherein the engineered wide-range nuclease comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 5 or SEQ ID NO:

6.

25. The engineered wide-range nuclease according to any one of claims 1-24, wherein the engineered wide-range nuclease comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO:

6.

26. The engineered wide-range nuclease according to any one of claims 1-25, wherein the engineered wide-range nuclease is encoded by a nucleic acid sequence having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 11 or SEQ ID NO:

12.

27. The engineered wide-range nuclease according to any one of claims 1-26, wherein the engineered wide-range nuclease is encoded by the nucleic acid sequence of SEQ ID NO: 11 or SEQ ID NO:

12.

28. The engineered wide-range nuclease according to any one of claims 1-27, wherein the engineered wide-range nuclease comprises a nuclear localization signal.

29. The engineered wide-range nuclease according to claim 28, wherein the nuclear localization signal is located at the N-terminus of the engineered wide-range nuclease.

30. The engineered wide-range nuclease according to claim 28, wherein the nuclear localization signal is located at the C-terminus of the engineered wide-range nuclease.

31. The engineered wide-range nuclease according to claim 28, wherein the engineered wide-range nuclease comprises a first nuclear localization signal located at the N-terminus and a second nuclear localization signal located at the C-terminus.

32. The engineered wide-range nuclease according to any one of claims 28-31, wherein the nuclear localization signal comprises an amino acid sequence having at least 80% or at least 90% sequence identity with SEQ ID NO:

17.

33. The engineered wide-range nuclease according to any one of claims 28-32, wherein the nuclear localization signal comprises SEQ ID NO:

17.

34. An engineered wide-range nuclease that binds to and cleaves a recognition sequence comprising SEQ ID NO: 3 within the HBV genome, wherein the engineered wide-range nuclease comprises the amino acid sequence of SEQ ID NO:

5.

35. An engineered wide-range nuclease that binds to and cleaves a recognition sequence comprising SEQ ID NO: 3 within the HBV genome, wherein the engineered wide-range nuclease comprises the amino acid sequence of SEQ ID NO:

6.

36. A polynucleotide comprising a nucleic acid sequence encoding the engineered wide-range nuclease of any one of claims 1-35.

37. The polynucleotide of claim 36, wherein the polynucleotide comprises a 5' ALB untranslated region (UTR) comprising the nucleic acid sequence shown in SEQ ID NO: 18 or SEQ ID NO:

82.

38. The polynucleotide of claim 36 or claim 37, wherein the polynucleotide comprises a 3' SNRPBUTR, the UTR comprising the nucleic acid sequence shown in SEQ ID NO: 19 or SEQ ID NO:

83.

39. The polynucleotide according to any one of claims 36-38, wherein the polynucleotide comprises a polyA terminator sequence.

40. The polynucleotide of claim 39, wherein the polyA terminator sequence comprises the nucleic acid sequence shown in SEQ ID NO:

20.

41. The polynucleotide according to any one of claims 36-40, wherein the polynucleotide comprises a Kozak sequence comprising the nucleic acid sequence shown in SEQ ID NO: 21 or SEQ ID NO:

84.

42. The polynucleotide according to any one of claims 36-41, wherein the nucleic acid sequence encoding the engineered wide-ranging nuclease is thymine or uracil depleted.

43. The polynucleotide according to any one of claims 36-42, wherein the nucleic acid sequence encoding the engineered wide-ranging nuclease is optimized for liver expression codons.

44. The polynucleotide according to any one of claims 36-43, wherein the polynucleotide is mRNA.

45. The polynucleotide of claim 44, wherein the polynucleotide comprises: (a) 5' ALB UTR, which contains the nucleic acid sequence shown in SEQ ID NO: 82; (b) The nucleic acid sequence encoding the engineered wide-range nuclease, wherein the nucleic acid sequence is uracil-depleted and optimized for liver expression codons; (c) 3' SNRPB UTR, which contains the nucleic acid sequence shown in SEQ ID NO: 83; and (d) polyA termination sequence.

