Engineered meganucleases specific for recognition sequences in hepatitis b virus genome

Abstract

To provide an engineered meganuclease useful for the treatment of Hepatitis B virus (HBV) infection.SOLUTION: There is provided an engineered meganuclease that recognizes and cleaves a recognition sequence within an open reading frame (ORF) of a genome of at least two genotypes of Hepatitis B virus. The engineered meganuclease comprises a first subunit and a second subunit. Therein: the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region; and the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the fields of oncology, molecular biology, and recombinant nucleic acid technology. In particular, the present invention , specificity for recognition sequences in at least two genotypes of the hepatitis B virus genome. The present invention relates to a genetically engineered meganuclease having the following structure: Hepatitis B virus infection and liver damage caused by hepatitis B virus The compounds are useful in methods for treating cell carcinomas.

[0002] Reference to sequence listings submitted as text files via EFS-WEB This application contains a sequence listing submitted in ASCII format via EFS-Web. , the entire contents of which are incorporated herein by reference. An ASCII copy of the above is P109070018WO00-SEQ-MJT.tx It is named t and is 169,011 bytes in size. [Background technology]

[0003] Hepatitis B virus (HBV) is a major health problem worldwide, affecting over 350 million people HBV infection is a serious and common infection of the liver in people who are chronic carriers. Chronic infection can lead to cirrhosis of the liver and hepatocellular carcinoma (HCC), one of the most common forms of cancer in humans. It is associated with an increased risk of developing serious liver disease, including HBV. The estimated risk of HCC in infected individuals is approximately 100 times greater than in uninfected individuals. Approximately one-third of the world's population has been infected at some point in their lives, or 240 million people. This includes 350 million people with chronic infections. Over 750,000 people die from hepatitis B each year. Of these, approximately 300,000 are due to liver cancer. Currently available anti-HBV drugs are limited. For example, administration of interferon alpha is associated with serious adverse reactions. Nucleoside analogues are virostatic and require long-term administration.

[0004] The HBV genome is 1.4–3.2 × 10 per site per year. -5 nucleotide This shows genetic diversity at the estimated rate of substitution by either viral polymerase. Replication occurs as a result of nucleotide misincorporation in the absence of proofreading. During this time, numerous viral variants arise. This diversity contributes to the well-recognized nature of the virus. HBV has evolved into a subtype characterized by a difference of more than 8% in the complete genome sequence. They are classified into distinct genotypes based on their genetic makeup, each with a distinct geographic distribution. Genotype A is widespread in sub-Saharan Africa, Northern Europe, and West Africa. Genotypes B and C are common in Asia. Genotype C is primarily observed in Southeast Asia. Genotype D is predominant in Africa, Europe, Mediterranean countries, and India. Genotype G has been reported in France, Germany, and the United States, and genotype H has been reported in Latin America. Genotype I has recently been reported in Vietnam and Laos. The offspring type, genotype J, has been identified in the Ryukyu Islands of Japan.

[0005] HBV is an enveloped DNA virus that belongs to the Hepadnaviridae family. A small, RNA intermediate that replicates by reverse transcription of pregenomic RNA (pgRNA). It contains a partially double-stranded (DS) relaxed circular DNA (rcDNA) genome. The DNA genome is unusual because the DNA is not entirely double-stranded. One end of the genome is bound to the viral DNA polymerase. nucleotides long (full-length strand), 1700-2800 nucleotides long (short-length strand). The negative sense (non-coding) is complementary to 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), whose initiation codon is The sequence is preceded by an in-frame AUG start codon from which the pre-core protein is produced. HBeAg is produced by proteolytic processing of the pre-core protein. The enzyme is encoded by the gene P. Gene S codes for the surface antigen (HBsAg). The HBsAg gene is one long open reading frame, but Three inflation steps that divide the child into three parts: pre-S1, pre-S2, and S. The start codon (ATG) of the nucleosome is included. In order, pre-S1 / pre-S2 / S), medium (pre-S2 / S), and small (S). Three different sized polypeptides called The function of the protein is not fully understood, but it is associated with the development of liver cancer. It stimulates genes that promote cell proliferation and inactivates growth-regulating molecules.

[0007] Viral DNA is found in the nucleus shortly after infection of a cell. Partially double-stranded DNA is , completion of the (+) sense strand and removal of protein molecules from the (-) sense strand; and It is made fully double-stranded by the removal of a short RNA sequence from the (+)-sense strand. bases are removed from the end of the (-) sense strand and the ends are religated.

[0008] The HBV life cycle begins when the virus attaches to and internalizes a host cell. Recent studies have shown that sodium taurocholate cotransporting polypeptide (NTCP) inhibits HBV infection. The virion relaxed circular DNA (rcDNA) is a functional receptor in A) is delivered to the nucleus, where it is repaired to form covalently closed circular DNA (cccDNA). Episomal cccDNA is formed by the pregenomic RNA polymerase II. It serves as a template for the transcription of NA (pgRNA) and other viral mRNAs. The product is then transported to the cytoplasm where translation of viral proteins occurs. T) binds to pgRNA and induces the core protein to enter the immature RNA-containing nucleocapsid. The immature nucleocapsid then undergoes a maturation process, resulting in p The gRNA is reverse transcribed by RT to produce mature rcDNA. A distinctive feature of transcription is the RT-primed initiation of minus-strand DNA synthesis, which This results in the covalent attachment of RT to the 5' end of the minus-strand DNA.

[0009] The mature rcDNA-containing nucleocapsid is then bound by viral surface proteins. They are enveloped and secreted as virions (secretory pathway) or, alternatively, by cccD The NA pool is then recycled back to the nucleus (recycling pathway). ccDNA persistence is a key factor in determining viral persistence, the resumption of viral replication after cessation of antiviral therapy. It plays an important role in activation and resistance to therapy.

[0010] Homing endonucleases are commonly found in plant and fungal genomes. Group 1 is a group of naturally occurring nucleases that recognize cleavage sites at 0 base pairs. Associated with parasitic DNA elements such as self-splicing introns and inteins They target specific regions of the host genome by creating double-strand breaks in the chromosome. naturally promotes homologous recombination or gene insertion at the site of Homing endonucleases are generally classified into four families: LAGLIDADG (SEQ ID NO: 2) family, GIY-YIG family, H The is-Cys box family and the HNH family. These families are They are characterized by structural motifs that influence catalytic activity and recognition sequences. For example, Members of the LAGLIDADG (SEQ ID NO: 2) family contain the conserved LAGLIDA DG (SEQ ID NO: 2) motif (Non-patented) (See reference 2.) LA has a single copy of the LAGLIDADG (SEQ ID NO: 2) motif. GLIDADG (SEQ ID NO: 2) homing endonuclease forms homodimers and On the other hand, members with two copies of the LAGLIDADG (SEQ ID NO: 2) motif are monomeric. Methods for producing homing endonucleases are known in the art. It is publicly known.

[0011] I-CreI (SEQ ID NO: 1) is a 22-base pair fragment located in the chloroplast chromosome of the green alga Chlamydomonas reinhardtii. The homing endonuclease LAGLIDADG (sequence (Column 2) is a member of the family. Using genetic selection techniques, wild-type I-CreI The cleavage site selectivity was modified (Non-Patent Documents 3 to 6). I-Cre is used to target a wide range of different DNA sites, including sites in the virus genome. Mono-L, which can comprehensively redesign I and other homing endonucleases A method for rationally designing the AGLIDADG (SEQ ID NO: 2) homing endonuclease was described (Patent Document 1).

[0012] As first described in US Pat. No. 5,629,999, I-CreI and its genetically engineered derivatives The conductor is usually a dimer, with the C-terminus of the first subunit connected to the N-terminus of the second subunit. Using short peptide linkers attached to the ends, they can be fused into a single polypeptide (Non-Patent Document 1). Therefore, functional "single-chain" meganucleases are those that encode a single transcript. It can be expressed from

[0013] The use of genetically engineered meganucleases for the treatment of HBV infection has been shown. For example, Patent Document 3 discloses a method for cleaving the genome of a non-genome-integrating virus. The authors suggest the use of genetically engineered meganucleases. The enzyme contains a mutant of I-CreI. Patent document 3 describes a gene present in the genome of one HBV strain. However, Patent Document 3 discloses a number of 22-base pair meganuclease recognition sequences. It does not identify any recognition sequences present in multiple genotypes of the BV genome. [Prior art documents] [Patent documents]

[0014]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Document

[0015]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

Problems to be Solved by the Invention

[0016] The present invention, in part, comprises a method for the detection of at least one open reading frame of the HBV genome. DNA sequences within the open reading frame (ORF) and within at least two different genotypes of the HBV genome Depends on the development of site-specific rare-cutting endonucleases that are engineered to recognize The present inventors have developed a recombinant meganuclease to inactivate multiple genotypes of viruses. Identifying specific recognition sequences conserved across genotypes that can be targeted by ATPases did.

[0017] The present invention improves upon the prior art in several ways. We found that sequences can be identified within the ORFs of several genotypes of the HBV genome. By targeting some genotypes of the virus, it is possible to identify the differences present in localized areas of the world. A single pharmaceutical composition can be prepared that can be used for multiple genotypes. Thus, the methods and compositions disclosed herein may contribute to the prevention of H. pylori infection in infected individuals worldwide. Useful for treating or reducing the proliferation of HBV. Deprivation of riboflavin leads to improvement of liver pathology and a reduction in progression to cirrhosis and hepatocellular carcinoma. There is clearly a need in the art for additional gene therapy approaches for HBV infection. Furthermore, the present invention provides a method for inserting a "suicide gene" into the HBV genome of HCC cells. Treating hepatocellular carcinoma by providing a means to target insertion by homologous recombination The suicide gene is a toxin or an apoptosis promoter that directly kills cancer cells. a protein, or cell surface antigen, that instructs the subject's own immune system to kill cancer cells It may encode a native or MHC class I antigen polypeptide.

[0018] The present invention provides a genetically engineered meganucleotide useful for the treatment of hepatitis B virus (HBV) infection. The engineered meganucleases of the present invention comprise at least two Recognition within the open reading frame (ORF) of the genome of hepatitis B virus genotypes The genetically engineered meganuclear protein disclosed herein recognizes and cleaves the recognition sequence. Cleavage at such recognition sequences by enzymes leads to non-homologous end joining (NHEJ) at the cleavage site. NHEJ can result in disruption of the expression of one or more viral proteins. frameshift mutations that may cause a mutation or prevent expression of a gene Therefore, by disrupting normal gene expression, HBV may Infection and proliferation of bacteria can be reduced or eliminated according to the methods disclosed herein. The present invention also provides a method for detecting a hepatitis B virus in an ORF of the genome of at least two genotypes of the virus. By utilizing genetically engineered meganucleases with specificity for recognition sequences located in The present invention further provides pharmaceutical compositions and methods for the treatment of HBV, including the treatment of HBV levels. and / or to reduce symptoms associated with HBV infection. The disclosed method for delivering engineered meganucleases to subjects infected with HBV is provide. [Means for solving the problem]

[0019] Thus, in one embodiment, the present invention provides a method for detecting at least two hepatitis B virus genotypes. Recognizes and cleaves recognition sequences within the genome open reading frame (ORF). The present invention provides a genetically engineered meganuclease. The genetically engineered meganuclease is a first subunit and a second subunit, the first subunit having a first recognition sequence; It binds to the recognition half site of the first hypervariable (HVR1) region and contains the second subunit. The nucleotide sequence binds to the second recognized half of the recognition sequence and binds to the second hypervariable (HVR2) region. Includes:

[0020] In some embodiments, the recognition sequences are at least three of the Hepatitis B virus are found within the ORFs of at least four, at least five, or at least six genotypes. In one such embodiment, the recognition sequence is the OR of Hepatitis B virus genotype A (SEQ ID NO: 3). F. Such embodiments may be any of the sequences different from SEQ ID NO: 3 but which are one or more of the sequences described herein. and a hepatitis B virus isolate of genotype A containing an HBV meganuclease recognition sequence of In further such embodiments, the recognition sequence is an ORF of genotype A as well as genotypes B, C , D, E, F, and G (SEQ ID NOs: 4 to 9, respectively) are within one or more ORFs. Such embodiments may include one or more HBV meganucleotides different from SEQ ID NOs: 3-9 but described herein. Hepatitis B virus of genotypes A, B, C, D, E, F, and G containing nuclease recognition sequences Includes Rus isolates.

[0021] In some embodiments, the recognition sequence is a sequence encoding a polymerase (P) protein, a large surface (pr eS1 / preS2 / S) proteins, mid-surface (preS2 / S) proteins, and subsurface At least one encoding a protein selected from the group consisting of surface (S) proteins In some embodiments, the recognition sequence is within the ORF of SEQ ID NO: 10 (i.e., H HBV1-2 recognition sequence), SEQ ID NO: 12 (i.e., HBV5-6 recognition sequence), SEQ ID NO: 14 ( i.e., HBV7-8 recognition sequence), or SEQ ID NO: 16 (i.e., HBV11-12 recognition sequence) may include:

[0022] In some embodiments, the recognition sequence comprises SEQ ID NO: 10 and the HVR1 region comprises SEQ ID NO: Residues 24-79 of SEQ ID NO: 18 or 19, or residues 215-27 of SEQ ID NO: 20 or 21 At least 80%, at least 85%, at least 9% of the amino acid sequence corresponding to 0 The amino acid sequence may comprise an amino acid sequence having 0%, at least 95%, or more sequence identity. In some such embodiments, the HVR1 region is selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 19. residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70 , 75, and 77, or residues 215, 217 of SEQ ID NO: 20 or 21 , 219, 221, 223, 224, 229, 231, 233, 235, 237, 259 , 261, 266, and 268. In certain embodiments, H The VR1 region is residues 24 to 79 of SEQ ID NO: 18 or 19, or residues 24 to 79 of SEQ ID NO: 20 or 21 may include residues 215-270.

[0023] In some such embodiments, the recognition sequence comprises SEQ ID NO: 10, and the HVR2 region comprises , residues 215 to 270 of SEQ ID NO: 18 or 19, or residues 215 to 270 of SEQ ID NO: 20 or 21 At least 80%, at least 85%, or at least amino acid sequences with at least 90%, at least 95%, or more sequence identity In some such embodiments, the HVR2 region may comprise SEQ ID NO: 18 or Residues 215, 217, 219, 221, 223, 224, 229, 231, and 232 are all nucleotides in the nucleotide sequence. Residues or sequences corresponding to 33, 235, 237, 259, 261, 266, and 268 Residues 24, 26, 28, 30, 32, 33, 38, 40, and 42 of numbers 20 and 21, In certain embodiments, the amino acid sequence may include residues corresponding to 44, 46, 68, 70, 75, and 77. wherein the HVR2 region is residues 215 to 270 of SEQ ID NO: 18 or 19, or It may comprise residues 24-79 of No. 20 or No. 21.

[0024] In such an embodiment, the recognition sequence comprises SEQ ID NO: 10 and the first subunit comprises the sequence Residues 7 to 153 of SEQ ID NO: 18 or 19, or residue 198 of SEQ ID NO: 20 or 21 At least 80%, at least 85%, at least 90%, at least The second subunit may comprise an amino acid sequence having 95% or more sequence identity. is residues 198 to 344 of SEQ ID NO: 18 or 19, or residues 20 or 21 At least 80%, at least 85%, at least 90%, or at least The amino acid sequence may have at least 95% or more sequence identity. In embodiments, the first subunit comprises residues 7 to 153 of SEQ ID NO: 18 or 19. or residues 198 to 344 of SEQ ID NO: 20 or 21. Similarly, some In embodiments, the second subunit comprises residues 198-3 of SEQ ID NO: 18 or 19. 44, or residues 7-153 of SEQ ID NO:20 or 21.

[0025] In certain such embodiments, the recognition sequence comprises SEQ ID NO: 10, and the genetically engineered The nuclease can include a linker, the linker connecting the first subunit and the second subunit. In certain embodiments, the engineered meganucleic acid is a nucleotide sequence that is covalently linked to the nucleotide sequence of the nucleotide. The ase may comprise the amino acid sequence of any one of SEQ ID NOs: 18-21.

[0026] In another embodiment, the recognition sequence comprises SEQ ID NO: 12 and the HVR1 region comprises SEQ ID NO: 22 Residues 215 to 270 of any one of SEQ ID NOs: 24, or residues 215 to 270 of any one of SEQ ID NOs: 25 to 28 At least 80%, at least 85%, or at least amino acid sequences with at least 90%, at least 95%, or more sequence identity In some such embodiments, the HVR1 region may comprise SEQ ID NO: 22-2 Any one of residues 215, 217, 219, 221, 223, 224, 229, 2 Residues corresponding to 31, 233, 235, 237, 259, 261, 266, and 268, or residues 24, 26, 28, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, It may include residues corresponding to 8, 40, 42, 44, 46, 68, 70, 75, and 77. In certain embodiments, the HVR1 region comprises residue 2 of any one of SEQ ID NOs: 22-24. 15 to 270, or residues 24 to 79 of any one of SEQ ID NOs: 25 to 28.

[0027] In some such embodiments, the recognition sequence comprises SEQ ID NO: 12, and the HVR2 region comprises residues 24 to 79 of any one of SEQ ID NOs: 22 to 24, or any one of SEQ ID NOs: 25 to 28 At least 80% of the amino acid sequence corresponding to residues 215-270 of one of the have at least 85%, at least 90%, at least 95%, or more sequence identity In some such embodiments, the HVR2 region may comprise an amino acid sequence Residues 24, 26, 28, 30, 32, 33, and 38 of any one of SEQ ID NOs: 22 to 24; Residues corresponding to 40, 42, 44, 46, 68, 70, 75, and 77, or SEQ ID NO: 2 Any one of residues 5-28 215, 217, 219, 221, 223, 224, 22 9, 231, 233, 235, 237, 259, 261, 266, and 268 In certain embodiments, the HVR2 region may comprise any one of SEQ ID NOs: 22-24. Residues 24 to 79 of any one of SEQ ID NOs: 25 to 28, or residues 215 to 27 of any one of SEQ ID NOs: 25 to 28 May contain 0.

[0028] In some such embodiments, the recognition sequence comprises SEQ ID NO: 12, and the first subunit The kit is a combination of residues 198 to 344 of any one of SEQ ID NOS: 22 to 24 or residues 25 to 26 of any one of SEQ ID NOS: 25 to 26 At least 80%, at least 85%, or at least 8% of residues 7-153 of any one of the eight containing amino acid sequences with at least 90%, at least 95%, or more sequence identity The second subunit may be a sequence of residues 7 to 153 of any one of SEQ ID NOs: 22 to 24, or At least 80%, at least 100% of residues 198 to 344 of any one of SEQ ID NOs: 25 to 28 at least 85%, at least 90%, at least 95%, or more sequence identity In some embodiments, the first subunit may comprise an amino acid sequence Residues 198 to 344 of any one of SEQ ID NOs: 22 to 24 or any one of SEQ ID NOs: 25 to 28 Similarly, in some embodiments, the second subunit may comprise one or more residues 7 to 153. The unit is composed of residues 7 to 153 of any one of SEQ ID NOs: 22 to 24 or residues 25 to 2 The amino acid sequence may comprise residues 198-344 of any one of the eight amino acid sequences.

[0029] In certain such embodiments, the recognition sequence comprises SEQ ID NO: 12, and the genetically engineered The nuclease can include a linker, the linker connecting the first subunit and the second subunit. In certain embodiments, the engineered meganucleic acid is covalently linked to the nucleotide subunits. The enzyme may comprise any one of the amino acid sequences of SEQ ID NOs: 22 to 28.