46. ​​The polynucleotide of claim 44, wherein the polynucleotide comprises: (a) 5' ALB UTR, which contains the nucleic acid sequence shown in SEQ ID NO: 82; (b) A Kozak sequence comprising the nucleic acid sequence shown in SEQ ID NO: 84; (c) The nucleic acid sequence encoding the engineered wide-range nuclease, wherein the nucleic acid sequence is uracil-depleted and optimized for liver expression codons; (d) 3' SNRPB UTR, which contains the nucleic acid sequence shown in SEQ ID NO: 83; and (e) polyA termination sequence.

47. A recombinant DNA construct comprising any one of claims 36-43.

48. The recombinant DNA construct of claim 47, wherein the recombinant DNA construct is plasmid DNA.

49. The recombinant DNA construct according to claim 47 or claim 48, wherein the polynucleotide comprises: (a) 5' ALB untranslated region (UTR) containing the nucleic acid sequence shown in SEQ ID NO: 18; (b) The nucleic acid sequence encoding the engineered wide-range nuclease; (c) 3' SNRPB UTR, which contains the nucleic acid sequence shown in SEQ ID NO: 19; and (d) polyA termination sequence.

50. The recombinant DNA construct according to claim 47 or claim 48, wherein the polynucleotide comprises: (a) 5' ALB UTR, which contains the nucleic acid sequence shown in SEQ ID NO: 18; (b) A Kozak sequence comprising the nucleic acid sequence shown in SEQ ID NO: 21; (c) The nucleic acid sequence encoding the engineered wide-range nuclease; (d) 3' SNRPB UTR, which contains the nucleic acid sequence shown in SEQ ID NO: 19; and (e) polyA termination sequence.

51. The recombinant DNA construct according to any one of claims 47-50, wherein the recombinant DNA construct encodes a recombinant virus comprising the polynucleotide.

52. The recombinant DNA construct according to claim 51, wherein the recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant adeno-associated virus (AAV).

53. The recombinant DNA construct according to claim 51 or claim 52, wherein the recombinant virus is a recombinant AAV.

54. The recombinant DNA construct according to any one of claims 49-53, wherein the polynucleotide comprises a promoter operatively linked to the nucleic acid sequence encoding the engineered wide-ranging nuclease.

55. The recombinant DNA construct according to claim 54, wherein the promoter is a liver-specific promoter.

56. A recombinant virus comprising the polynucleotide of any one of claims 36-43.

57. The recombinant virus according to claim 56, wherein the recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant AAV.

58. The recombinant virus according to claim 56 or claim 57, wherein the recombinant virus is a recombinant AAV.

59. The recombinant virus according to any one of claims 56-58, wherein the polynucleotide comprises a promoter operatively linked to the nucleic acid sequence encoding the engineered wide-ranging nuclease.

60. The recombinant virus according to claim 59, wherein the promoter is a liver-specific promoter.

61. A lipid nanoparticle composition comprising lipid nanoparticles of the polynucleotide of any one of claims 36-46.

62. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the engineered wide-range nuclease of any one of claims 1-35.

63. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the polynucleotide of any one of claims 36-46.

64. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the recombinant DNA construct of any one of claims 47-55.

65. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the recombinant virus of any one of claims 56-60.

66. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the lipid nanoparticle composition of claim 61.

67. A host cell comprising the polynucleotide of any one of claims 36-46.

68. A method for inactivating a polymerase (pol) gene for HBV genome or HBV genome fragment, the method comprising introducing it into a eukaryotic cell containing the HBV genome or HBV genome fragment: (a) A polynucleotide comprising a nucleic acid sequence encoding the engineered wide-ranging nuclease of any one of claims 1-35, wherein the engineered wide-ranging nuclease is expressed in the eukaryotic cell; or (b) The engineered wide-range nuclease according to any one of claims 1-35; The engineered large-scale nuclease wherein the pol gene generates a cleavage site at the recognition sequence containing SEQ ID NO: 3, wherein the pol gene is inactivated by introducing an insertion or deletion at the cleavage site, or wherein the pol gene is inactivated by eliminating the HBV genome or the HBV genome fragment.

69. The method of claim 68, wherein the HBV genome or the HBV genome fragment is contained in a covalently closed circular DNA (cccDNA).