[0030] In some embodiments, the recognition sequence comprises SEQ ID NO: 14 and the HVR1 region comprises SEQ ID NO: At least one amino acid sequence corresponding to residues 215 to 270 of any one of 29 to 32 At least 80%, at least 85%, at least 90%, at least 95%, or more In some such embodiments, the amino acid sequence may include an amino acid sequence having sequence identity. The HVR1 region is a region consisting of residues 215, 217, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 21, 223, 224, 229, 231, 233, 235, 237, 259, 261, 2 In certain embodiments, the HVR1 region may comprise residues corresponding to SEQ ID NO: 66, 67, and 68. , may comprise residues 215 to 270 of any one of SEQ ID NOs: 29 to 32.

[0031] In some such embodiments, the recognition sequence comprises SEQ ID NO: 14, and the HVR2 region comprises , for the amino acid sequence corresponding to residues 24 to 79 of any one of SEQ ID NOs: 29 to 32 At least 80%, at least 85%, at least 90%, at least 95%, or In some such embodiments, the amino acid sequence may include an amino acid sequence having at least one amino acid sequence identity greater than or equal to 100% of the amino acid sequence. The HVR2 region is a sequence of residues 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, Corresponding to 0, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 In certain embodiments, the HVR2 region may comprise any of SEQ ID NOs: 29-32. Any one of residues 24 to 79 may be included.

[0032] In some such embodiments, the recognition sequence comprises SEQ ID NO: 14, and the first subunit The set contains at least 8 sequences for residues 198 to 344 of any one of SEQ ID NOs: 29 to 32. 0%, at least 85%, at least 90%, at least 95% or more sequence identity The second subunit may comprise an amino acid sequence having the same identity as that of SEQ ID NOs: 29 to 32. At least 80%, at least 85%, at least 9% of any one of residues 7-153 The amino acid sequence may comprise an amino acid sequence having 0%, at least 95%, or more sequence identity. In some embodiments, the first subunit is any one of SEQ ID NOs: 29-32. Similarly, in some embodiments, the second subunit may comprise residues 198-344. The unit may comprise residues 7-153 of any one of SEQ ID NOs: 29-32.

[0033] In certain such embodiments, the recognition sequence comprises SEQ ID NO: 14, and the genetically engineered The nuclease can include a linker, the linker connecting the first subunit and the second subunit. In certain embodiments, the engineered meganucleic acid is covalently linked to the nucleotide subunits. The enzyme may comprise any one of the amino acid sequences of SEQ ID NOs: 29 to 32.

[0034] In some embodiments, the recognition sequence comprises SEQ ID NO: 16 and the HVR1 region comprises SEQ ID NO: At least 80% of residues 215-270 of any one of 33-39, Amino acids with 85%, at least 90%, at least 95%, or more sequence identity In some such embodiments, the HVR1 region may comprise the amino acid sequence of SEQ ID NO: residues 215, 217, 219, 221, 223, and 224 of any one of Nos. 33 to 39; Compatible with 229, 231, 233, 235, 237, 259, 261, 266, and 268 In certain embodiments, the HVR1 region may comprise residues corresponding to any of SEQ ID NOS: 33-39. It may include any one of residues 215-270.

[0035] In some such embodiments, the recognition sequence comprises SEQ ID NO: 16, and the HVR2 region comprises , at least 80%, at least have at least 85%, at least 90%, at least 95%, or more sequence identity In some such embodiments, the HVR2 region may comprise an amino acid sequence Residues 24, 26, 28, 30, 32, 33, and 38 of any one of SEQ ID NOs: 33 to 39; It may include residues corresponding to 40, 42, 44, 46, 68, 70, 75, and 77. In one embodiment, the HVR2 region comprises residues 24-39 of any one of SEQ ID NOs: 33-39. 79 may be included.

[0036] In such an embodiment, the recognition sequence comprises SEQ ID NO: 16 and the first subunit comprises the sequence At least 80% of residues 198 to 344 in any one of sequences 33 to 39, have at least 85%, at least 90%, at least 95%, or more sequence identity The second subunit may comprise an amino acid sequence of any one of SEQ ID NOs: 33 to 39. At least 80%, at least 85%, at least 90%, or at least The amino acid sequence may have at least 95% or more sequence identity. In an embodiment, the first subunit comprises residue 1 of any one of SEQ ID NOs: 33-39. 98 to 344. Similarly, in some embodiments, the second subunit may comprise , may comprise residues 7 to 153 of any one of SEQ ID NOs: 33 to 39.

[0037] In certain embodiments, the engineered meganuclease can include a linker. The linker covalently bonds the first subunit to the second subunit. In this embodiment, the engineered meganuclease is selected from the group consisting of SEQ ID NOs: 33-39. It may comprise one amino acid sequence.

[0038] In another aspect, the present invention provides a method for producing a medicament comprising the steps of: An isolated polynucleotide comprising a nucleic acid sequence encoding a nuclease is provided. In this embodiment, the isolated polynucleotide may be mRNA.

[0039] In a further embodiment, the mRNA is derived from one or more genetically engineered mRNAs described herein. In certain embodiments, the mRNA may be a polycistronic mRNA encoding a ganucleases. In this regard, the polycistronic mRNA of the present invention is not limited to, but includes, but is not limited to, the polycistronic mRNAs described herein. A bicistronic mRNA encoding the two engineered meganucleases described in this paper A tricistronic m encoding the three engineered meganucleases described herein. RNA or clones encoding the four engineered meganucleases described herein. In such an embodiment, two or more genetic manipulations may be involved. The resulting meganucleases are encoded by polycistronic mRNAs, each The encoded engineered meganucleases have specificity for different HBV recognition sequences. Furthermore, in such an embodiment, the polycistronic mRNA of the present invention has may encode any combination of the engineered meganucleases described herein In certain embodiments, the polycistronic mRNA is an HBV1-2 meganuclear HBV5-6 meganuclease, HBV7-8 meganuclease, and HBV11 In another specific embodiment, the polycistronic nucleotide sequence may encode a -12 meganuclease. The mRNA contains HBV5-6 meganuclease and HBV11-12 meganuclease. It may be a bicistronic mRNA.

[0040] In a further embodiment, the polycistronic mRNA of the invention comprises the polycistronic mRNA of any one of the sequences described herein. One or more genetically engineered meganucleases and HBV-infected cells and / or HBV-infected cells. The gene may encode one or more additional proteins that induce a therapeutically beneficial effect in the infected subject.

[0041] In another aspect, the present invention provides a method for producing a medicament comprising administering to a subject the steps of: In some embodiments, the recombinant DNA construct comprises a nucleic acid sequence encoding the The DNA construct comprises a promoter and an engineered meganuclease as described herein. and a nucleic acid sequence encoding the comprises two or more cassettes, each cassette containing a promoter and a genetic manipulation as described herein. each engineered meganuclease comprising a nucleic acid sequence encoding the engineered meganuclease; The enzymes have specificity for the different HBV recognition sequences disclosed herein. In some embodiments, the recombinant DNA construct may comprise two cassettes, three cassettes, four cassettes, or , or more.

[0042] In another embodiment, the recombinant DNA construct comprises a promoter and a polycistronic nucleic acid. a cassette containing a polycistronic nucleic acid sequence, and a promoter driving expression of the polycistronic nucleic acid sequence. to produce the polycistronic mRNA described herein in the target cell.

[0043] In certain embodiments, the recombinant DNA construct comprises any of the genes disclosed herein. Encoding a viral vector containing a nucleic acid sequence encoding an engineered meganuclease In such embodiments, the viral vector may be a retrovirus, a lentivirus, an adenovirus, or a virion. In certain embodiments, the vector may be a virion, a virion, or an adeno-associated virus (AAV) vector. Alternatively, the viral vector may be a recombinant AAV vector.

[0044] In some embodiments, the viral vector comprises a promoter and a gene described herein. and a cassette containing a nucleic acid sequence encoding an engineered meganuclease. In embodiments, the viral vector comprises two or more cassettes, each cassette comprising a promoter. and a nucleic acid sequence encoding the engineered meganuclease described herein. Each engineered meganuclease is directed against a different HBV recognition sequence as disclosed herein. It has specificity.

[0045] In another embodiment, the viral vector comprises a promoter and a polycistronic nucleic acid sequence. wherein the promoter drives expression of the polycistronic nucleic acid sequence. to produce the polycistronic mRNA described herein in the target cell.

[0046] In another aspect, the present invention provides a method for producing a medicament comprising administering to a subject the steps of: In some embodiments, viral vectors are provided that include a nucleic acid sequence encoding the viral vector. The vector may be a retrovirus, lentivirus, adenovirus, or adeno-associated virus. In certain embodiments, the viral vector may be a recombinant AAAV vector. In some embodiments, the viral vector may be a V vector. and a nucleic acid sequence encoding the engineered meganuclease described herein. In other embodiments, the viral vector comprises two or more cassettes, each cassette The fragment contains a promoter and a gene encoding the engineered meganuclease described herein. and a nucleic acid sequence, wherein each engineered meganuclease comprises a different H It has specificity for the BV recognition sequence.

[0047] In a further embodiment, the viral vector comprises a promoter and a polycistronic nucleic acid. a promoter for directing expression of the polycistronic nucleic acid sequence; This drives the production of the polycistronic mRNA described herein in the target cell.

[0048] In another aspect, the present invention provides a method for treating hepatitis B virus (HBV) caused by HBV. or a pharmaceutical composition for treating a subject with hepatocellular carcinoma, comprising a pharmaceutically acceptable a carrier; and (a) a nucleic acid encoding an engineered meganuclease described herein; or (b) a pharmaceutical comprising an engineered meganuclease protein as described herein. The invention provides a composition, wherein the engineered meganuclease inhibits at least one of the hepatitis B virus. Recognition sequences within the open reading frames (ORFs) of the genomes of the two genotypes In some embodiments, the recognition sequence is at least one of the sequences of Hepatitis B virus. at least three, at least four, at least five, or at least six genotype ORFs In one such embodiment, the recognition sequence is an OR of HBV genotype A (SEQ ID NO: 3). F or recognition sequence, and at least one, at least two, at least three of HBV a single genotype that includes one, at least four, at least five, or at least six other genotypes; In various embodiments, the additional HBV genotypes are genotypes B, C, D, E, F, and / or G (SEQ ID NOS: 4-9, respectively), or one or more of the amino acids described herein It may include an isolate thereof that contains an HBV recognition sequence.

[0049] In some embodiments, the engineered meganuclease or the pharmaceutical composition Encoded by the nucleic acid sequence of the pharmaceutical composition is a polymerase (P) protein. surface (preS1 / preS2 / S) proteins, mid-surface (preS2 / S) proteins, and small surface (S) proteins, In some embodiments, both have specificity for a recognition sequence within a single ORF. The sequence may comprise SEQ ID NO: 10, 12, 14, or 16.

[0050] In one embodiment, the engineered meganucleases disclosed herein are The nucleic acid sequence encoding the pharmaceutical composition can be an mRNA as described herein. In some such embodiments, the mRNA comprises a polycistronic amino acid sequence as described herein. and the resulting mRNA may be a mixture of two or more engineered meganucleic acids as described herein. The ase is expressed in the target cells in vivo.

[0051] In another embodiment, the pharmaceutical composition comprises the genetically engineered megagene disclosed herein. and a recombinant DNA construct as described herein that contains a nucleic acid sequence encoding a nuclease. In some such embodiments, the recombinant DNA construct comprises a promoter and a and a cassette containing a nucleic acid sequence encoding the engineered meganuclease. In embodiments, the recombinant DNA construct of the pharmaceutical composition comprises two or more cassettes, each cassette The promoter and the nucleic acid encoding the engineered meganuclease described herein. and a nucleic acid sequence, wherein each engineered meganuclease comprises a different HBV sequence disclosed herein. In certain embodiments, the recombinant DNA of the pharmaceutical composition has specificity for the V recognition sequence. The A construct may contain two cassettes, three cassettes, four cassettes, or more. do.

[0052] In another embodiment, the recombinant DNA construct of the pharmaceutical composition comprises a promoter and a polycistronic acid sequence. a cassette containing a polycistronic nucleic acid sequence, wherein the promoter controls the expression of the polycistronic nucleic acid sequence; to drive expression of the polycistronic mRNA described herein in vivo in target cells. A, and two or more engineered meganucleases described herein are administered to a target cell. to be expressed in the

[0053] In another embodiment, the pharmaceutical composition comprises the genetically engineered megagene disclosed herein. In one such embodiment, the nucleic acid sequence encoding the nuclease is a viral vector. So, viral vectors can be retroviruses, lentiviruses, adenoviruses, or AA V. In certain embodiments, the viral vector may be a recombinant AAV vector. do.

[0054] In some such embodiments, the viral vector comprises a promoter and a vector as described herein. and a cassette comprising a nucleic acid sequence encoding the engineered meganuclease of the present invention. In other embodiments, the viral vector comprises two or more cassettes, each cassette comprising a promoter. a motor and a nucleic acid sequence encoding an engineered meganuclease as described herein; each engineered meganuclease comprising a different HBV recognition sequence disclosed herein. It has specificity for columns.

[0055] In other such embodiments, the viral vector comprises a promoter and a polycistronic amino acid sequence. a cassette containing a polycistronic nucleic acid sequence, wherein the promoter is a to drive expression of the polycistronic m The two or more engineered meganucleases described herein produce RNA and transfect the target cells. The gene is expressed in the cells.

[0056] In one such embodiment, the pharmaceutical composition comprises a compound according to the present invention that recognizes and cleaves SEQ ID NO: 10. The invention also includes the genetically engineered meganuclease (or nucleic acid encoding it) disclosed in the document. In certain embodiments, the engineered meganuclease can be selected from the group consisting of SEQ ID NOs: 18 to 19. It may contain any one of the 21 amino acid sequences.

[0057] In another embodiment, the pharmaceutical composition comprises a compound disclosed herein that recognizes and cleaves SEQ ID NO: 12. The invention can include a genetically engineered meganuclease (or a nucleic acid encoding the same) of In certain embodiments, the engineered meganuclease is selected from the group consisting of SEQ ID NOs: 22-28. It may contain any one of the amino acid sequences.

[0058] In another embodiment, the pharmaceutical composition comprises a compound disclosed herein that recognizes and cleaves SEQ ID NO: 14. The invention can include a genetically engineered meganuclease (or a nucleic acid encoding the same) of In certain embodiments, the engineered meganuclease is any of SEQ ID NOs: 29-32. It may contain any one of the amino acid sequences.

[0059] In another embodiment, the pharmaceutical composition comprises a compound disclosed herein that recognizes and cleaves SEQ ID NO: 16. The invention can include a genetically engineered meganuclease (or a nucleic acid encoding the same) of In certain embodiments, the engineered meganuclease is any of SEQ ID NOs: 33-39. It may contain any one of the amino acid sequences.

[0060] In various embodiments, the pharmaceutical composition comprises two or more genetically engineered compounds described herein. The engineered meganuclease can comprise a meganuclease protein, In other embodiments, the antibody has specificity for the different HBV recognition sequences described herein. The pharmaceutical composition may comprise two or more nucleic acids encoding the engineered meganucleases described herein. The engineered meganucleases can comprise nucleic acids, and can be any of the different meganucleases described herein. In such an embodiment, the two or more nucleic acids have specificity for the HBV recognition sequence. The mRNA described herein, the recombinant DNA construct described herein, and / or the In other embodiments, the pharmaceutical composition may be comprised in a viral vector as described herein. One or more of the engineered meganuclease proteins described herein, and One or more nucleic acids (i.e., mRNA, recombinant DNA) encoding the engineered meganuclease. A construct, or viral vector), and The engineered meganucleases used herein are capable of inhibiting the different HBV receptors described herein. It has specificity for the recognition sequence.

[0061] In a further embodiment, the pharmaceutical composition comprises an engineered meganuclear compound as described herein. combinations of enzyme proteins, including the engineered meganucleases described herein; Combinations of nucleic acids (i.e., mRNA, recombinant DNA constructs, viral vectors) or a genetically engineered meganuclease protein and a genetically engineered meganuclease The genetically engineered pharmaceutical compositions may contain a combination of nucleic acids encoding enzyme proteins. Combinations of ganucleases and / or encoded engineered meganucleases are the HBV genotypes A, B, C, D, E, F, and G (SEQ ID NOs: 3 to 9), respectively. The present invention also recognizes isolates of each genotype containing the recognition sequences in the present invention, as well as HBV recognition sequences described herein. and can be cut.

[0062] In some embodiments, the pharmaceutical composition comprises a pharmaceutical composition as described herein encapsulated within a lipid nanoparticle. In certain embodiments, the lipid composition of the pharmaceutical composition may comprise one or more mRNAs of the The polymer nanoparticles of the present invention have specificity for the different HBV recognition sequences described herein. Two or more m as described herein, each encoding an engineered meganuclease. In certain embodiments, the lipid nanoparticles each contain a different HBV recognition sequence. The present invention relates to a method for producing a meganuclease having specificity for a target sequence. The pharmaceutical composition may comprise two, three, or four mRNAs as described herein. The lipid nanoparticles of the composition have specificity for the different HBV recognition sequences described herein. The present invention provides a method for producing a method for producing a nuclease comprising the steps of: In certain embodiments, the lipid nanoparticles may comprise one or more polycistronic mRNAs as described above. The child may contain two, three, or four of the engineered meganucleases described herein. In certain other embodiments, the polypeptide may comprise a polycistronic mRNA encoding a lipid. The nanoparticles can comprise two or more polycistronic mRNAs described herein, Each encodes two or more engineered meganucleases of the invention. In this form, lipid nanoparticles have compositions that enhance delivery and uptake into the liver, particularly hepatocytes. Has.

[0063] In another aspect, the present invention provides a method for treating a subject with HBV. Also provided herein are methods for reducing the level and / or proliferation of HBV, Alternatively, the present invention relates to a method for reducing symptoms associated with HBV. The method comprises administering to a subject target cells (a) a nucleic acid encoding an engineered meganuclease, (b) a nucleic acid by which the selected meganuclease is expressed in vivo in a target cell; or and delivering an engineered meganuclease protein. The cleavage enzyme recognizes ORFs in the genomes of at least two genotypes of hepatitis B virus. The sequence-specific, genetically engineered meganuclease recognizes the recognition sequence and This method results in the HBV genome being cleaved in the target cell. The present invention can reduce or eliminate infection and / or proliferation of bacteria.

[0064] In another embodiment, the present invention provides a method for treating a subject with HCC caused by HBV. The present invention provides a method for treating a target cell of a subject, the method comprising: (1) (a) administering to the target cell of a subject a genetically engineered a nucleic acid encoding a meganuclease, wherein the engineered meganuclease is a target (b) a nucleic acid expressed in vivo in a target cell, or (b) a genetically engineered meganuclear (2) polynucleotide sequences and meganucleotides encoding suicide genes; and delivering a nucleic acid containing a sequence homologous to a sequence adjacent to the cleavage site of the ribosomal enzyme. The meganucleases developed have been shown to be capable of synthesizing the genomes of at least two genotypes of hepatitis B virus. The engineered meganuclease has specificity for a recognition sequence in the ORF. The suicide gene recognizes and cleaves the HBV genome in the target cell. Inserted into the truncated HBV genome by recombination, the suicide gene expression kills the target cell To make.