70. The method of claim 69, wherein the cccDNA is eliminated after being generated at the cleavage site.

71. The method of claim 69, wherein the pol gene is inactivated in the cccDNA by introducing the indel at the cleavage site.

72. The method of claim 71, wherein the indel is introduced via a non-homologous end join (NHEJ).

73. The method according to claim 71 or claim 72, wherein the inactivated pol gene does not encode active and / or full-length HBV polymerase protein.

74. The method of claim 68, wherein the HBV genome or the HBV genome fragment is contained in the genome of the eukaryotic cell.

75. The method of claim 74, wherein the genome is a nuclear genome.

76. The method of claim 74, wherein the genome is a mitochondrial genome.

77. The method according to any one of claims 74-76, wherein the indel is introduced via NHEJ.

78. The method according to any one of claims 74-77, wherein the inactivated pol gene does not encode active and / or full-length HBV polymerase protein.

79. The method according to any one of claims 68-77, wherein the method inactivates the HBV S antigen (HBsAg) gene in the HBV genome or the HBV genome fragment.

80. The method according to any one of claims 71-79, wherein the eukaryotic cell is a human cell.

81. The method of claim 80, wherein the human cell is a liver cell.

82. The method of claim 81, wherein the liver cells are hepatocytes.

83. The method according to any one of claims 68-82, wherein the polynucleotide is the polynucleotide of any one of claims 36-46.

84. The method according to any one of claims 68-83, wherein the polynucleotide is introduced into the eukaryotic cell via mRNA or recombinant virus.

85. The method of claim 84, wherein the mRNA is the mRNA of any one of claims 42-44.

86. The method of claim 84 or claim 85, wherein the mRNA is introduced into the eukaryotic cell by contacting the eukaryotic cell with lipid nanoparticles containing the mRNA.

87. The method of claim 84, wherein the recombinant virus is the recombinant virus of any one of claims 56-60.

88. The method of claim 87, wherein the recombinant virus is a recombinant AAV.

89. A method for inactivating the pol gene, or an HBV genome fragment, in target cells of a subject, the method comprising delivering to the target cells containing the HBV genome or HBV genome fragment: (a) A polynucleotide comprising a nucleic acid sequence encoding the engineered wide-range nuclease of any one of claims 1-35, wherein the engineered wide-range nuclease is expressed in the target cells; or (b) The engineered wide-range nuclease according to any one of claims 1-35; The engineered large-scale nuclease wherein the pol gene generates a cleavage site at the recognition sequence containing SEQ ID NO: 3, wherein the pol gene is inactivated by introducing an indel at the cleavage site, or wherein the pol gene is inactivated by eliminating the HBV genome or the HBV genome fragment.

90. The method of claim 89, wherein the HBV genome or the HBV genome fragment is contained in cccDNA.

91. The method of claim 90, wherein the cccDNA is eliminated after being generated at the cleavage site.

92. The method of claim 91, wherein the pol gene is inactivated in the cccDNA by introducing the indel at the cleavage site.

93. The method of claim 92, wherein the indel is introduced via NHEJ.

94. The method according to claim 92 or claim 93, wherein the inactivated pol gene does not encode active and / or full-length HBV polymerase protein.

95. The method of claim 89, wherein the HBV genome or the HBV genome fragment is contained in the genome of the target cell.

96. The method of claim 95, wherein the genome is a nuclear genome.

97. The method of claim 95, wherein the genome is a mitochondrial genome.

98. The method according to any one of claims 95-97, wherein the indel is introduced via NHEJ.

99. The method according to any one of claims 95-97, wherein the inactivated pol gene does not encode active and / or full-length HBV polymerase protein.

100. The method according to any one of claims 89-99, wherein the method inactivates the HBsAg gene in the HBV genome or the HBV genome fragment.

101. The method according to any one of claims 89-100, wherein the serum HBsAg concentration in the subject is reduced.

102. The method according to any one of claims 92-101, wherein the target cell is a liver cell.

103. The method of claim 102, wherein the liver cells are hepatocytes.

104. The method according to any one of claims 89-103, wherein the polynucleotide is the polynucleotide of any one of claims 36-46.