[0065] In some embodiments, the suicide gene is directly lethal to the target cell. In such embodiments, the directly lethal suicide gene is a toxic polypeptide or an apoptotic polypeptide. In some embodiments, the suicide gene encodes a cis-facilitating protein. It is indirectly lethal to the host, instructing the subject's own immune system to kill the targeted cells. In some such embodiments, the indirectly lethal suicide gene is activated by the subject's immune system. Cell surface proteins that are recognized as foreign and targeted by humoral or cellular immune responses In other such embodiments, the indirectly lethal suicide gene encodes a MH Presented by C class I molecules, they are recognized as foreign by the subject's immune system and induce cell damage. The polypeptide encodes a polypeptide that is targeted by the harmful immune response.

[0066] In some embodiments of the methods for treating HBV infection or HCC, the recognition sequence is at least 3 one, at least four, at least five, or at least six genotypes of hepatitis B virus In one such embodiment, the recognition sequence is found within the ORF of the hepatitis B virus gene. Within the ORF of genotype A (SEQ ID NO: 3). Such embodiments may be different from SEQ ID NO: 3. of genotype A, which comprises one or more HBV meganuclease recognition sequences described herein. In further such embodiments, the recognition sequence is a genotype A hepatitis B virus isolate. and one or more of the ORFs of genotypes B, C, D, E, F, and G (SEQ ID NOs: 4 to 9, respectively). Such embodiments may differ from SEQ ID NOS: 3-9 but are not included herein. HBV meganuclease recognition sequences comprising one or more of the following: Includes E, F, and G hepatitis B virus isolates.

[0067] In such embodiments of the method of treating HBV infection or HCC, the recognition sequence is enzyme (P) proteins, large surface (preS1 / preS2 / S) proteins, middle surface ( proteins selected from the group consisting of preS2 / S proteins, and small surface (S) proteins. In some embodiments, the nucleotide sequence may be present within at least one ORF encoding a protein. In this case, the recognition sequence is SEQ ID NO: 10 (i.e., HBV1-2 recognition sequence), SEQ ID NO: 12 (i.e., SEQ ID NO: 14 (i.e., HBV5-6 recognition sequence), SEQ ID NO: 14 (i.e., HBV7-8 recognition sequence), or SEQ ID NO: It may contain sequence number 16 (i.e., the HBV11-12 recognition sequence).

[0068] In certain embodiments of the method for treating HBV infection or HCC, a genetically engineered meganuclear The enzyme protein or the encoded engineered meganuclease is defined herein as 1 is a genetically engineered meganuclease described.

[0069] In a further embodiment, the method for treating HBV infection or HCC comprises at least one pharmaceutically An acceptable carrier and (a) a carrier containing the engineered meganuclease described herein. a nucleic acid encoding the target nucleic acid, wherein the engineered meganuclease is capable of encoding the target nucleic acid in vivo in a target cell. (b) the nucleic acid to be expressed, or (b) the engineered meganuclease protein described herein. The method includes administering to a subject any of the pharmaceutical compositions of the present invention described herein that contain the substance.

[0070] In some embodiments of the methods for treating HBV infection or HCC, the genetically engineered megagene targeting hepatocytes with nucleic acids encoding nucleases or engineered meganucleases In certain embodiments, an effective amount of engineered meganuclei can be delivered to The nucleic acid encoding the meganuclease or the engineered meganuclease is delivered to the target hepatocyte cells. can be achieved.

[0071] In certain embodiments, delivery of the hepatocytes to the cells occurs ex vivo and involves the use of genetically engineered mediators. A nucleic acid encoding a ganucleases or engineered meganucleases is delivered to the An effective amount of hepatocyte cells is administered to the subject.

[0072] In some embodiments, a hepatotoxic protein or a nucleic acid encoding a hepatotoxic protein Alternatively, the AAV is administered in conjunction with a pharmaceutical composition disclosed herein.

[0073] In certain embodiments of the method, the first recognition sequence may comprise SEQ ID NO: 10. In such an embodiment, the engineered meganuclease recognizes SEQ ID NO: 10. The meganuclease may be any engineered meganuclease of the present invention that recognizes and cleaves a specific In embodiments, the engineered meganuclease is any of SEQ ID NOs: 18-21. It may contain any one of the amino acid sequences.

[0074] In another embodiment of the method, the first recognition sequence may comprise SEQ ID NO: 12. In such embodiments, the engineered meganuclease recognizes SEQ ID NO: 12. The cleavage may be any engineered meganuclease of the invention. In one embodiment, the engineered meganuclease is selected from the group consisting of SEQ ID NOs: 22-28. It may comprise one amino acid sequence.

[0075] In other embodiments of the method, the first recognition sequence may comprise SEQ ID NO: 14. In such embodiments, the engineered meganuclease recognizes SEQ ID NO: 14. The cleavage may be any engineered meganuclease of the invention. In one embodiment, the engineered meganuclease is selected from the group consisting of SEQ ID NOs: 29-32. It may comprise an amino acid sequence.

[0076] In other embodiments of the method, the first recognition sequence may comprise SEQ ID NO: 16. In such embodiments, the engineered meganuclease recognizes SEQ ID NO: 16. The cleavage may be any engineered meganuclease of the invention. In one embodiment, the engineered meganuclease is selected from the group consisting of SEQ ID NOs: 33 to 39. It may comprise an amino acid sequence.

[0077] In certain embodiments of the method, the subject may be a mammal, such as a human.

[0078] In another aspect, the present invention provides a method for producing a genetically engineered compound as described herein for use as a pharmaceutical. The present invention further provides a meganuclease for treating HBV by reducing the level of HBV. or to reduce the proliferation of HBV, to reduce symptoms associated with HBV, or to prevent HCC In the manufacture of a medicament for treating The present invention provides for the use of enzymes.

[0079] In another aspect, the present invention provides an isolated polynucleotide for use as a pharmaceutical. The isolated polynucleotides may be used in the genetically engineered megakaryons disclosed herein. The present invention further provides a method for treating HBV by administering to a mammalian subject the HBV nuclease. to reduce the level or proliferation of HBV, to reduce symptoms associated with HBV, and The present invention relates to the use of the isolated polynucleotide in the manufacture of a medicament for treating HCC. This provides:

[0080] In another aspect, the present invention provides a recombinant AAV vector for use as a pharmaceutical. The recombinant AAV vector comprises an isolated polynucleotide, The peptide comprises a nucleic acid sequence encoding the engineered meganuclease disclosed herein. The present invention further provides a method for treating HBV, reducing the level or proliferation of HBV, and in the manufacture of medicines for the relief of symptoms associated with HBV or for the treatment of HCC The recombinant AAV vector is used in the method of the present invention. The isolated polynucleotides include those disclosed herein. It comprises a nucleic acid sequence encoding an engineered meganuclease.

[0081] These and other aspects and embodiments of the invention are described in detail below with reference to the detailed description and claims. The present invention can be more fully understood by referring to the accompanying drawings, in which separate embodiments are provided for clarity. Certain features of the invention which are described in the context of embodiments may also be used in combination in a single embodiment. All combinations of the embodiments are specifically encompassed by the present invention. as if each and every combination were separately and expressly disclosed Conversely, for the sake of brevity, the context of a single embodiment is The various features of the invention described herein may be used separately or in any suitable subcombination. All subcombinations of the features listed in the embodiments may also be provided. Also specifically encompassed by the present invention, as if each and every such subcomponent The same combinations are used herein as if they were individually and expressly disclosed herein. Embodiments of each aspect of the invention disclosed herein are intended to be illustrative, mutatis mutandis. This applies to each of the other aspects of the invention. [Brief explanation of the drawings]

[0082] [Figure 1] A genomic map of the HBV genome is shown, identifying all open reading frames (ORFs). The virus particle has a partially double-stranded genome (shown by dashed lines) with cohesive overlaps flanked by direct repeats (DR1 and DR2) spanning the 5' region of each strand. Gene S encodes the major hepatitis B surface antigen (HBsAg) protein, a transmembrane protein within the viral envelope, and its glycosylation partners. An in-frame sequence upstream of the S gene encodes the pre-S domain, which, when translated with the S sequence, generates pre-S and S polypeptides (medium and large proteins) that contain the viral receptor for hepatocyte infection. Gene C encodes the hepatitis B core antigen (HBcAg), which forms the viral nucleocapsid. The P region encodes the viral reverse transcriptase, which also possesses DNA-dependent DNA polymerase and RNase H activities required for viral replication. Although HBV is a DNA virus, it replicates via a pregenomic RNA intermediate. Finally, the X gene encodes the viral small regulatory protein, the hepatitis B x (HBx) antigen. HBx is a transactivator protein that stimulates viral gene expression and replication, protects virus-infected cells from immune-mediated destruction, and contributes to the development of hepatocellular carcinoma. [Figure 2]Engineered meganuclease recognition sequences of the HBV genome. A) Each recognition sequence targeted by the engineered meganuclease of the present invention contains two recognition half sites. Each recognition half site contains 9 base pairs separated by a 4 base pair central sequence. The HBV1-2 recognition sequence (SEQ ID NO: 10) contains two recognition half sites designated HBV1 and HBV2. The HBV5-6 recognition sequence (SEQ ID NO: 12) contains two recognition half sites designated HBV5 and HBV6. The HBV7-8 recognition sequence (SEQ ID NO: 14) contains two recognition half sites designated HBV7 and HBV8. The HBV11-12 recognition sequence (SEQ ID NO: 16) contains two recognition half sites designated HBV11 and HBV12. [Figure 3] Diagram of the HBV genotype A genome and the location of the recognition sequences of HBV1-2, HBV5-6, HBV7-8, and HBV11-12 within the genome. The HBV1-2 and HBV5-6 recognition sequences are both located within four ORFs of the HBV genome: P, preS1 / preS2 / S, preS2 / S, and S. The HBV7-8 and HBV11-12 recognition sequences are each located within the ORFs encoding the polymerase. [Figure 4]Alignment of HBV recognition sequences in HBV genotypes A to G. The recognition sequences targeted by the present invention are conserved across multiple HBV genotypes. The HBV1-2 recognition sequence spans residues 185-206 of HBV genotype A as shown in SEQ ID NO: 3, and this recognition sequence is completely conserved in genotypes B, C, E, F, and G. Genotype D contains a single nucleotide difference, G to T, at position -4 of the first half site. The HBV5-6 recognition sequence spans residues 742-763 of HBV genotype A as shown in SEQ ID NO: 3, and this recognition sequence is completely conserved in genotypes B, C, D, E, and G. Genotype F contains a single nucleotide difference, G to C, at position -3 of the first half site. The HBV7-8 recognition sequence spans residues 1183-1204 of HBV genotype A as shown in SEQ ID NO: 3, and this recognition sequence is completely conserved in genotypes B, C, D, F, and G. Genotype E contains a single nucleotide difference of G to C at position -1 of the first half site. The HBV11-12 recognition sequence spans residues 1259 to 1280 of HBV genotype A as shown in SEQ ID NO: 3, and this recognition sequence is completely conserved in genotypes B, C, D, E, F, and G. [Figure 5] The engineered meganucleases of the present invention comprise two subunits, with a first subunit comprising the HVR1 region binding to a first recognition half-site (e.g., HBV1, HBV5, HBV7, or HBV11) and a second subunit comprising the HVR2 region binding to a second recognition half-site (e.g., HBV2, HBV6, HBV8, or HBV12). In embodiments where the engineered meganuclease is a single-chain meganuclease, the first subunit comprising the HVR1 region can be positioned as either the N-terminal or C-terminal subunit. Similarly, the second subunit comprising the HVR2 region can be positioned as either the N-terminal or C-terminal subunit. [Figure 6]Schematic diagram of a reporter assay in CHO cells for evaluating engineered meganucleases targeting ORFs in the genome of at least two genotypes of Hepatitis B virus. For the engineered meganucleases described herein, CHO cell lines were generated in which a reporter cassette was stably integrated into the cellular genome. The reporter cassette contained, in 5' to 3' order, an SV40 early promoter; the 5' two-thirds of the GFP gene; a recognition sequence for the engineered meganuclease of the present invention (e.g., the HBV1-2 recognition sequence); a recognition sequence for the CHO-23 / 24 meganuclease (WO 2012 / 167192); and the 3' two-thirds of the GFP gene. Cells stably transfected with this cassette did not express GFP in the absence of a DNA cleavage-inducing agent. Meganucleases were introduced by transduction of plasmid DNA or mRNA encoding the respective meganuclease. When DNA cleavage was induced at either of the meganuclease recognition sequences, the overlapping regions of the GFP gene recombined with each other to generate a functional GFP gene.The percentage of GFP-expressing cells could then be determined by flow cytometry as an indirect measure of the frequency of genome cleavage by the meganuclease. [Figure 7]Efficiency of engineered meganucleases to recognize and cleave recognition sequences in the ORFs of the genomes of at least two genotypes of hepatitis B virus in a CHO cell reporter assay. The engineered meganucleases set forth in SEQ ID NOS: 18-39 were engineered to target the HBV1-2 recognition sequence (SEQ ID NOS: 10), HBV5-6 recognition sequence (SEQ ID NOS: 12), HBV7-8 recognition sequence (SEQ ID NOS: 14), or HBV11-12 recognition sequence (SEQ ID NOS: 16) and were screened for efficacy in a CHO cell reporter assay. The results shown provide the percentage of GFP-expressing cells observed in each assay, indicating the efficacy of each meganuclease for cleaving the target recognition sequence or the CHO-23 / 24 recognition sequence. A negative control (bs) was also included in each assay. Figure 7A shows a meganuclease targeting the HBV1-2 recognition sequence. Figure 7B shows a meganuclease targeting the HBV5-6 recognition sequence. Figure 7C shows a meganuclease targeting the HBV7-8 recognition sequence. Figure 7D shows a meganuclease targeting the HBV11-12 recognition sequence. [Figure 8]Figure 8 shows the efficiency of engineered meganucleases to recognize and cleave recognition sequences in the ORFs of the genomes of at least two genotypes of Hepatitis B virus in a CHO cell reporter assay. The engineered meganucleases set forth in SEQ ID NOS: 18-39 were engineered to target the HBV1-2 recognition sequence (SEQ ID NOS: 10), HBV5-6 recognition sequence (SEQ ID NOS: 12), HBV7-8 recognition sequence (SEQ ID NOS: 14), or HBV11-12 recognition sequence (SEQ ID NOS: 16) and were screened for efficacy in a CHO cell reporter assay at multiple time points over a 12-day period following nucleofection. The results shown provide the percentage of GFP-expressing cells observed in each assay over the 12-day analysis period, indicating the efficacy of each meganuclease for cleaving the target recognition sequence or the CHO-23 / 24 recognition sequence as a function of time. Figure 8A shows a meganuclease targeting the HBV1-2 recognition sequence. Figure 8B shows a meganuclease targeting the HBV5-6 recognition sequence. Figure 8C shows a meganuclease targeting the HBV7-8 recognition sequence. Figure 8D shows a meganuclease targeting the HBV11-12 recognition sequence. [Figure 9] Evaluation of the ability of HBV meganuclease to recognize and cleave the recognition sequence within an episomal DNA plasmid in an E. coli reporter system. Figure 9A shows the pARCUS and pHBVa plasmids. Figure 9B shows the number of colonies present on each selection plate, providing evidence that HBV meganuclease can cleave the pHBVa plasmid. [Figure 10] Evaluation of HBV meganuclease in AD38 cells. AD38 cells, which express the HBV genome and secrete HBsAg, were transfected with plasmid DNA encoding HBV5-6×.33 or HBV11-12×.26 meganuclease. Plasmid DNA encoding red fluorescent protein was used as a control in this experiment. Cell supernatants were collected 3 and 7 days after transfection and assayed for the presence of HBsAg by ELISA. [Figure 11]Evaluation of HBV meganuclease in AD38 cells. AD38 cells, which express the HBV genome and secrete HBsAg, were transduced with lentivirus encoding HBV5-6×.33 meganuclease or HBV11-12×.26 meganuclease, or a combination of the two. A lentivirus encoding red fluorescent protein was used as a control in this experiment. MOIs of 1, 2, and 4 were examined. Seven days after transduction, cell supernatants were collected and assayed for the presence of HBsAg by ELISA. [Figure 12] Evaluation of HBV meganuclease in AD38 cells. AD38 cells expressing the HBV genome and secreting HBsAg were transduced with lentivirus encoding HBV5-6×0.33 meganuclease (LV224), HBV11-12×0.26 meganuclease (LV225), or red fluorescent protein (LV212). An MOI of 4 was tested, and cell supernatants were collected 7 days post-transduction and assayed for the presence of HBsAg and the copy number of extracellular HBV DNA. Additionally, cell lysates were obtained 7 days post-transduction and analyzed for the intracellular copy number of HBV cccDNA. Figure 12A shows the HBsAg concentration in the cell culture medium. Figure 12B shows the copy number of extracellular HBV DNA in the cell culture medium. Figure 12C shows the copy number of intracellular HBV cccDNA in the cell lysates. [Figure 13]Evaluation of HBV meganuclease activity in HBV-infected primary human hepatocytes. Lentiviruses expressing RFP, HBV5-6×.33, or HBV11-12×.26 were generated and used to transduce HBV-infected primary human hepatocytes to determine the effect of meganucleases on HBsAg and HBeAg production. Primary human hepatocytes were seeded and infected with HBV 24 hours later. One day postinfection, the cells were washed and transduced 24 hours later (day 2 postinfection) with either lentivirus encoding RFP, HBV5-6×.33, or HBV11-12×.26, or a 1:1 mixture of lentivirus encoding HBV meganuclease. As an additional control, infected cells were treated with DMSO. Cell supernatants were collected, and the medium was replaced on days 4, 8, 11, and 13 posttransduction. HBsAg and HBeAg were measured in the cell supernatants by ELISA at each time point. In addition, extracellular DNA in the supernatant was measured 13 days after infection. To determine whether lentiviral transduction generally affected the secretion of either HBsAg or HBeAg, cell supernatants from cells transduced with RFP-encoding lentivirus were compared with cells treated with DMSO. Figure 13A shows that transduction with RFP-encoding lentivirus had little effect on HBsAg expression at MOIs of 1.25, 2.5, or 5. Figure 13B shows that transduction with RFP-encoding lentivirus had little effect on HBeAg expression at MOIs of 1.25, 2.5, or 5. Figure 13C shows that transduction with RFP-encoding lentivirus had little effect on HBV DNA expression at MOIs of 1.25, 2.5, or 5. [Figure 14]Evaluation of HBV meganuclease activity in HBV-infected primary human hepatocytes. Lentiviruses expressing RFP, HBV5-6×.33, or HBV11-12×.26 were generated and used to transduce HBV-infected primary human hepatocytes to determine the effect of meganucleases on HBsAg and HBeAg production. Primary human hepatocytes were seeded and infected with HBV 24 hours later. One day postinfection, the cells were washed and transduced 24 hours later (day 2 postinfection) with either lentivirus encoding RFP, HBV5-6×.33, or HBV11-12×.26, or a 1:1 mixture of lentivirus encoding HBV meganuclease. As an additional control, infected cells were treated with DMSO. Cell supernatants were collected, and the medium was replaced on days 4, 8, 11, and 13 posttransduction. HBsAg and HBeAg were measured in the cell supernatants by ELISA at each time point. Figure 14A shows HBsAg in cell culture medium after transduction at an MOI of 2.5. Figure 14B shows HBsAg in cell culture medium after transduction at an MOI of 1.25. Figure 14C shows HBeAg in cell culture medium after transduction at an MOI of 2.5. Figure 14D shows HBeAg in cell culture medium after transduction at an MOI of 1.25. [Figure 15] Lipid nanoparticle-encapsulated mRNA delivery to the liver. We expressed a genetically engineered meganuclease with specificity for the recognition sequence of the mouse CMP-NeuAc hydrolase (Cmah) gene, which is expressed in mouse liver. ARCA-capped mRNA encoding the Cmah meganuclease was encapsulated in three different commercially available LNP formulations. LNP-encapsulated mRNA was administered to CD-1 mice via IV injection, and livers were harvested 6 days later. Total liver genomic DNA (gDNA) was isolated, and the frequency of insertion / deletion (indel) mutations in the Cmah gene was determined using a T7 endonuclease I (T7E) assay and deep sequencing.

[0083] A brief description of arrays SEQ ID NO: 1 is the amino acid sequence of the wild-type I-CreI meganuclease from the green alga Chlamydomonas reinhardtii. The amino acid sequence is shown.

[0084] SEQ ID NO: 2 shows the amino acid sequence of LAGLIDADG.

[0085] SEQ ID NO: 3 shows the amino acid sequence of HBV genotype A.

[0086] SEQ ID NO: 4 shows the amino acid sequence of HBV genotype B.

[0087] SEQ ID NO: 5 shows the amino acid sequence of HBV genotype C.

[0088] SEQ ID NO: 6 shows the amino acid sequence of HBV genotype D.

[0089] SEQ ID NO: 7 shows the amino acid sequence of HBV genotype E.

[0090] SEQ ID NO: 8 shows the amino acid sequence of HBV genotype F.

[0091] SEQ ID NO: 9 shows the amino acid sequence of HBV genotype G.

[0092] SEQ ID NO: 10 shows the amino acid sequence of the HBV1-2 recognition sequence (sense).

[0093] SEQ ID NO: 11 shows the amino acid sequence of the HBV1-2 recognition sequence (antisense).

[0094] SEQ ID NO: 12 shows the amino acid sequence of the HBV5-6 recognition sequence (sense).

[0095] SEQ ID NO: 13 shows the amino acid sequence of the HBV5-6 recognition sequence (antisense).

[0096] SEQ ID NO: 14 shows the amino acid sequence of the HBV7-8 recognition sequence (sense).

[0097] SEQ ID NO: 15 shows the amino acid sequence of the HBV7-8 recognition sequence (antisense).

[0098] SEQ ID NO: 16 shows the amino acid sequence of the HBV11-12 recognition sequence (sense).

[0099] SEQ ID NO: 17 shows the amino acid sequence of the HBV11-12 recognition sequence (antisense).

[0100] SEQ ID NO: 18 shows the amino acid sequence of HBV1-2x.2 meganuclease.

[0101] SEQ ID NO: 19 shows the amino acid sequence of HBV1-2x.14 meganuclease.

[0102] SEQ ID NO: 20 shows the amino acid sequence of HBV1-2x.68 meganuclease.

[0103] SEQ ID NO: 21 shows the amino acid sequence of HBV1-2x.93 meganuclease.

[0104] SEQ ID NO: 22 shows the amino acid sequence of HBV5-6x.33 meganuclease.

[0105] SEQ ID NO: 23 shows the amino acid sequence of HBV5-6x.84 meganuclease.

[0106] SEQ ID NO: 24 shows the amino acid sequence of HBV5-6x.90 meganuclease.

[0107] SEQ ID NO: 25 shows the amino acid sequence of HBV5-6x.4 meganuclease.

[0108] SEQ ID NO: 26 shows the amino acid sequence of HBV5-6x.5 meganuclease.

[0109] SEQ ID NO: 27 shows the amino acid sequence of HBV5-6x.68 meganuclease.

[0110] SEQ ID NO: 28 shows the amino acid sequence of HBV5-6x.79 meganuclease.

[0111] SEQ ID NO: 29 shows the amino acid sequence of HBV7-8x.2 meganuclease.

[0112] SEQ ID NO: 30 shows the amino acid sequence of HBV7-8x.9 meganuclease.

[0113] SEQ ID NO: 31 shows the amino acid sequence of HBV7-8x.17 meganuclease.

[0114] SEQ ID NO: 32 shows the amino acid sequence of HBV7-8x.44 meganuclease.

[0115] SEQ ID NO: 33 shows the amino acid sequence of HBV11-12x.26 meganuclease.

[0116] SEQ ID NO: 34 shows the amino acid sequence of HBV11-12x.9 meganuclease.

[0117] SEQ ID NO: 35 shows the amino acid sequence of HBV11-12x.13 meganuclease.

[0118] SEQ ID NO: 36 shows the amino acid sequence of HBV11-12x.16 meganuclease.

[0119] SEQ ID NO: 37 shows the amino acid sequence of HBV11-12x.27 meganuclease.

[0120] SEQ ID NO: 38 shows the amino acid sequence of HBV11-12x.41 meganuclease.

[0121] SEQ ID NO: 39 shows the amino acid sequence of HBV11-12x.48 meganuclease.

[0122] SEQ ID NO: 40 is the amino acid sequence of the HBV1-2x.2 meganuclease HBV1 binding subunit The amino acid sequence is shown.

[0123] SEQ ID NO: 41 is the sequence of the HBV1-2x.14 meganuclease HBV1 binding subunit The amino acid sequence is shown.

[0124] SEQ ID NO: 42 is the sequence of the HBV1-2x.68 meganuclease HBV1 binding subunit The amino acid sequence is shown.

[0125] SEQ ID NO: 43 is the sequence of the HBV1-2x.93 meganuclease HBV1 binding subunit The amino acid sequence is shown.

[0126] SEQ ID NO: 44 is the amino acid sequence of the HBV1-2x.2 meganuclease HBV2 binding subunit. The amino acid sequence is shown.

[0127] SEQ ID NO: 45 is the HBV1-2x.14 meganuclease HBV2-binding subunit The amino acid sequence of

[0128] SEQ ID NO: 46 is the sequence of the HBV1-2x.68 meganuclease HBV2 binding subunit The amino acid sequence is shown.

[0129] SEQ ID NO: 47 is the sequence of the HBV1-2x.93 meganuclease HBV2 binding subunit The amino acid sequence is shown.

[0130] SEQ ID NO: 48 is the sequence of the HBV5-6x.33 meganuclease HBV5 binding subunit The amino acid sequence is shown.

[0131] SEQ ID NO: 49 is the sequence of the HBV5-6x.84 meganuclease HBV5 binding subunit The amino acid sequence is shown.

[0132] SEQ ID NO: 50 is the sequence of the HBV5-6x0.90 meganuclease HBV5 binding subunit The amino acid sequence is shown.

[0133] SEQ ID NO: 51 is the amino acid sequence of the HBV5-6x.4 meganuclease HBV5 binding subunit The amino acid sequence is shown.

[0134] SEQ ID NO: 52 is the amino acid sequence of the HBV5-6x.5 meganuclease HBV5 binding subunit The amino acid sequence is shown.

[0135] SEQ ID NO: 53 is the sequence of the HBV5-6x.68 meganuclease HBV5 binding subunit The amino acid sequence is shown.

[0136] SEQ ID NO: 54 is the sequence of the HBV5-6x.79 meganuclease HBV5 binding subunit The amino acid sequence is shown.

[0137] SEQ ID NO: 55 is the sequence of the HBV5-6x.33 meganuclease HBV6 binding subunit The amino acid sequence is shown.

[0138] SEQ ID NO: 56 is the sequence of the HBV5-6x.84 meganuclease HBV6 binding subunit The amino acid sequence is shown.

[0139] SEQ ID NO: 57 is the sequence of the HBV5-6x.90 meganuclease HBV6 binding subunit The amino acid sequence is shown.

[0140] SEQ ID NO: 58 is the amino acid sequence of the HBV5-6x.4 meganuclease HBV6 binding subunit The amino acid sequence is shown.

[0141] SEQ ID NO: 59 is the amino acid sequence of the HBV5-6x.5 meganuclease HBV6 binding subunit The amino acid sequence is shown.

[0142] SEQ ID NO: 60 is the sequence of the HBV5-6x.68 meganuclease HBV6 binding subunit The amino acid sequence is shown.

[0143] SEQ ID NO: 61 is the sequence of the HBV5-6x.79 meganuclease HBV6 binding subunit The amino acid sequence is shown.

[0144] SEQ ID NO: 62 is the amino acid sequence of the HBV7-8x.2 meganuclease HBV7 binding subunit The amino acid sequence is shown.

[0145] SEQ ID NO: 63 is the amino acid sequence of the HBV7-8x.9 meganuclease HBV7 binding subunit The amino acid sequence is shown.

[0146] SEQ ID NO: 64 is the sequence of the HBV7-8x.17 meganuclease HBV7 binding subunit The amino acid sequence is shown.

[0147] SEQ ID NO: 65 is the sequence of the HBV7-8x.44 meganuclease HBV7 binding subunit The amino acid sequence is shown.

[0148] SEQ ID NO: 66 is the amino acid sequence of the HBV7-8x.2 meganuclease HBV8 binding subunit The amino acid sequence is shown.

[0149] SEQ ID NO: 67 is the amino acid sequence of the HBV7-8x.9 meganuclease HBV8 binding subunit The amino acid sequence is shown.

[0150] SEQ ID NO: 68 is the HBV7-8x.17 meganuclease HBV8-binding subunit The amino acid sequence of

[0151] SEQ ID NO: 69 is the sequence of the HBV7-8x.44 meganuclease HBV8 binding subunit The amino acid sequence is shown.

[0152] SEQ ID NO: 70 is the HBV11-12x.26 meganuclease HBV11 binding subunit The amino acid sequence of TT is shown.

[0153] SEQ ID NO: 71 is the HBV11-12x.9 meganuclease HBV11 binding subunit The amino acid sequence of the

[0154] SEQ ID NO: 72 is the HBV11-12x.13 meganuclease HBV11 binding subunit The amino acid sequence of TT is shown.

[0155] SEQ ID NO: 73 is the HBV11-12x.16 meganuclease HBV11 binding subunit The amino acid sequence of TT is shown.

[0156] SEQ ID NO: 74 is the HBV11-12x.27 meganuclease HBV11 binding subunit The amino acid sequence of TT is shown.

[0157] SEQ ID NO: 75 is the HBV11-12×.41 meganuclease HBV11 binding subunit The amino acid sequence of TT is shown.

[0158] SEQ ID NO: 76 is the HBV11-12×.48 meganuclease HBV11 binding subunit The amino acid sequence of TT is shown.

[0159] SEQ ID NO: 77 is the HBV11-12x.26 meganuclease HBV12 binding subunit The amino acid sequence of TT is shown.

[0160] SEQ ID NO: 78 is the HBV11-12x.9 meganuclease HBV12 binding subunit The amino acid sequence of the

[0161] SEQ ID NO: 79 is the HBV11-12x.13 meganuclease HBV12 binding subunit The amino acid sequence of TT is shown.

[0162] SEQ ID NO: 80 is the HBV11-12x.16 meganuclease HBV12 binding subunit The amino acid sequence of TT is shown.

[0163] SEQ ID NO: 81 is the HBV11-12x.27 meganuclease HBV12 binding subunit The amino acid sequence of TT is shown.

[0164] SEQ ID NO: 82 is the HBV11-12×.41 meganuclease HBV12 binding subunit The amino acid sequence of TT is shown.

[0165] SEQ ID NO: 83 is the HBV11-12×.48 meganuclease HBV12 binding subunit The amino acid sequence of TT is shown.

[0166] SEQ ID NO: 84 is the HBV gene with a G to C substitution at position -4 of the first half site. The nucleic acid sequence of the recognition sequence present in HBV genotype D, which corresponds to the HBV type A recognition sequence for HBV1-2. Indicates a column.

[0167] SEQ ID NO: 85 is the HBV gene with a G to C substitution at position -3 of the first half site. The nucleic acid sequence of the recognition sequence present in HBV genotype F corresponding to the HBV 5-6 recognition sequence of type A Indicates a column.

[0168] SEQ ID NO: 86 is the HBV gene with a C to T substitution at position -1 of the first half site. The nucleic acid sequence of the recognition sequence present in HBV genotype E corresponding to the HBV 7-8 recognition sequence of type A Indicates a column. DETAILED DESCRIPTION OF THE INVENTION

[0169] 1.1 Criteria and Definitions

[0170] The patent and scientific literature referred to herein establishes knowledge that is available to those skilled in the art. Issued U.S. patents, allowed applications, published foreign applications, and G References containing sequences from the enBank database are treated as if each were a specific and individual reference. The inventions incorporated herein by reference to the extent indicated are incorporated by reference. can be.

[0171] This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, it is intended that this disclosure be thorough and complete and that the present invention be fully understood and construed as constituting an entire disclosure. These embodiments are provided so as to fully convey the scope to those skilled in the art. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and specific implementations may be Features illustrated with respect to an embodiment may be deleted from that embodiment. Numerous variations and additions to the embodiments suggested herein will occur to those skilled in the art in view of this disclosure. Obviously, they do not depart from the invention.

[0172] Unless otherwise defined, all technical and scientific terms used herein are defined by the It has the same meaning as commonly understood by a person skilled in the art to which the invention pertains. The terminology used in the description is for the purpose of describing particular embodiments only. and are not intended to limit the invention.

[0173] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. No. 6,299,499, which is incorporated herein by reference.

[0174] As used herein, "a," "an," or "the" means one or more. For example, "a cell" can mean a single cell or many cells. do.

[0175] As used herein, unless specifically indicated otherwise, the word "or" means "or" "and / or" is used in the inclusive sense, not the exclusive sense of "either / or." Not used.

[0176] As used herein, the terms "nuclease" and "endonuclease" refer to naturally occurring or genetically engineered enzymes that cleave phosphodiester bonds within polynucleotide chains The terms "anticoagulant" and "anticoagulant" are used interchangeably to refer to enzymes engineered to inhibit the growth of inflammatory cytokines.

[0177] As used herein, the term "meganuclease" refers to a nucleic acid having a length greater than 12 base pairs. It refers to an endonuclease that binds to double-stranded DNA at a recognition sequence. The recognition sequence of the meganuclease is 22 base pairs. I-derived endonucleases, e.g., DNA binding specificity, DNA cleavage with respect to the native I-CreI in terms of activity, DNA binding affinity, or dimerization properties. It can refer to genetically engineered variants of I-CreI that have been modified. Methods for producing modified I-CreI variants are known in the art. (See, for example, International Publication No. WO 2007 / 047859). When activated, meganucleases bind to double-stranded DNA as heterodimers. The enzyme also utilizes a peptide linker to bind a pair of DNA-binding domains to a single polypeptide. It may also be a "single-chain meganuclease" bound to a homing end. The term "nuclease" is synonymous with the term "meganuclease." The nuclease was demonstrated to be effective when expressed intracellularly without any observed adverse effects on cell viability. Qualitatively non-toxic or non-toxic to meganuclease cleavage as measured using the methods described herein. There is a significant decrease in cleavage activity.

[0178] As used herein, the term "single-chain meganuclease" refers to a single-chain meganuclease that is connected by a linker. A single-chain meganuclease refers to a polypeptide comprising a pair of nuclease subunits linked by a single chain. The enzyme has the structure of N-terminal subunit-linker-C-terminal subunit The two meganuclease subunits are generally not identical in amino acid sequence and are non- Therefore, single-chain meganucleases are typically pseudo-pairs. Cleave palindromic or non-palindromic recognition sequences. Single-stranded meganuclease Although it is not actually a dimer, it is referred to as a "single-chain heterodimer" or "single-chain heterodimer meganuclear gene." For clarity, unless otherwise stated, The term "meganuclease" can refer to a dimeric or single-chain meganuclease.

[0179] As used herein, the term "linker" refers to a linker that connects two meganuclease subunits. The term "phosphoryl" refers to an exogenous peptide sequence used to link multiple units into a single polypeptide. Cars have sequences found in natural proteins and are not found in any natural proteins. The linker may be an artificial sequence that does not have any secondary structure. or may have a tendency to form a particular three-dimensional structure under physiological conditions. Examples include those included in U.S. Patent No. 8,445,251 and U.S. Patent No. 9,434,931. Examples of suitable ion exchange agents include, but are not limited to, those listed below. wherein the linker is an amino acid sequence comprising residues 154 to 195 of any one of SEQ ID NOs: 18 to 39. The amino acid sequence may be:

[0180] As used herein, "recombinant" or "genetically engineered" refers to a protein. The term "derived from" refers to a nucleic acid that encodes a protein and to a cell or organism that expresses the protein. It means that the amino acid sequence has been altered as a result of applying genetic engineering techniques. The terms "recombinant" or "genetically engineered" in reference to nucleic acids refer to the application of genetic engineering techniques. As a result of the genetic engineering technique, it means that the nucleic acid sequence has been modified. , PCR and DNA cloning techniques; transfection, transformation, and other gene transfer techniques; These include, but are not limited to, homologous recombination; site-directed mutagenesis; and gene fusion. According to this definition, a protein has the same amino acid sequence as a naturally occurring protein, but Proteins produced by cloning and expression in a heterologous host are considered recombinant. Not considered.

[0181] As used herein, the term "wild type" refers to a population of alleles of the same type of gene. The most common naturally occurring alleles (i.e., polynucleotide sequences) in In this case, the polypeptide encoded by the wild-type allele does not perform its intended function. The term "wild-type" also refers to a polypeptide encoded by a wild-type allele. The wild-type alleles (i.e., polynucleotides) and polypeptides are also shown. A mutant or variant allele containing one or more mutations and / or substitutions relative to A wild-type allele or polypeptide is a polypeptide that is normally present in an organism. Although mutant or variant alleles or polypeptides can confer a specific phenotype, In some cases, the wild-type nuclease can confer an altered phenotype. "Wild-type" is distinguishable from a recombinant or non-naturally occurring nuclease. The term also refers to cells, organisms, and / or subjects that have a wild-type allele of a particular gene, Alternatively, cells, organisms, and / or subjects used for comparative purposes can be indicated. .

[0182] As used herein, the term "genetic modification" refers to the modification of a cell or organism. or their ancestors, whose genomic DNA sequences have been intentionally altered by recombinant technology As used herein, the term "genetically modified" refers to a cell or organism. This term encompasses the term "scientifically competent."

[0183] As used herein with respect to recombinant proteins, the term "modified" refers to a modification of a reference sequence Any insertion, deletion or substitution of amino acid residues in the recombinant sequence relative to (e.g., the wild-type or naturally occurring sequence) It means the loss or replacement of

[0184] As used herein, the term "recognition sequence" refers to a sequence that is bound by an endonuclease. In the case of meganucleases, the recognition sequence is a sequence of four base pairs. It contains a pair of inverted 9 base pair "half sites" separated by a single stranded meganucleotide. In the case of a proteinase, the N-terminal domain of the protein contacts the first half-site, and the protein The C-terminal domain of the nuclease contacts the second half site. This results in a 3' "overhang" of the base pair. The "overhang" or "sticky end" is , a short single-stranded DNA that can be produced by endonucleolytic cleavage of a double-stranded DNA sequence The segments of I-CreI-derived meganucleases and single-chain meganucleases In this case, the overhang contains 10 to 13 bases of the 22 base pair recognition sequence.

[0185] As used herein, the term "target site" or "target sequence" refers to a site that is targeted by a nuclease. A region of a cell's chromosomal DNA that contains a recognition sequence for a specific gene.