105. The method according to any one of claims 89-104, wherein the polynucleotide is mRNA or is contained in the genome of a recombinant virus.

106. The method of claim 105, wherein the mRNA is the mRNA of any one of claims 44-46.

107. The method of claim 105 or claim 106, wherein the mRNA is delivered to the target cell using lipid nanoparticles containing the mRNA.

108. The method of claim 104 or claim 105, wherein the polynucleotide is delivered to the target cell using a recombinant virus containing the polynucleotide in its genome.

109. The method of claim 105, wherein the recombinant virus is the recombinant virus of any one of claims 56-60.

110. The method of claim 109, wherein the recombinant virus is recombinant AAV.

111. A method for treating hepatitis B virus (HBV) infection or a disease associated with hepatitis B virus infection, the method comprising delivering to target cells in the subject: (a) A therapeutically effective amount of a polynucleotide comprising a nucleic acid sequence encoding the engineered macronuclease of any one of claims 1-35, wherein the engineered macronuclease is expressed in the target cells; or (b) A therapeutically effective amount of the engineered wide-range nuclease of any one of claims 1-35; The target cell contains an HBV genome or an HBV genome fragment containing a pol gene, wherein the engineered large-scale nuclease generates a cleavage site at the recognition sequence of SEQ ID NO: 3 within the pol gene, wherein the pol gene is inactivated by introducing an indel at the cleavage site, or wherein the pol gene is inactivated by eliminating the HBV genome or the HBV genome fragment.

112. The method according to claim 111, wherein the disease is chronic hepatitis B, hepatocellular carcinoma, and / or cirrhosis.

113. The method of claim 111 or claim 112, wherein the HBV genome or the HBV genome fragment is contained in cccDNA.

114. The method of claim 113, wherein the cccDNA is eliminated after being generated at the cleavage site.

115. The method of claim 113, wherein the pol gene is inactivated in the cccDNA by introducing the indel at the cleavage site.

116. The method of claim 115, wherein the indel is introduced via NHEJ.

117. The method of claim 115 or claim 116, wherein the inactivated pol gene does not encode active and / or full-length HBV polymerase protein.

118. The method of claim 111 or claim 112, wherein the HBV genome or the HBV genome fragment is contained in the genome of the target cell.

119. The method of claim 118, wherein the genome is a nuclear genome.

120. The method of claim 118, wherein the genome is a mitochondrial genome.

121. The method according to any one of claims 118-120, wherein the indel is introduced via NHEJ.

122. The method according to any one of claims 118-121, wherein the inactivated pol gene does not encode active and / or full-length HBV polymerase protein.

123. The method according to any one of claims 111-122, wherein the method inactivates the HBsAg gene in the HBV genome or the HBV genome fragment.

124. The method according to any one of claims 111-123, wherein the serum HBsAg concentration in the subject is reduced.

125. The method according to any one of claims 115-124, wherein the target cell is a liver cell.

126. The method of claim 125, wherein the liver cells are hepatocytes.

127. The method according to any one of claims 111-126, wherein the polynucleotide is the polynucleotide of any one of claims 36-46.

128. The method according to any one of claims 111-127, wherein the polynucleotide is mRNA or is contained in the genome of a recombinant virus.

129. The method of claim 128, wherein the mRNA is the mRNA of any one of claims 44-46.

130. The method of claim 128 or claim 129, wherein the mRNA is delivered to the target cell using lipid nanoparticles containing the mRNA.

131. The method of claim 127 or claim 128, wherein the polynucleotide is delivered to the target cell using a recombinant virus containing the polynucleotide in its genome.

132. The method of claim 128, wherein the recombinant virus is the recombinant virus of any one of claims 56-60.

133. The method of claim 131, wherein the recombinant virus is a recombinant AAV.

134. The method according to any one of claims 111-133, wherein the subject is further administered an antiviral drug or an immunomodulator.

135. The method according to any one of claims 111-134, wherein the subject is further administered one or more drugs selected from tenofovir disoproxil fumarate, buleviride, tenofovir alafenamide, entecavir, telbivudine, adefovir dipivoxil, lamivudine, pegylated interferon, and interferon alpha.

Images