[0186] As used herein, the term "DNA binding affinity" or "binding affinity" refers to The tendency of the ganucleases to associate non-covalently with the reference DNA molecule (e.g., recognition sequence or The term "binding affinity" refers to any sequence. Binding affinity is measured by the dissociation constant, Kd. As used herein, When d but for the reference nuclease A nuclease is considered "unchanged" if it increases or decreases by a statistically significant amount (p<0.05). It has a binding affinity of

[0187] As used herein, the term "specificity" refers to the ability of a specific base pair, called a recognition sequence, to bind to a specific target. The meganuclease recognizes only double-stranded structures at the sequence of This means that the DNA molecule can be recognized and cleaved. The set of recognition sequences is a set of specific conserved positions. They share a common position or sequence motif, but may be degenerate at one or more positions. A different meganuclease can cleave only one or only a few recognition sequences. Specificity can be determined by any method known in the art. When in use, the meganuclease exhibits a similar activity to a reference meganuclease (e.g., If it binds to and cleaves a recognition sequence that is not bound to or cleaved by a wild-type antibody, or if it The rate of cleavage of the array is increased by a biologically significant amount (e.g., a small amount) compared to the reference meganuclease. If the amount of ATP is increased or decreased (at least 2-fold, or 2-fold to 10-fold), the meganuclease is considered to be "modified." " has specificity.

[0188] As used herein, the term "homologous recombination" or "HR" refers to the process of repairing a gene by A natural cellular process by which double-stranded DNA breaks are repaired using homologous DNA sequences as targets. (e.g., Cahill et al. (2006), Front. Biosc i.11:1958-1976). Homologous DNA sequences may be endogenous chromosomal sequences or cellular The nucleic acid may be an exogenous nucleic acid delivered to the host.

[0189] As used herein, the term "non-homologous end joining" or "NHEJ" refers to the process of joining a double-stranded A natural process in which DNA breaks are repaired by the direct joining of two nonhomologous DNA segments Refers to cellular processes (e.g., Cahill et al. (2006), Front (See Biosci. 11:1958-1976.) DNA repair by non-homologous end joining The repair is error-prone and involves the untemplated DNA sequence at the repair site. ) additions or deletions frequently result. In some instances, cleavage at the target recognition sequence NHEJ occurs at the target recognition site. The enzyme-induced breaks and subsequent DNA repair by NHEJ result in the formation of a fragment that disrupts gene function. Mutations, such as sequence-shift mutations, can be introduced into the coding sequence. Engineered meganucleases can be used to effectively knock out genes in cell populations. Can be used for.

[0190] As used herein with respect to both amino acid sequences and nucleic acid sequences, "percent Terms such as "sequence identity," "sequence identity," "percent similarity," and "sequence similarity" are used herein to refer to , aligning sequences to maximize similarity between aligned amino acid residues or nucleotides; or the number of similar residues or nucleotides, the total number of residues or nucleotides, and The alignment of two sequences based on the presence and length of gaps in the sequence alignment. This shows a measure of the degree of similarity between the two sequences. Standard parameters are used to determine sequence similarity. Various algorithms and computer programs are available for this purpose. When using the BLASTp program for amino acid sequences, the sequence similarity is calculated using the BLASTp program. and nucleic acid sequences, both of which were determined using the BLASTn program. , through the National Center for Biotechnology Information ( www.ncbi.nlm.nih.gov / ). See, for example, Altschul et al. (1990), J. Mol. Biol. l.215:403-410;Gish and States (1993), Nat ure Genet.3:266-272;Madden et al.(1996), Meth.Enzymol.266:131-141;Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402); Zhang et al.(2000),J.Comput.Biol.7(1-2): As used herein, a pair of two amino acid sequences is described in The similarity of the markers is calculated for the BLASTp algorithm, with word size = 3 and gap Opening penalty = -11, Gap extension penalty = -1, score The score is based on the parameters of the BLOSUM62 matrix. As used herein, the percent similarity of two nucleic acid sequences is determined by BLASTn algorithm. For the algorithm, word size = 11, gap opening penalty = -5, Chance extension penalty = -2, matched reward = 1, and mismatch penalty = The score is based on the parameter Luti = -3.

[0191] As used herein in reference to alterations in the sequence of two proteins or amino acids, " The term "corresponding" refers to the fact that a particular modification in a first protein corresponds to a modification in a second protein. The modification is a substitution of the same amino acid residue as the modification in the first protein, and The acid positions are determined when the two proteins are subjected to a standard sequence alignment (e.g., BLAS Tp program) corresponds to the amino acid position of the modification in the second protein. Therefore, residues X and Y are used to indicate that the sequence is aligned. Despite the fact that X and Y can be different numbers, First, the modification of residue "X" in the first protein to amino acid "A" results in the formation of a second protein. This corresponds to the modification of residue "Y" to amino acid "A" in the target protein.

[0192] As used herein, "recognized half site," "recognition sequence half site," or The term "half site" simply refers to a monomer of a homodimeric or heterodimeric meganuclease. recognized by the body or by one subunit of a single-chain meganuclease It refers to the sequence of nucleic acids in a double-stranded DNA molecule.

[0193] As used herein, the term "hypervariable region" refers to an amino acid sequence having a relatively high degree of variability. refers to a localized sequence within a monomer or subunit of a meganuclease, containing amino acids The hypervariable region may comprise about 50 to 60 contiguous residues, about 53 to 57 contiguous residues, or preferably about In some embodiments, the hypervariable region residues may comprise residues 18-56. The hypervariable region may correspond to positions 24 to 79 or 215 to 270 of any one of the following: It may contain one or more residues that contact a DNA base in the recognition sequence, and may be a monomer or subunit. The hypervariable region can also be modified to change the base selectivity of the unit. When a nuclease associates with a double-stranded DNA recognition sequence, it binds to one or more DNA backbones. Such residues may include the DNA backbone and the meganuclear target recognition sequence. In a different embodiment of the invention, the enzyme can be modified to alter its binding affinity. The hypervariable region can contain 1 to 20 residues, which exhibits variability and base preference. and / or can be modified to affect DNA binding affinity. In some embodiments, the variable residues within the hypervariable region are selected from the group consisting of 24 of any one of SEQ ID NOS: 18-39. 1st place, 26th place, 28th place, 30th place, 32nd place, 33rd place, 38th place, 40th place, 42nd place, 44th place, 46th place 1st, 49th, 50th, 54th, 64th, 68th, 70th, 75th, and 77th place In another embodiment, the variable residues in the hypervariable region correspond to any of SEQ ID NOS: 18-39. Or one of 215th, 217th, 219th, 221st, 223rd, 224th, 229th, 2 31st, 233rd, 235th, 237th, 240th, 241st, 245th, 255th, 2 It corresponds to one or more of the 59th, 261st, 266th, and 268th positions.

[0194] "recombinant DNA construct", "recombinant construct", "expression cassette", "expression construct", The terms "chimeric construct," "construct," and "recombinant DNA fragment" are used interchangeably herein. Recombinant constructs are fragments of nucleic acids that contain regulatory and coding sequences that are not found together in nature. This includes any artificial combination of nucleic acid fragments, including, but not limited to, sequences such as: Recombinant DNA constructs may contain regulatory and coding sequences derived from different sources, or from the same source. Regulatory and coding sequences that are arranged in a manner different from that found in nature from the source. Such constructs may be used by themselves or in conjunction with vectors. stomach.

[0195] As used herein, a "vector" or "recombinant DNA vector" refers to a vector capable of replication. system, and capable of transcribing and translating polypeptide-encoding sequences in a given host cell. When a vector is used, the choice of vector depends on the As is well known to those skilled in the art, the method used to transform the host cell will determine the vector. The present invention also includes, without limitation, plasmid vectors and recombinant AAV vectors, or meganuclease vectors of the present invention. Any other suitable method known in the art for delivering a gene encoding a lyase to a target cell. Those skilled in the art will appreciate that any of the isolated nucleotide or nucleic acid sequences of the present invention may be used in a variety of applications. In order to successfully transform, select and grow host cells containing either We are well aware of the genetic factors that must be taken into account.

[0196] As used herein, "vector" can also refer to a viral vector. Viral vectors include retroviral vectors, lentiviral vectors, and adenoviral vectors. viral vectors, and adeno-associated viral vectors (AAV), Not limited.

[0197] As used herein, a "polycistronic" mRNA is a mRNA containing two or more codons. A single message containing a sequence (i.e., a cistron) that encodes two or more proteins. Polycistronic mRNA refers to a polycistronic mRNA that contains an IRES element, a T2A element, a P2A element, E2A element, and F2A element, on the same mRNA molecule. Any element known in the art that allows translation of two or more genes from the It may include.

[0198] As used herein, a "control" or "control cell" refers to the genetically modified cells. A control cell is a cell that provides a reference point for measuring genetic or phenotypic changes. For example, (a) wild-type cells, i.e., the same as the starting material for the genetic modification that resulted in the genetically altered cells; (b) of the same genotype as the genetically modified cells but with a null construct (i.e. (c) a cell transformed with a construct that has no known effect on the trait of interest; or Genetically identical to the genetically modified cell, but induces the expression of an altered genotype or phenotype It may include cells that have not been exposed to a condition or stimulus or further genetic modification.

[0199] As used herein in reference to alterations in the sequence of two proteins or amino acids, " The term "corresponding" refers to the fact that a particular modification in a first protein corresponds to a modification in a second protein. The modification is a substitution of the same amino acid residue as the modification in the first protein, and The acid positions are determined when the two proteins are subjected to a standard sequence alignment (e.g., BLAS Tp program) corresponds to the amino acid position of the modification in the second protein. Therefore, residues X and Y are used to indicate that the sequence is aligned. Despite the fact that X and Y can be different numbers, First, the modification of residue "X" in the first protein to amino acid "A" results in the formation of a second protein. This corresponds to the modification of residue "Y" to amino acid "A" in the target protein.

[0200] As used herein, the term "treatment" or "treating a subject" refers to at least By cutting the genome of one HBV particle, the rate of HBV proliferation is slowed or The genetically engineered meganuclease of the present invention or the gene of the present invention is used to terminate the administering a nucleic acid encoding the engineered meganuclease to a subject infected with HBV. Such treatment reduces or prevents HBV transfection and replication in a subject; Partially or completely relieves one or more symptoms of HBV in a subject. A means to assess the reduction in Hepatic seroconversion, i.e., disappearance of HBeAg, can be assessed by determining the level of hepatic seroconversion. Additionally, the alleviation or reduction of HBV symptoms may be assessed by liver biopsy and the results may include measurements of liver function. This can be determined by measuring the level of tissue fibrosis using methods well known in the art. The number of particles can be determined by measuring the level of HBV DNA using PCR, for example. This can be determined by detecting the level of HBsAg in the blood. The terms "treating a subject" or "treating a subject" further include administering to a subject a therapeutically effective amount of ... This may refer to the administration of cells (e.g., hepatocytes) containing nucleic acid, where the cells are delivered to the target tissue (e.g., the liver) in an amount sufficient to treat HBV infection in the subject. and producing a meganuclease engineered to partially or completely alleviate one or more symptoms of HBV. In some embodiments, the engineered meganucleases of the present invention The nucleic acid encoding it or the genetically modified cell of the invention may be in the form of a pharmaceutical composition of the invention, It is administered during treatment.

[0201] The term "hepatitis B virus infection" refers to chronic liver disease / injury, inflammatory, fibrotic conditions, and liver damage. Proliferative disorders, including cancer, related to or resulting from infection with the hepatitis B virus Persistent chronic HBV infection can cause fatigue, liver damage, cirrhosis, and can cause hepatocellular carcinoma, a primary liver cancer.

[0202] As used herein, the terms "proliferate" and "proliferation" refer to cells that are actively dividing and multiplying. Thus, the reduction in proliferation is indicative of the genetic manipulations disclosed herein. The engineered meganuclease or nucleic acid encoding the engineered meganuclease at least 1%, 2%, 3%, 4%, 5%, 1% or more when compared with an appropriate untreated control 0%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 9 Any reduction in HBV proliferation is shown, including a 0%, 95%, or 100% reduction. Therefore, the term "proliferative disorder" refers to a disorder characterized by unwanted or abnormal growth of tissue. As used herein, the term "proliferative disorder" refers to any disease / disorder that is characterized by a proliferative disorder. The term refers to unregulated and / or abnormal growth of cells, which may be cancerous or non-cancerous. This refers to an unwanted condition that may result in the onset of disease or illness.

[0203] The terms "effective amount" or "therapeutically effective amount" refer to an amount that provides beneficial or desirable biological and / or clinical benefits. A therapeutically effective amount refers to an amount sufficient to produce a therapeutically effective result. The nature of the disease, its severity, and the age, weight, health, and responsiveness of the subject being treated. In certain embodiments, an effective amount of the genetically engineered molecules disclosed herein is administered. The ganucleases or pharmaceutical compositions may be used to reduce levels of HBV in subjects infected with HBV. or reduce proliferation or at least one symptom of HBV.

[0204] The term "lipid nanoparticles" refers to typically spherical nanoparticles with an average diameter of 10 to 1000 nanometers. In some formulations, the lipid nanoparticles have at least another cationic lipid, at least one non-cationic lipid, and at least one poly The present invention relates to a method for encapsulating nucleic acids, such as mRNA, in a polymerizable liquid. Any known lipid nanoparticle is contemplated for use in the present invention.

[0205] As used herein, the recitation of a numerical range for a variable includes any value within that range. The intent is to convey that the invention can be practiced with any number of variables. For a general variable, the variable can be equal to any integer value within the numeric range, including the range endpoints. Similarly, for variables that are continuous in nature, the variables may be It can be equal to any real number within the given range. Examples include, but are not limited to, 0 Variables described as having values ​​of ∼2 are 0, 1 if the variable is discrete in nature. or 2 values, 0.0, 0.1, 0.01 if the variable is continuous in nature. , 0.001, or any other real number between 0 and 2 inclusive.

[0206] 2.1 Principles of the invention

[0207] The present invention relates, in part, to a method for the detection of HBV by a genetically engineered meganuclease that targets an ORF (Open Reading Frame) within the HBV genome. To reduce levels of HBV or slow the growth of HBV by cleaving More specifically, meganucleases are based on the hypothesis that they can be used to Genetic engineering to recognize and cleave the recognition sequence present in the genome of the V genotype Therefore, it is possible to utilize a single genetically engineered meganuclease to suppress the level of HBV. It reduces the proliferation or proliferation of hepatitis B virus and reduces the symptoms of HBV infection with multiple genotypes of the virus. It can be reduced.

[0208] Therefore, the present invention provides a method for identifying recognition sequences within the ORFs of at least two genotypes of the HBV genome. The present invention also encompasses engineered meganucleases that recognize and cleave the Such engineered meganucleases can be used in pharmaceutical compositions and to treat HBV infection. The present invention further encompasses the use of genetically engineered megakaryocytes in methods for producing the same. Nuclease proteins or nucleic acids encoding engineered meganucleases. and the use of such compositions for treating HBV infection. Contains.

[0209] 2.2 Meganuclease for recognizing and cleaving recognition sequences in the HBV genome

[0210] It is possible to cut DNA in the viral genome using site-specific nucleases and that such DNA breaks result in permanent genome alterations via NHEJ. This allows HBV virions to divide and infect human cells. It is well known in the art that this is no longer possible. In embodiments, the present invention is practiced using genetically engineered recombinant meganucleases. This can be done.

[0211] In a preferred embodiment, the nuclease used to practice the present invention is a single-stranded megakaryon The single-chain meganuclease is a nuclease consisting of a pair of nucleotides linked by a linker peptide. It contains an N-terminal subunit and a C-terminal subunit. Each of the two domains has a recognition domain. It recognizes half of the sequence (i.e., the recognized half site), and the site of DNA cleavage is divided into two subunits. The DNA strand break is counterbalanced by four base pairs. The meganuclease cleaves DNA at a pair of four-base-pair, 3' single-stranded overhangs. to generate the following.

[0212] In some instances, the engineered meganucleases of the present invention inhibit HBV1-2 It has been genetically engineered to recognize and cleave the recognition sequence (SEQ ID NO: 10). The recognition sequences are P protein, S, preS2 / S, and preS proteins of multiple HBV genotypes. The HBV1-2 recognition sequence is located within the ORF of 1 / preS2. Multiple HBV genotypes, including HBV, C, E, F, and G (e.g., SEQ ID NOS: 3-5 and 6, respectively) These can be found at least in the genomes of the genotype ... The nucleases are collectively referred to herein as "HBV1-2 meganucleases." Exemplary HBV1-2 meganucleases are provided in SEQ ID NOs: 18-21.

[0213] In a further example, the engineered meganuclease of the present invention comprises the HBV5-6 recognition sequence It has been genetically engineered to recognize and cleave (SEQ ID NO: 12) HBV5-6 recognition sequence The P proteins, S, preS2 / S, and preS1 / pr of multiple HBV genotypes The HBV5-6 recognition sequence is located within the ORF of eS2. Multiple HBV genotypes, including HBV, E, and G (e.g., SEQ ID NOS: 3-7 and 9, respectively) Such engineered meganucleases can be found at least in the genome. are collectively referred to herein as "HBV5-6 meganucleases." The HBV5-6 meganuclease is provided in SEQ ID NOs: 22-28.

[0214] In a further example, the engineered meganuclease of the invention comprises the HBV7-8 recognition sequence (SEQ ID NO: 14). is located within the ORF of the P protein of several HBV genotypes. The identification sequence identifies multiple HBV genotypes, including genotypes A, B, C, D, F, and G (e.g., They can be found at least in the genomes of SEQ ID NOS: 3 to 6, 8, and 9, respectively. Such engineered meganucleases are collectively referred to herein as "HBV7-8 An exemplary HBV7-8 meganuclease is represented by SEQ ID NO:2. Available from 9 to 32.

[0215] In a further example, the engineered meganuclease of the present invention is capable of recognizing HBV11-12. It has been genetically engineered to recognize and cleave the sequence (SEQ ID NO: 16). The recognition sequence is located within the P protein ORF of several HBV genotypes. The 1-12 recognition sequence recognizes multiple HBV genotypes, including genotypes A, B, C, D, E, F, and G. These can be found at least in the genomes of the various types (e.g., SEQ ID NOS: 3 to 9, respectively). Such engineered meganucleases are collectively referred to herein as "HBV11 An exemplary HBV11-12 meganuclease is Provided in SEQ ID NOs: 34-40.

[0216] The engineered meganucleases of the present invention comprise a first hypervariable (HVR1) region. a first subunit and a second subunit comprising a second hypervariable (HVR2) region; Furthermore, the first subunit recognizes the first half of the recognition sequence (e.g., HBV1 , HBV5, HBV7, or HBV11 half-site), and the second subunit The second recognized half site of the recognition sequence (e.g., HBV2, HBV6, HBV8, or H The engineered meganuclease binds to the single-chain meganuclease (the half-site of BV12). In embodiments where the first and second subunits are cleavage enzymes, the first and second subunits comprise an HVR1 region and The first subunit, which binds to the half site of 1, is located as the N-terminal subunit and The second subunit, which contains the VR2 region and binds to the second half-site, is the C-terminal subunit. In another embodiment, the first and second subunits can be oriented so as to be positioned as a pair. The first subunit, which contains the HVR1 region and binds to the first half-site, is the C-terminal subunit. The second subunit is positioned as a unit and contains the HVR2 region and binds to the second half site. The HBV1 gene can be oriented so that the HBV1 gene is located as the N-terminal subunit. Exemplary HBV5-6 meganucleases of the present invention are listed in Table 1. Exemplary HBV7-8 meganucleases of the present invention are presented in Table 2. Exemplary HBV7-8 meganucleases of the present invention are presented in Table 3. Exemplary HBV11-12 meganucleases of the present invention are provided in Table 4.

[0217] Table 1: Genetically engineered to recognize and cleave the HBV1-2 recognition sequence (SEQ ID NO: 10) Exemplary Engineered Meganucleases [Table 1]

[0218] *"HBV1 subunit (%)" and "HBV2 subunit (%)" are the same as those of each meganuclease. HBV1-binding and HBV2-binding subunit regions of HBVase and HBV1-2×.2 megaproteins The amino acid alignment between the HBV1-binding and HBV2-binding subunit domains of the nuclease Represents column identity.

[0219] Table 2: Genetically engineered to recognize and cleave the HBV5-6 recognition sequence (SEQ ID NO: 12) Exemplary Engineered Meganucleases [Table 2]

[0220] *"HBV5 subunit (%)" and "HBV6 subunit (%)" are the percentages of each meganuclease. The HBV5-binding and HBV6-binding subunit regions of the HBVase and the HBV5-6×.33 membrane The amino acid sequence between the HBV5-binding and HBV6-binding subunit regions of the ganucleases Indicates sequence identity.

[0221] Table 3: Genetically engineered to recognize and cleave the HBV7-8 recognition sequence (SEQ ID NO: 14) Exemplary Engineered Meganucleases [Table 3]

[0222] *"HBV7 subunit (%)" and "HBV8 subunit (%)" are the same as those of each meganuclease. HBV7-binding and HBV8-binding subunit regions of HBVase and HBV7-8×.2 megakaryon The amino acid alignment between the HBV7-binding and HBV8-binding subunit domains of the nuclease Represents column identity.

[0223] Table 4: Genetically engineered to recognize and cleave the HBV11-12 recognition sequence (SEQ ID NO: 16) Exemplary engineered meganucleases [Table 4]

[0224] *"HBV11 subunit (%)" and "HBV12 subunit (%)" are the subunits of each megavirus. HBV11-binding and HBV12-binding subunit regions of the nuclease and HBV11-1 HBV11-binding and HBV12-binding subunit regions of 2x.26 meganuclease This represents the identity of the amino acid sequence between

[0225] 2.3 Methods for delivery and expression of endonucleases

[0226] Disclosed herein are methods for treating HBV infection or HCC in a subject. Similarly, the symptoms of HBV infection and the amount of HBV in the subject may be reduced, and the increase in HBV may be prevented. A method for reducing the growth rate or treating HCC is provided, comprising administering to a subject a pharmaceutically acceptable carrier and the subject a method for treating HCC. The engineered meganucleases (or engineered meganucleases) disclosed herein The method of the present invention includes administering a pharmaceutical composition containing a nucleic acid encoding a leukemia enzyme. The engineered meganucleases disclosed herein are The meganuclease is delivered to the DNA / RNA of the target cell, where it can be delivered to the HBV genome. It may be achieved and / or expressed therefrom.

[0227] The engineered meganucleases disclosed herein may be used in the form of proteins or is preferably delivered intracellularly as a nucleic acid encoding an engineered meganuclease Such nucleic acids may be DNA (e.g., circular or linearized plasmid DNA or P The gene may be a nucleic acid (e.g., a nucleic acid product) or RNA (e.g., mRNA). For embodiments in which the enzyme coding sequence is delivered in the form of DNA, it may be a nuclease gene. The gene should be operably linked to a promoter to promote transcription of the gene. Mammalian promoters include the cytomegalovirus early (CMV) promoter (Thoms en et al. (1984), Proc Natl Acad Sci USA.8 1(3):659-63), or the SV40 early promoter (Benoist and C Hambon (1981), Nature. 290 (5804): 304-10), and Inducible promoters, such as the tetracycline-inducible promoter (Dingermann et al. (1992), Mol Cell Biol. 12(9):4038-4 5) and other constitutive promoters. The enzyme may be operably linked to a synthetic promoter. Synthetic promoters include, but are not limited to, , JeT promoter (International Publication No. 2002 / 012514, pamphlet In certain embodiments, the genetically engineered meganuclear cells as disclosed herein The nucleic acid sequence encoding the enzyme may be operably linked to a liver-specific promoter. Examples of heterologous promoters include, but are not limited to, the human alpha-1 antitrypsin promoter. Examples include the α and apolipoprotein A-II promoters.

[0228] In certain embodiments, a gene encoding at least one engineered meganuclease is The nucleic acid sequence is delivered in a recombinant DNA construct or expression cassette. The A construct encodes a promoter and an engineered meganuclease as described herein. and an expression cassette (i.e., a "cassette") containing a nucleic acid sequence encoding the desired gene. In embodiments, the recombinant DNA construct comprises two or more cassettes, each cassette containing a promoter. and a nucleic acid sequence encoding the engineered meganuclease described herein. Each engineered meganuclease is directed against a different HBV recognition sequence as disclosed herein. In a particular embodiment, the recombinant DNA construct comprises two cassettes The cassette or cassettes may include three cassettes, four cassettes, or more. The set includes HBV1-2 meganuclease and HBV5-6 meganuclease. HBV7-8 meganuclease, and HBV11-12 meganuclease. In some embodiments, a single cassette HBV1-2 Meganuclease, HBV5-6 Meganuclease, HBV7-8 Meganuclease nuclease, and HBV11-12 meganuclease. In the form, the cassette or combination of cassettes comprises HBV5-6 meganuclease and H It may encode the BV11-12 meganuclease.

[0229] In another embodiment, the recombinant DNA construct comprises a promoter and a polycistronic nucleic acid. a cassette containing a polycistronic nucleic acid sequence, and a promoter driving expression of the polycistronic nucleic acid sequence. to produce the polycistronic mRNA described herein in the target cell.

[0230] In some embodiments, the mRNA encoding the engineered meganuclease The gene encoding the engineered meganuclease is delivered to the cell. This reduces the chance that the offspring will be integrated into the cell's genome. Such mRNA encoding a ganucleases can be prepared by methods known in the art, such as in vitro transcription. In some embodiments, the mRNA can be produced using methods known in the art. The NA is capped with 7-methyl-guanosine. The mRNA may be polyadenylated.

[0231] In certain embodiments, the mRNA encoding the engineered nuclease of the invention is , a polycistronic mRNA encoding two or more nucleases that are simultaneously expressed in a cell In some embodiments, the polycistronic mRNA may be HB Targeting different recognition sequences in the HBV genome so that the V genome is cleaved at multiple sites In some embodiments, the nucleotide sequence may encode two or more meganucleases described herein. In this regard, a polycistronic mRNA may comprise two or more meganuclei as described herein. and at least one further protein that induces a therapeutically beneficial effect in the cell. The polycistronic mRNA of the present invention may contain an IRES element, a T2A element, The same mRNA, including but not limited to the P2A element, the E2A element, and the F2A element Any element known in the art that allows for the translation of two or more genes from a molecule. In certain embodiments, the polycistronic mRNA may comprise any of the elements described herein. A bicistronic mRNA encoding the two meganucleases described herein, A tricistronic mRNA encoding one meganuclease or four meganucleases as described herein. In this case, each mRNA is a quadcistronic mRNA encoding a meganuclease. The nuclease encoded by A has specificity for different recognition sequences in the HBV genome. For example, polycistronic mRNAs can be used to encode HBV1-2 meganuclease, H BV5-6 meganuclease, HBV7-8 meganuclease, and HBV11-12 meganuclease Any number or combination of ganucleases can be encoded. In this state, polycistronic mRNA is used to encode HBV1-2 meganuclease, HBV5 -6 meganuclease, HBV7-8 meganuclease, and HBV11-12 meganuclease In another specific embodiment, the polycistronic mRNA may encode a H Bis-encoding BV5-6 meganuclease and HBV11-12 meganuclease It may be stron mRNA.

[0232] In another specific embodiment, the nucleic acid encoding the endonuclease of the invention is a single-stranded D Single-stranded DNA can be introduced into cells using a DNA template. 5' and / or 3' AAV reverse transcription upstream and / or downstream of the ganucleases-encoding sequence In other embodiments, the single-stranded DNA may further comprise an indirect terminal repeat (ITR). 5' and / or 3' phases upstream and / or downstream of the sequence encoding the meganuclease It may further comprise a homosexual arm.

[0233] In another specific embodiment, the gene encoding the endonuclease of the present invention is linearized. A DNA template can be used to introduce the gene into a cell. The plasmid DNA encoding the enzyme can be digested with one or more restriction enzymes, The circular plasmid DNA is linearized before being introduced into cells.

[0234] The purified nuclease protein is as further detailed herein below. cleavage of genomic DNA by a variety of different mechanisms known in the art, including The compound can be delivered intracellularly to

[0235] The target tissue for delivery of the engineered meganucleases of the present invention is not limited. and / or liver cells, e.g., hepatocytes, or preferably primary hepatocytes, more preferably human hepatocytes. These include human primary hepatocytes, HepG2.2.15 or HepG2-hNTCP cells. As discussed, the meganucleases of the present invention can be obtained as purified proteins or as meganucleases. In one embodiment, the enzyme can be delivered as RNA or DNA encoding the enzyme. mRNA or DNA encoding a meganuclease protein or endonuclease A vector can be delivered to target cells (e.g., liver cells) by direct injection into the target tissue. Alternatively, the endonuclease protein, mRNA, or DNA may be delivered to the circulating It can be delivered systemically via the system.

[0236] In some embodiments, an endonuclease protein, or an endonuclease The DNA / mRNA encoding the enzyme can be administered systemically or to a target tissue using known methods. They are formulated in a pharmaceutically acceptable carrier according to the art. The Science And Practice of Pharmacy(21s In the preparation of the pharmaceutical preparation according to the present invention, the protein The / RNA / mRNA is typically mixed with a pharmaceutically acceptable carrier. The carrier may, of course, It must be acceptable in the sense of being compatible with any other ingredients of the formulation and The carrier may be solid or liquid, or both. , can be formulated with the compound as a unit dose formulation.

[0237] In some embodiments, an endonuclease protein, or an endonuclease The DNA / mRNA encoding the enzyme is transfected with a cell-permeable peptide to facilitate cellular uptake. Examples of cell-penetrating peptides known in the art include: Polyarginine (Jearawiriyapaisarn, et al. (200 8) Mol Ther. 16:1624-9), TAT peptide derived from HIV virus ( Hudecz et al.(2005),Med.Res.Rev.25:679-7 36), MPG (Simeoni, et al. (2003) Nucleic Acid s 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 Lif e Sci. 62:1839-49). The DNA / mRNA encoding the endonuclease protein is then transferred to the target cell. Covalently or non-covalently bound to an antibody that recognizes a specific cell surface receptor expressed on The endonuclease protein / DNA / mRNA binds to the target cell and Alternatively, the endonuclease protein / DNA / mRNA to the natural ligand (or one of the natural ligands) of such cell surface receptors. The hydroxylase moiety can be covalently or non-covalently bound to the hydroxylase moiety (McCall, et al. (2014)Tissue Barriers.2(4):e944449;Dinda , et al.(2013)Curr Pharm Biotechnol.14:1 264-74;Kang,et al.(2014)Curr Pharm Biote chnol.15(3):220-30;Qian et al.(2014)Expe rt Opin Drug Metab Toxicol.10(11):1491-5 08).

[0238] In some embodiments, the endonuclease protein or endonuclease The encoding DNA / mRNA is expressed in (e.g., liver sinusoidal endothelial cells or hemogenic endothelial cells, or for injection or transplantation into the desired region of the liver (or in the vicinity of the precursor cells that differentiate into it). The hydrogel allows the drug to reach the target tissue without the need for frequent injections. It can provide sustained and tunable release of a therapeutic payload to a desired area of ​​tissue, Thermoresponsive materials (e.g., temperature-responsive and pH-responsive hydrogels) are environmentally or externally applied materials. The payload can be designed to be released in response to a given signal (Kang Derwe nt et al.(2008)Trans Am Ophthalmol Soc.1 06:206-214).

[0239] In some embodiments, an endonuclease protein, or an endonuclease The DNA / mRNA encoding the enzyme is covalently or, preferably, non-covalently attached to the nanoparticles. or encapsulated within such nanoparticles using methods known in the art ( Sharma, et al. (2014) Biomed Res Int.2014). Nanoparticles are nanoscale particles whose length scale is <1 μm, preferably <100 nm. Such nanoparticles may be metal, lipid, polymer, or biopolymer. The core can be designed to contain endonuclease proteins, mRNAs, and A, or multiple copies of DNA can be attached or encapsulated in the nanoparticle core. This increases the number of copies of protein / mRNA / DNA delivered to each cell, thus The intracellular expression of each endonuclease increases, maximizing the possibility of cleaving the target recognition sequence. The surface of such nanoparticles is coated with polymers or lipids (e.g., chitosan, cationic The surface can be further modified with a polymer (e.g., a cationic polymer, or a cationic lipid) to enhance cell delivery and payload. Core-shell nanoparticles can be formed that impart additional functionality to enhance drug uptake (J ian et al.(2012)Biomaterials.33(30):7621 -30) To target nanoparticles to the appropriate cell type and / or to increase the likelihood of cellular uptake, To enhance the efficacy, the nanoparticles can be advantageously further conjugated to targeting molecules. Examples of such targeting molecules include antibodies specific for cell surface receptors and molecules specific for cell surface receptors. Natural ligands (or portions of natural ligands) are included.

[0240] In some embodiments, an endonuclease protein, or an endonuclease The DNA / mRNA encoding the enzyme is encapsulated in liposomes or transported using cationic lipids. complexed with LIPOFECTAMINE transfection reagent,Life Technologies Corp.,Carlsba d,CA;Zuris et al.(2015)Nat Biotechnol.33 :73-80;Mishra et al.(2011)J Drug Deliv.2 Liposome and lipoplex formulations deliver payloads in separate compartments. protects the target cell from degradation, enhances accumulation and retention at the target site, and inhibits fusion with the plasma membrane of the target cell These can increase the efficiency of cellular uptake and delivery through disruption of the cellular membrane and / or cell membrane.

[0241] In some embodiments, an endonuclease protein, or an endonuclease The DNA / mRNA encoding the enzyme is encapsulated within a polymer scaffold (e.g., PLGA). , complexed with cationic polymers (e.g., PEI, PLL) (Tamboli et al. (2011) Ther Deliv. 2(4):523-536). Poly The polymer carrier also provides tunable drug release rates through control of polymer erosion and drug diffusion. The drug can be designed to deliver the desired drug to the desired target cell population with high drug encapsulation efficiency. This can provide protection for the therapeutic payload until delivery within the cell.

[0242] In some embodiments, an endonuclease protein or a genetically engineered megakaryon The DNA / mRNA encoding the nuclease is combined with amphiphilic molecules that self-assemble into micelles. (Tong et al. (2007) J Gene Med. 9(11) :956-66). Polymeric micelles prevent aggregation, mask charge interactions, and Hydrophilic polymers (e.g., polyethylene glycol) that can reduce heterogeneous interactions The shell may comprise a micellar shell formed from a polymer (such as a methyl methacrylate).

[0243] In some embodiments, the endonuclease protein or endonuclease The encoding DNA / mRNA is then formulated into an emulsion for administration and / or delivery to target cells. They are formulated into emulsions or nanoemulsions (i.e., with an average particle size of <1 nm). The term "solution" refers, without limitation, to the formation of a non-polar solution when a water-immiscible phase is mixed with an aqueous phase. Hydrophobicity, which directs polar residues (e.g., long hydrocarbon chains) away from water and polar head groups toward water Any oil-in-water or water-in-oil containing lipid structures that can form as a result of the forces These other lipid structures include dispersions or droplets of the water-in-oil-in-water type, or the oil-in-water-in-oil type. Examples include unilamellar, sublamellar, and multilamellar lipid vesicles, micelles, and lamellar phases. Emulsions are composed of an aqueous phase and a lipophilic phase (typically containing oil and an organic solvent). Emulsions also frequently contain one or more surfactants. Lution formulations are described, for example, in U.S. Patent Application Publication Nos. 2002 / 0045667 and 20 04 / 0043041, and U.S. Pat. Nos. 6,015,832 and 6,506,80 3, 6,635,676, and 6,559,189. No. 6,299,133, each of which is incorporated herein by reference in its entirety.

[0244] In some embodiments, an endonuclease protein, or an endonuclease The DNA / mRNA encoding the enzyme was synthesized by a multifunctional polymer conjugate, a DNA dendrimer. and can be covalently attached or non-covalently associated with the polymer dendrimer ( Mastorakos et al.(2015)Nanoscale.7(9):38 45-56;Cheng et al.(2008)J Pharm Sci.97(1 ):123-43). The generation of dendrimers allows for control of payload capacity and size, It can also provide high drug payload capacity. Furthermore, the display of multiple surface groups can be utilized. It can be used to improve stability, reduce non-specific interactions, and facilitate cell-specific targeting and drug The release can be enhanced.

[0245] In some embodiments, the gene encoding the endonuclease is expressed as a viral vector. Such vectors are known in the art and are used in the delivery of vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viruses AAV vectors are included (Vannucci, et al. (2013) Ne In some embodiments, the virus The vector is injected directly into the target tissue (e.g., liver tissue). AAV vectors are delivered systemically via the circulatory system. Different AAV vectors are delivered to different tissues. It is known in the art that the target tissues of the liver tend to be located in the liver. In this context, efficient transduction of hepatocytes has been demonstrated with, for example, AAV serotypes 2, 8, and 9. (Sands(2011)Methods Mol.Biol.807:141-15 7) In addition, AAV vectors do not require second-strand DNA synthesis in the host cell. It can be self-complementary, as in the case of Ther.8:1248-54).

[0246] In one embodiment, the viral vector used for endonuclease gene delivery is Self-limiting viral vectors are vectors that are genetically engineered within the vector. Due to the presence of a recognition sequence for the designated meganuclease, Therefore, self-limiting viral vectors can have a limited duration of action. The endonuclease described in the document and the endonuclease recognition site in the ITR are encoded by the Self-limiting viral vectors can be engineered to The enzyme is expressed and is able to cleave the cell's genome at its endogenous recognition sequence. The endonuclease gene is delivered to a cell, tissue, or organism. The lyase also finds and binds to target sites within the self-limiting viral vector itself. Once cleaved, the 5' and 3' ends of the viral genome are exposed. and degraded by exonucleases, thus killing the virus and Stops the production of creatine.

[0247] The endonuclease gene may be in the form of DNA (e.g., a plasmid) and / or in a virus. When delivered via a vector (e.g., AAV), they are promoter-operable. In some embodiments, this is a viral vector. Endogenous promoter (e.g., LTR of lentiviral vector) or known site A viral promoter such as the megalovirus or SV40 virus early promoter. In a preferred embodiment, the meganuclease gene is preferentially expressed in the target cell. The liver-specific promoter is operably linked to a promoter that drives gene expression. Examples include, but are not limited to, the human alpha-1 antitrypsin promoter and the apolipoprotein B1 promoter. An example is the protein A-II promoter.

[0248] In certain embodiments, the viral vector comprises a promoter and a gene manipulation vector as described herein. and a cassette containing a nucleic acid sequence encoding the constructed meganuclease. A vector may contain two or more cassettes, each cassette containing a promoter and a Each of the engineered meganucleases comprises a nucleic acid sequence encoding the engineered meganuclease. The meganucleases have specificity for different HBV recognition sequences as disclosed herein. In some embodiments, the viral vector comprises a promoter and a polycistronic nucleic acid. a promoter for directing expression of the polycistronic nucleic acid sequence; to induce the production of polycistronic mRNAs, e.g., genes, as described herein, in target cells. Generate polycistronic mRNAs encoding engineered meganucleases.

[0249] Methods for delivering meganucleases disclosed herein to the liver of a subject infected with HBV In one embodiment, native hepatocytes extracted from a mammal are , can be transduced with a vector encoding an engineered meganuclease Alternatively, native hepatocytes from HBV-infected subjects may be transfected with engineered meganucleases and / or an adenoviral vector encoding a molecule that stimulates liver regeneration, such as a hepatotoxin, Preferably, the hepatotoxin is uPA and the viral vector is It has been modified to inhibit its secretion from hepatocytes when expressed by the In an embodiment, the vector encodes tPA, which stimulates hepatocyte regeneration de novo. The transduced hepatocytes removed from the mammal can then be returned to the mammal. where conditions conducive to expression of the engineered meganuclease are provided. Typically, transduced hepatocytes are injected through the spleen or portal vasculature. The patient can then be administered a single or multiple doses over a period of 1 to 5 days or more. It could be.

[0250] In an in vivo embodiment of the method of the present invention, a gene encoding an engineered meganuclease is provided, A retroviral, pseudotyped or adenoviral-associated vector is constructed and administered to a subject. Administration of a vector encoding a genetically engineered meganuclease results in secretion of the enzyme. In conjunction with the administration of an adenoviral vector encoding a hepatotoxin or encoding tPA. It stimulates liver cell regeneration without acting as a hepatotoxin.

[0251] The appropriate dose will depend, among other factors, on the specifics of any AAV vector selected (e.g., serotype, etc.), route of administration, subject to be treated (i.e., age, weight, sex, and general condition of the subject) ) and the mode of administration. Therefore, the appropriate dosage may vary from patient to patient. Effective amounts can be readily determined by one of ordinary skill in the art. Dosage treatment can be a single dose regimen or multiple doses. Furthermore, the subject can be administered as many doses as possible. The appropriate number of doses can be readily determined. The dosage may be adjusted to take into account alternative routes of administration or Adjustments may need to be made to balance the benefits of treatment with any side effects.

[0252] 2.4 Pharmaceutical Compositions

[0253] In some embodiments, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a gene of the present invention. The engineered meganuclease or a pharmaceutically acceptable carrier and the genetically engineered meganuclease of the present invention. and a pharmaceutical composition comprising an isolated polynucleotide comprising a nucleic acid encoding a meganuclease. In another embodiment, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the pharmaceutical composition of the present invention. to target tissues where cells express the engineered meganucleases disclosed in The pharmaceutical composition of the present invention comprises the cells of the present invention, which can Treating a subject with HBV, reducing the level or proliferation of HBV, Useful for reducing at least one symptom of BV or treating HCC. obtain.

[0254] Pharmaceutical compositions can be designed or selected according to the genotype of the target HBV strain. As described in detail in the specification, the meganuclease of the present invention targets a specific gene of HBV. It is genetically engineered to recognize and cleave the recognition sequence in the HBV type. -2 meganuclease (e.g., SEQ ID NOs: 18-21) is a nuclease that inhibits at least HBV genotype A. A, B, C, E, F, and G (e.g., SEQ ID NOS: 3-5 and 7-9, respectively) Furthermore, the gene manipulations disclosed herein recognize and cleave the HBV1-2 recognition sequence. The recognition sequences of the constructed meganucleases are shown in SEQ ID NOs: 3 to 9. For example, HBV genotypes A, B, C, D, and It can be found in isolates E, F, and G. As used herein, HBV "Isolates" of the present invention are examples of the corresponding genotypes provided in any of SEQ ID NOS: 3-9. and 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 more sequence identity. In some embodiments, the pharmaceutical compositions disclosed herein comprise a compound selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16. The present invention can be administered to a subject having any genotype of HBV containing the recognition sequence defined in do.

[0255] Such pharmaceutical compositions can be prepared according to known techniques. ngton,The Science And Practice of Pharma In the manufacture of pharmaceutical formulations according to the present invention, The endonuclease polypeptide (or the DNA / RNA encoding it) is typically In this case, the composition is mixed with a pharmaceutically acceptable carrier and the resulting composition is administered to a subject. Naturally, it must be acceptable in the sense of being compatible with any other ingredients of the formulation, and In some embodiments, the pharmaceutical compositions of the present invention may be administered in a manner that is not harmful to the subject. The composition may further comprise one or more additional drugs or biomolecules useful for treating a disease in a subject. Similarly, additional drugs and / or biomolecules can be co-administered as separate compositions. It is possible.

[0256] In certain embodiments, the pharmaceutical compositions of the present invention comprise the genetically engineered megakaryocytes described herein. Combinations of nucleases (or nucleic acids encoding engineered meganucleases) wherein each engineered meganuclease comprises a different H The BV recognition sequence has specificity for the BV, so that a single pharmaceutical composition can be used to treat a wide range of BV infections in a subject. This will be useful for the treatment of a wide array of HBV genotypes and / or genotypic isolates. In another embodiment, the pharmaceutical compositions of the present invention have specificity for different HBV recognition sequences. A polynucleotide encoding a plurality of engineered meganucleases described herein, cistronic mRNA (or a cassette that produces polycistronic mRNA upon expression) Such pharmaceutical compositions may also contain recombinant DNA constructs or viral vectors. and broad spectrum of therapeutic options for the treatment of a broad array of HBV genotypes and / or genotype isolates in subjects. In any case, such pharmaceutical compositions may be useful for specific HBV genotypes or The isolate may be useful as a single treatment when known or unknown in the subject.

[0257] For example, a recombinant meganuclease comprising a plurality of different recombinant meganucleases disclosed herein, or Pharmaceutical compositions comprising nucleic acid molecules encoding multiple different recombinant meganucleases as disclosed in , administered to patients infected with multiple genotypes of HBV or infected with an unknown genotype of HBV. Therefore, it is possible to prepare a pharmaceutical composition containing multiple different recombinant meganucleases. and / or a plurality of different recombinant meganuclease-encoding nucleic acid molecules, thereby This makes it difficult to accurately determine the genotype of HBV, and a rapid and comprehensive treatment solution is desired. It provides flexible options for the treatment and control of V infections.

[0258] In certain embodiments of the present invention, the pharmaceutical composition comprises the pharmaceutical compositions described herein encapsulated in lipid nanoparticles. The mRNA may comprise one or more of the mRNAs described elsewhere herein. In certain embodiments, the lipid nanoparticles are labeled with different HBV recognition sequences as described herein. Each encodes an engineered meganuclease of the invention having specificity for In certain embodiments, the lipid nanoparticles may comprise two or more mRNAs described herein. The genetically engineered HBV receptors of the present invention each have specificity for a different HBV recognition sequence. It may contain two, three, or four mRNAs encoding the ganucleases described herein. In other embodiments, the lipid nanoparticles may be directed against different HBV recognition sequences as described herein. and a gene encoding each of two or more engineered meganucleases of the present invention each having a specificity. The polycistronic mRNA may comprise one or more of the polycistronic mRNAs described herein. In the present invention, the lipid nanoparticles are composed of two, three, or four genetically engineered meganucleic acids as described herein. The polycistronic mRNA encoding the enzyme may be included. In some embodiments, the lipid nanoparticles contain two or more polycistronic mRNAs as described herein. Each of the genes can contain two or more engineered meganucleases of the invention.

[0259] Some lipid nanoparticles contemplated for use in the present invention contain at least one cationic lipid, at least one non-cationic lipid, and at least one complex lipid. In certain examples, the lipid nanoparticles comprise about 50 mol % to about 85 mol % cationic lipids, about 13 mol % mol% to about 49.5 mol% non-cationic lipid, and about 0.5 mol% to about 10 mol% lipid The polymer may contain a polymer conjugate and may be prepared in such a way that it has a non-lamellar (i.e., non-bilayer) morphology. In another particular example, the lipid nanoparticles are about 40 mol % to about 85 mol % cationic lipids, from about 13 mol % to about 49.5 mol % non-cationic lipids, and from about 0.5 mol % to about 1 0 mol % lipid conjugate and have a non-lamellar (i.e., non-bilayer) morphology. It is manufactured in a manner that

[0260] The cationic lipids may include, for example, one or more of the following: palmitoyl-oleoyl- Arginine (PONA), MPDACA, GUADACA, ((6Z, 9Z, 28Z , 31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-( Dimethylaminobutanoic acid (MC3); LenMC3, CP-LenMC3, γ-Le nMC3, CP-γ-LenMC3, MC3MC, MC2MC, MC3 ether, MC4 Ether, MC3 Amide, Pan-MC3, Pan-MC4, and PanMC5, 1,2 -Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) 1,2-dilinoleyloxy-N,N-dimethylaminopropane Nolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-diamino Leyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K -C2-DMA; "XTC2"), 2,2-dilinoleyl-4-(3-dimethylaminopropanediol; "XTC2"); DLin-K-C3-DMA)-[1,3]-dioxolane, 2,2-dilinol Leyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K -C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-di Oxane (DLin-K6-DMA), 2,2-Dilinoleyl-4-N-methylbenzidine No-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4- Dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2- Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP) ), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin -DAC), 1,2-dilinoleoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-di Linoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleo DLin-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP) , 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin- TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt ( DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino ) propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propane Pandiol (DLinAP), 3-(N,N-dioleylamino)-1,2-propane Dio(DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino) Ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethyl Ammonium chloride (DODAC), 1,2-dioleoyloxy-N,N-dimethyl Aminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylamino Propane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N ,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N -Dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy) (Di)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-( N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-C hol), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl- N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleoyloxy cis-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanol Ammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylsperm amine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxidase (cis,cis-9,12-octadecadienoxy)propan-4-oxy lopane (CLinDMA), 2-[5´-(cholest-5-ene-3-beta-oxy) -3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'- Octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-di Oleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamate Dimethyl-3-aminopropane (DOcarbDAP), 1,2-N,N'-dilinole ylcarbamyl-3-dimethylaminopropane (DLincarbDAP), or Cationic lipids are also available as DLinDMA, DLin-K-C2-DMA ("X TC2''), MC3, LenMC3, CP-LenMC3, γ-LenMC3, CP-γ -LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 A Pan-MC3, Pan-MC4, Pan-MC5, or a mixture thereof. stomach.

[0261] In various embodiments, the cationic lipid is about 50 mol % of the total lipid present in the particle. ~90mol%, approximately 50mol%~approximately 85mol%, approximately 50mol%~approximately 80mol% , about 50 mol% to about 75 mol%, about 50 mol% to about 70 mol%, about 50 mol% It may contain about 65 mol % or about 50 mol % to about 60 mol %.

[0262] In other embodiments, the cationic lipid comprises from about 40 mol % to about 9 mol % of the total lipid present in the particle. 0 mol%, about 40 mol% to about 85 mol%, about 40 mol% to about 80 mol%, about 4 0mol%~about 75mol%, about 40mol%~about 70mol%, about 40mol%~about 6 It may contain 5 mol %, or about 40 mol % to about 60 mol %.

[0263] Non-cationic lipids can include, for example, one or more anionic lipids and / or neutral lipids. In a preferred embodiment, the non-cationic lipid comprises one of the following components of a neutral lipid: (1) cholesterol or its derivatives; (2) phospholipids; or (3) phospholipids and Mixtures with cholesterol or its derivatives. Examples of cholesterol derivatives include cholesterol Coprostanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxybenzoate cholesteryl-4'-hydroxybutyl ether, and their Phospholipids include, but are not limited to, neutral lipids, such as, but not limited to, mixtures of phospholipids. , dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine Phosphate (DSPC), Dioleoylphosphatidylethanolamine (DOPE), Palmitate Palmitoyloleoyl-phosphatidylcholine (POPC), Palmitoyloleoyl-phosphatidylcholine Photidylethanolamine (POPE), palmitoyloleoyl-phosphatidylcholine Glycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE ), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl- Phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanol Amine, Dimethyl-Phosphatidylethanolamine, Dielidoyl-Phosphatidyl Ethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine phosphatidylcholine (SOPE), egg phosphatidylcholine (EPC), and mixtures thereof. In a preferred embodiment, the phospholipid is DPPC, DSPC, or a mixture thereof. is.

[0264] In some embodiments, a non-cationic lipid (e.g., one or more phospholipids and / or Cholesterol accounts for approximately 10 mol% to 60 mol% of the total lipids present in the particles, and approximately 1 5mol%~about 60mol%, about 20mol%~about 60mol%, about 25mol%~about 6 0mol%, about 30mol% to about 60mol%, about 10mol% to about 55mol%, about 1 5mol%~about 55mol%, about 20mol%~about 55mol%, about 25mol%~about 5 5mol%, about 30mol% to about 55mol%, about 13mol% to about 50mol%, about 1 It may contain 5 mol % to about 50 mol %, or about 20 mol % to about 50 mol %. When the ionic lipid is a mixture of phospholipid and cholesterol or a cholesterol derivative The mixture may comprise up to about 40, 50, or 60 mole % of the total lipid present in the particle.

[0265] Conjugated lipids that inhibit particle aggregation include, for example, polyethylene glycol (PEG)-lipids. Conjugates, Polyamide (ATTA)-Lipid Conjugates, Cationic Polymers The present invention may comprise one or more of a lipid conjugate (CPL), a lipid conjugate (CPL), or a mixture thereof. In embodiments, the nucleic acid-lipid particles comprise a PEG-lipid conjugate or an ATTA-lipid conjugate. In certain embodiments, the PEG-lipid conjugate or TTA-lipid conjugates are used in conjunction with CPLs to inhibit particle aggregation. The substrates include, for example, PEG-diacylglycerol (DAG), PEG dialkyloxypropanol (PEG-DAG), and Pill (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof The PEG-DAA conjugate may comprise a PEG-lipid comprising PEG-dilauryl Oxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG- Dipalmityloxypropyl (C16), PEG-distearyloxypropyl (C18 ), or a mixture thereof.

[0266] Additional PEG-lipid conjugates suitable for use in the present invention include mPEG2 000-1,2-di-O-alkyl-sn3-carbomoyl glyceride (PEG-CD The synthesis of PEG-C-DOMG can be carried out by the method of PC Further suitable compounds for use in the present invention are described in PCT Application No. PCT / US08 / 88676. Additional PEG-lipid conjugates include, but are not limited to, 1-[8'-(1,2 -Dimyristoyl-3-propanoxy)-carboxamido-3',6'-dioxaocta Nyl]carbamoyl-ω-methyl-poly(ethylene glycol) (2KPEG-DMG) The synthesis of 2KPEG-DMG is described in U.S. Pat. No. 7,404,969. It is being done.

[0267] In some cases, conjugated lipids (e.g., PEG-lipid conjugates) are used to inhibit particle aggregation. The lipid content of the particles is approximately 0.1 mol% to 2 mol%, and approximately 0.5 mol% to 10 mol% of the total lipid content of the particles. Approx. 2 mol%, approx. 1 mol% to approx. 2 mol%, approx. 0.6 mol% to approx. 1.9 mol%, approx. 0.7mol%~about 1.8mol%, about 0.8mol%~about 1.7mol%, about 1mol % to approximately 1.8 mol%, approximately 1.2 mol% to approximately 1.8 mol%, approximately 1.2 mol% to approximately 1 .7mol%, about 1.3mol%~about 1.6mol%, about 1.4mol%~about 1.5mo 1%, 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 or range thereof). Typically, in such cases, the PEG moiety has an average molecular weight of about 2,000 daltons. In other cases, conjugated lipids (e.g., PEG-lipid conjugates) are used to inhibit particle aggregation. The lipid content of the particles is about 5.0 mol% to about 10 mol%, and about 5 mol% to about 10 mol% of the total lipid content of the particles. Approximately 9 mol%, approximately 5 mol% to approximately 8 mol%, approximately 6 mol% to approximately 9 mol%, approximately 6 mol % to approximately 8 mol%, approximately 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, Typically, the amount of hydroxybenzoates used may be 10 mol % (or any fraction or range thereof). In such cases, the PEG moieties have an average molecular weight of about 750 daltons.

[0268] In other embodiments, the composition comprises at least one positive charge carrier and at least one other charge carrier that is different from the positive charge. The liposome may contain amphoteric liposomes containing at least one negative charge carrier, and the isoelectric point of the liposome may be The purpose of this is to prepare liposomes with a pH-dependent variable charge. It is achieved by facts.

[0269] The structure of liposomes with desired properties can be determined, for example, by membrane-forming or membrane-based cationic charge. The amount of carriers exceeds the amount of anionic charge carriers at low pH, and the ratio reverses at higher pH. This is always the case when the pKa value of the ionic 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 become more charged. The charge carrier loses its charge.

[0270] Cationic compounds useful in amphoteric liposomes include those already mentioned hereinabove. Strongly cationic compounds include, but are not limited to, compounds with strong cationic properties, such as , DC-Chol 3-β-[N-(N',N'-dimethylmethane)carbamoyl]chol Sterol, TC-Chol 3-β-[N-(N',N',N'-trimethylaminoethyl ester Tan) Carbamoyl cholesterol, BGSC bisguanidinium-spermidine-cholesterol Sterol, BGTC bis-guanidinium-tren-cholesterol, DOTAP(1, 2-Dioleoyloxypropyl)-N,N,N-trimethylammonium chloride, D OSPER (1,3-dioleoyloxy-2-(6-carboxy-spermyl)-pro pyramide), DOTMA (1,2-dioleoyloxypropyl)-N,N,N-trimethylsilyl Methylammonium chloride) (Lipofectin®), DORIE 1,2-di oleoyloxypropyl)-3-dimethylhydroxyethylammonium bromide, D OSC (1,2-dioleoyl-3-succinyl-sn-glyceryl choline ester), DOGSDSO (1,2-dioleoyl-sn-glycero-3-succinyl-2-hydrogen Dimethyl disulfide ornithine (omithine), DDAB dimethyl dioctadecanoate Decylammonium bromide, DOGS((C18)2GlySper3+)N,N-di Octadecylamide-glycol-spermine (Transfectam®) (C 18) 2Gly + N,N-Dioctadecylamido-glycine, CTAB cetyltrimethyl Ammonium bromide, CpyC cetylpyridinium chloride, DOEPC 1,2-dioxide leoyl-sn-glycero-3-ethylphosphocholine or other O-alkyl-phosphatidylinositol diethylcholine or ethanolamine, amides from lysine, arginine or ornithine (o mithine), and phosphatidylethanolamine.

[0271] Examples of weakly cationic compounds include, but are not limited to, His-Chol( Histaminyl-cholesterol hemisuccinate), Mo-Chol (morpholine-N- ethylamino-cholesterol hemisuccinate), or histidinyl-PE do.

[0272] Examples of neutral compounds include cholesterol, ceramide, phosphatidylcholine, phosphatidylcholine, and phosphatidylcholine. acetidylethanolamine, tetraether lipid, or diacylglycerol. However, it is not limited to these.

[0273] Anionic compounds useful in amphoteric liposomes include the non-cationic compounds previously described herein. Non-limiting examples of weakly anionic compounds include CHEMS ( Cholesterol hemisuccinate), alkyl carboxylic acid with 8 to 25 carbon atoms or diacylglycerol hemisuccinate. Additional weakly anionic compounds These include amides of aspartic acid or glutamic acid with PE and PS, as well as glycine, Lanine, glutamine, asparagine, serine, cysteine, threonine, tyrosine, glutamine Amino acid, aspartic acid or other amino acids, or their amino acids with aminodicarboxylic acids Following the same principle, hydroxycarboxylic acids or hydroxydicarboxylic acids may be used. The ester of methyl methyl ester with PS is also a weakly anionic compound.

[0274] In some embodiments, the amphoteric liposomes are complexes such as those described herein above. Specific examples of useful conjugated lipids include PEG-modified phosphatidylethanol. amines and phosphatidic acids, PEG-ceramide conjugates (e.g., PEG-Ce rC14 or PEG-CerC20), PEG-modified dialkylamine and PEG-modified 1, Particularly preferred are, but not limited to, 2-diacyloxypropan-3-amine. Preferred are PEG-modified diacylglycerols and dialkylglycerols.

[0275] In some embodiments, the neutral lipid is about 10 mol% to about 10 mol% of the total lipid present in the particle. Approximately 60 mol%, approximately 15 mol% to approximately 60 mol%, approximately 20 mol% to approximately 60 mol%, Approx. 25mol%~Approx. 60mol%, Approx. 30mol%~Approx. 60mol%, Approx. 10mol%~ Approximately 55 mol%, approximately 15 mol% to approximately 55 mol%, approximately 20 mol% to approximately 55 mol%, Approx. 25mol%~Approx. 55mol%, Approx. 30mol%~Approx. 55mol%, Approx. 13mol%~ About 50 mol%, about 15 mol% to about 50 mol%, or about 20 mol% to about 50 mol May contain %.

[0276] In some cases, conjugated lipids (e.g., PEG-lipid conjugates) are used to inhibit particle aggregation. The lipid content of the particles is approximately 0.1 mol% to 2 mol%, and approximately 0.5 mol% to 10 mol% of the total lipid content of the particles. Approx. 2 mol%, approx. 1 mol% to approx. 2 mol%, approx. 0.6 mol% to approx. 1.9 mol%, approx. 0.7mol%~about 1.8mol%, about 0.8mol%~about 1.7mol%, about 1mol % to approximately 1.8 mol%, approximately 1.2 mol% to approximately 1.8 mol%, approximately 1.2 mol% to approximately 1 .7mol%, about 1.3mol%~about 1.6mol%, about 1.4mol%~about 1.5mo 1%, 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 or range thereof). Typically, in such cases, the PEG moiety has an average molecular weight of about 2,000 daltons. In other cases, conjugated lipids (e.g., PEG-lipid conjugates) are used to inhibit particle aggregation. The lipid content of the particles is about 5.0 mol% to about 10 mol%, and about 5 mol% to about 10 mol% of the total lipid content of the particles. Approximately 9 mol%, approximately 5 mol% to approximately 8 mol%, approximately 6 mol% to approximately 9 mol%, approximately 6 mol % to approximately 8 mol%, approximately 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, Typically, the amount of hydroxybenzoates used may be 10 mol % (or any fraction or range thereof). In such cases, the PEG moieties have an average molecular weight of about 750 daltons.

[0277] Considering the total amount of neutral and complex lipids, the remaining residue of amphoteric liposomes is The cationic lipids and anionic compounds may be formulated as a mixture. The ratio of anionic lipids can be adjusted to suit the desired properties of nucleic acid encapsulation, zeta potential, pKa, or charged lipid components. selected to achieve other physicochemical properties that depend at least in part on the presence of obtain.

[0278] In some embodiments, lipid nanoparticles are capable of delivering and uptake into the liver, particularly within hepatocytes. It has a composition that specifically enhances

[0279] 2.5 Methods for producing recombinant AAV vectors

[0280] In some embodiments, the present invention provides recombinant AAV vectors for use in the methods of the present invention. Recombinant AAV vectors are typically expressed in mammalian cells such as HEK-293. The viral cap and rep genes are removed from the vector. to provide space for delivery of therapeutic genes (e.g., endonuclease genes). These are packaged in trans in cell lines to prevent self-renewal. rans) and any "helpers" needed to support replication (e.g. It is necessary to provide components (e.g., adenovirus) (Cots D, Bosch A, Chillon M(2013)Curr.Gene Ther.13(5):370- 81) Recombinant AAV vectors often contain a first promoter encoding a “helper” component. A second plasmid containing the plasmid, the cap and rep genes, and the a third plasmid containing the viral ITRs containing the intervening DNA sequence to be transfected into the cells; The strain is produced using triple transfection, where the encapsidated vector is then transfected. Viral particles containing the genome (ITRs and intervening genes of interest) are purified by freeze-thaw cycles, They are isolated from cells by sonication, detergents, or other means known in the art. The particles are then separated by cesium chloride density gradient centrifugation or affinity chromatography. and subsequently injecting the gene of interest into a cell, tissue, or organism, such as a human patient. Serve the child.

[0281] Recombinant AAV particles are typically produced in cells, allowing for the site-specific endonuclease To ensure that the enzyme is not expressed in the packaging cells, the present invention is carried out Care must be taken when translating the viral genome of the present invention. Any endonuclease expressed in the packaging cell line will contain a recognition sequence The viral genome can be cleaved before it can be packaged into a viral particle. This can result in reduced packaging efficiency and / or packaging of fragmented genomes. To prevent expression of the endonuclease in the packaging cells, Several approaches can be used.

[0282] The endonuclease inhibits the regulation of tissue-specific promoters that are not active in packaging cells. For example, a viral vector can deliver an endonuclease gene to muscle cells. If developed for delivery to tissue, muscle-specific promoters can be used. An example of a muscle-specific promoter is C5-12 (Liu, et al. (2004) Hu m Gene Ther.15:783-92), muscle-specific creatine kinase (MC K) promoter (Yuasa, et al. (2002) Gene Ther. 9:15 76-88), or the smooth muscle 22 (SM22) promoter (Haase, et al. (2 013)BMC Biotechnol.13:49-54) including CNS (neuro) Examples of gene-specific promoters include the NSE, synapsin, and MeCP2 promoters. (Lentz, et al. (2012) Neurobiol Dis.48:17 9-88). Examples of liver-specific promoters include the albumin promoter (Palb, etc.), α1-antitrypsin (Pa1AT, etc.) and hemopexin (Phpx, etc.) (Kramer, MG et al., (2003) Mol. Therapy 7:3 75-85). Examples of eye-specific promoters include opsin and the corneal epithelium-specific K12 promoter. Motor included (Martin KRG, Klein RL, and Quigley HA(2002)Methods(28):267-75)(Tong Y,et a l., (2007) J Gene Med, 9:956-66). Other tissue-specific promoters known in the art are highly expressed in HEK-293 cells. Therefore, when incorporated into the viral vector of the present invention, It is not expected to result in significant levels of endonuclease gene expression in caged cells. Similarly, the viral vectors of the present invention may be designed to express incompatible tissue-specific promoters (i.e., Use of the well-known HeLa cell line (human epithelial cells) and the liver-specific hemopexin promoter The use of other cell lines employing tissue-specific promoters (such as the use of promoters from other cell lines) is contemplated. Other examples of tissue-specific promoters include promoters from smooth muscle. PDZD4 (cerebellum), C6 (liver), ASB5 (muscle), PPP1R12B (heart) ), SLC5A12 (kidney), cholesterol-regulating APOM (liver), and ADPRHL 1 (heart), and the monogenic malformation syndrome TP73L (muscle). (Jacox E,et al.,(2010)PLoS One v.5(8):e12274).

[0283] Alternatively, the vector may be derived from a different species in which the endonuclease is unlikely to be expressed. For example, viral particles can be packaged in non-mammalian packaging. The well-known cytomegalovirus or SV40 virus early process is not active in immunizing cells. The promoters can be used to produce the gene in microbial, insect, or plant cells. In a preferred embodiment, the viral particles can be prepared as described in Gao et al. al. (2007) J. Biotechnol. 131(2):138-43 Produced in insect cells using the baculovirus system as described. Endonuclease under the control of the promoter can be expressed in these cells. Low possibility Airenne, KJ, et al. (2013) Mol. Ther. 21( 4):739-49). Furthermore, insect cells have different mRNA splicing sequences than mammalian cells. Therefore, the intron of human growth hormone (HGH) or SV40 Mammalian introns, such as the intron of the large T antigen, are inserted into the coding sequence of the endonuclease. These introns can be incorporated into pre-mRNA transcripts in insect cells. Since the endonuclease is not efficiently spliced ​​from the nuclease, insect cells cannot produce a functional endonuclease. In contrast, the resulting recombinant AAV particles do not express the full-length genome. Mammalian cells splice pre-mRNA properly and produce functional endonucleases. Haifeng Chen has demonstrated that the expression of ribosomal enzymes in insect packaging cells In order to reduce the expression of the toxic proteins barnase and diphtheria toxin fragment A in reported the use of HGH and SV40 large T antigen intron for this purpose, and these toxins It enables the production of recombinant AAV vectors carrying genes (Chen, H (2012) Mo l Ther Nucleic Acids.1(11):e57).

[0284] The endonuclease gene requires a small molecule inducer for endonuclease expression. It can be operably linked to an inducible promoter as needed. Examples of inducible promoters include The Tet-On system (Clontech; Chen H., et al., (20 15)BMC Biotechnol.15(1):4)) and RheoSwitch System (Intrexon; Sowa G., et al., (2011) Spine , 36(10):E623-8). Both systems and those known in the art Similar systems use small molecule activators (doxycycline or ecdysone, respectively). Ligand-inducible transcription factors (Tet repressor and Tet receptor, respectively) activate transcription in response to These ligand-inducible transcriptional activators are dependent on the ecdysone receptor (and mutants of the ecdysone receptor). Practicing the invention using a gene encoding a transcription factor involves: 1) placing the gene under the control of a promoter that responds to the corresponding transcription factor; The endonuclease gene is located, and the endonuclease gene binds to the transcription factor. and 2) the packaged viral genome contains a gene encoding a transcription factor. If a transcriptional activator is not also provided to the same cell, The latter is because the nuclease is not expressed in target cells or tissues after recombinant AAV delivery. The transcriptional activator is then treated with its cognate small molecule activator. This approach induces the expression of the endonuclease gene only in the cells or tissues that contain the endonuclease. by choosing when and to which tissues to deliver small molecule inducers. , to allow the expression of the endonuclease gene to be regulated in a spatiotemporal manner, However, the need to include an inducer in the viral genome significantly reduces delivery capacity. This is quite limiting and creates drawbacks to this approach.

[0285] In another preferred embodiment, a transcriptional repressor that prevents expression of the endonuclease is expressed. Recombinant AAV particles are produced in mammalian cell lines that express the transcriptional repressor. Known in the art, Tet repressor, Lac repressor, Cro repressor, and Many nuclear hormone receptors, such as the ecdysone receptor, also In the absence of their cognate hormone ligands, they also act as transcriptional repressors. To perform this procedure, packaging cells are transfected with a vector encoding a transcriptional repressor. Transfection / transduction of the endonuclease gene of the viral genome (packaging vector) operably linked to a promoter modified to contain a binding site for a repressor The repressor silences the promoter. The encoding gene can be placed in various locations. It can be encoded by a separate vector. It can be incorporated into the packaging vector outside the ITR sequences. It can be incorporated into a cap / rep vector or an adenovirus helper vector. Alternatively, and most preferably, it can be packaged so that it is constitutively expressed. It can be stably integrated into the genome of the host cell. Methods for modifying common mammalian promoters for this purpose are known in the art. For example, Chang and Roninson described strong, constitutive CMV and RSV promoters. The gene was modified to include an operator for the Lac repressor, and the gene was expressed by the modified promoter. showed that gene expression was greatly attenuated in cells expressing the repressor (Chang BD, and Roninson IB (1996) Gene183:137-42). By using a non-human transcriptional repressor, transcription of the endonuclease gene is suppressed by the repressor. The resulting recombinant AAV vector was suppressed only in packaging cells that express the . This ensures that the gene is not suppressed in target cells or tissues transduced with the target gene.

[0286] 2.6 Genetically engineered meganuclease variants

[0287] Embodiments of the present invention include the engineered meganucleases and variants thereof described herein. Further embodiments of the present invention include those comprising the endonucleases described herein. and isolated polynucleotides comprising nucleic acid sequences encoding such polynucleotides. Includes variants.

[0288] As used herein, "variant" is intended to mean a substantially similar sequence. A "variant" polypeptide is one that has one or more internal modifications of the native protein. deletions or additions of one or more amino acids, and / or at one or more sites in the native polypeptide A polypeptide derived from a "native" polypeptide by substitution of one or more amino acids As used herein, "natural" polynucleotides are intended to mean The peptide or polypeptide includes the parent sequence from which the variant is derived. The polypeptides are biologically active, i.e., they do not affect the desired function of the native protein. It retains biological activity against at least two genotypes of hepatitis B virus. Recognition sequences within the genome ORF, such as the HBV1-2 recognition sequence (SEQ ID NO: 10), HBV5 -6 recognition sequence (SEQ ID NO: 12), HBV7-8 recognition sequence (SEQ ID NO: 14), or HBV1 It still retains the ability to recognize and cleave the 1-12 recognition sequence (SEQ ID NO: 16). Such variants may result, for example, from human manipulation. or a biologically active variant thereof (e.g., SEQ ID NOS: 18-39), or a recognized fragment thereof as described herein. Biologically active variants of the binding subunits of the nucleotide site are described elsewhere herein. Naturally occurring sequences, as determined by the sequence alignment program and parameters used at least about 40% to the amino acid sequence of the polypeptide or naturally occurring subunit of Approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, Approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, The polypeptides of the embodiments have about 97%, about 98%, or about 99% sequence identity. A biologically active variant of a subunit is a variant of the polypeptide or subunit that is about 1 to 2 times as strong as the polypeptide or subunit. Approximately 40 amino acid residues, approximately 1 to 20, approximately 1 to 10, approximately 5, 4, 3, 2, or can differ by as little as one amino acid residue.

[0289] Polypeptides of the embodiments may be modified in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be generated by DNA mutations. Methods for mutagenesis and modification of polynucleotides are well known in the art. For example, Kunkel (1985) Proc. Natl. Acad. Sci. U SA82:488-492;Kunkel et al.(1987)Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; W alker and Gaastra, eds. (1983) Techniques i n 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 protein of interest. The needles were prepared according to the method of Dayhoff et al. (1978), which is incorporated herein by reference. )Atlas of Protein Sequence and Structure (Natl.Biomed.Res.Found.,Washington,DC) This can be seen in the model of replacing one amino acid with another with similar properties. Conservative substitutions such as:

[0290] In some embodiments, the engineered meganucleases of the present invention are The parent HVR regions may include variants of the disclosed HVR1 and HVR2 regions. , residues 24-79 or residues 215-270 of exemplary engineered meganucleases Thus, mutant HVRs may be included in the engineered meganuclear sequences exemplified herein. A sequence that is at least 80% identical to the amino acid sequence corresponding to residues 24-79 or residues 215-270 of ATP , at least 85%, at least 90%, at least 95%, or more sequence identity and the variant HVR region can comprise an amino acid sequence having the following structure: The biological activity of the meganuclease (i.e., binding to and cleavage of the recognition sequence) is maintained. Furthermore, in some embodiments of the present invention, a mutated HVR1 region or a mutated HVR The two regions may contain residues that correspond to amino acid residues found at particular positions in the parent HVR. In this context, "corresponding to" means that the amino acid residues of the variant HVR are at the same corresponding positions. the same amino acid present in the parent HVR sequence at the same position (i.e., relative to the remaining amino acids in the parent sequence) residue (i.e., another identical residue). For example, if the parent HVR sequence has If the HVR contains a serine residue at residue 26, then the variant HVR "containing a residue corresponding to" residue 26 also contains a serine residue at residue 26 of the parent Contains serine at the position corresponding to position 26.

[0291] A significant number of amines for the DNA recognition domain of wild-type I-CreI meganuclease were identified. Modifications of the amino acid have been previously identified (e.g., U.S. Pat. No. 8,021,867), and These can be used alone or in combination to identify individual bases within the DNA recognition half-sequence. Genetically engineered meganucleases with altered specificity are generated and the resulting rationally designed The engineered meganuclease has a different half-site specificity than the wild-type enzyme. Table 5 shows the salts present at positions (-1 to -9) of each half site of the recognized half site. Meganuclease monomers or subunits engineered to enhance specificity based on the Provides potential substitutions that can be made in the unit.

[0292] [Table 5]

[0293] Entries in bold are wild-type contact residues and constitute the "modification" as used herein. An asterisk indicates that the residue is in contact with a base in the antisense strand.

[0294] In the case of polynucleotides, a "variant" is a variant of one or more sites within the native polynucleotide. Those skilled in the art will appreciate that the nucleic acid sequences of the present invention may be modified by the deletion and / or addition of one or more nucleotides. It is recognized that variants are constructed to maintain the open reading frame. With respect to polynucleotides, conservative variants are examples of polynucleotides of the embodiments due to the degeneracy of the genetic code. The variant polypeptides include those sequences that encode the amino acid sequence of one of the polypeptides. Nucleotides may include those generated, for example, by using site-directed mutagenesis. and the like, but still encoding the engineered meganucleases of the embodiments. In general, variants of the particular polynucleotides of the embodiments are also included. is a sequence alignment program and parameters described elsewhere herein. At least about 40%, about 45%, about 50%, or %, approx. 55%, approx. 60%, approx. 65%, approx. 70%, approx. 75%, approx. 80%, approx. 85%, approx. 90 %, approx. 91%, approx. 92%, approx. 93%, approx. 94%, approx. 95%, approx. 96%, approx. 97%, approx. 98 %, about 99%, or more sequence identity. A variant of (i.e., a reference polynucleotide) may also be coded by the variant polynucleotide. and the polypeptide encoded by the reference polynucleotide. This can be assessed by comparing the percent sequence identity between

[0295] Deletions, insertions, and substitutions in the sequences of the proteins encompassed herein do not affect the specificity of the polypeptide. However, the exact nature of the substitutions, deletions, or insertions is not expected to fundamentally alter the gene's characteristics. If it is difficult to predict the desired effect before implementation, a person skilled in the art may Preferentially recognizes recognition sequences within the ORFs of the genomes of at least two genotypes of the virus. The ability to cleave the polypeptide is evaluated by screening the polypeptide. Recognize that. [Example]

[0296] This invention is further illustrated by the following examples which should not be construed as limiting. Those skilled in the art will be able to identify, using only routine experimentation, the specific substances described herein. Numerous equivalents to the methods and procedures are recognized or can be identified. are intended to be encompassed within the scope of the claims that follow the examples below.

[0297] Example 1: Characterization of meganucleases that recognize and cleave HBV recognition sequences

[0298] Meganuclease that recognizes and cleaves the HBV1-2 recognition sequence

[0299] The genetically engineered HBV1-2 meganucleases are collectively referred to herein as "HBV1-2 meganucleases." The designed meganucleases (SEQ ID NOs: 18 to 21) contain the HBV1-2 recognition sequence (SEQ ID NO: 1 0) and genotypes A, B, C, E, F, and G ( For example, P proteins of multiple HBV genotypes containing SEQ ID NOs: 3 to 5 and 7 to 9, respectively Each of these sequences is located within the S, S, preS2 / S, and preS1 / preS2 ORFs. HBV1-2 engineered meganuclease is an N-terminal nuclease derived from SV40 A localization signal, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit of each HBV1-2 meganuclease The first subunit binds to the recognition half site of HBV1 of SEQ ID NO: 10, while the second subunit binds to the recognition half-site of HBV2 (see Figure 2).

[0300] The HBV1 binding subunit and the HBV2 binding subunit are HVR1 and HVR2, respectively. Each HBV-binding subunit contains a 56-base pair hypervariable region called VR2. The HVR1 region is highly conserved. Similarly, the HBV2 binding subunit is also highly conserved. The HBV1 binding regions of SEQ ID NOs: 18 to 21 are highly conserved outside the VR2 region. Each of SEQ ID NOs: 40 to 43 is provided as a meganuclease. SEQ ID NO: 40, which is the HBV1 binding domain of HBV1-2x.2 (SEQ ID NO: 18) The HBV2 binding regions of SEQ ID NOs: 18 to 21 share at least 90% sequence identity. They are provided as SEQ ID NOs: 44 to 47, respectively. Each of SEQ ID NOs: 44 to 47 is SEQ ID NO: 18 is the HBV2 binding region of meganuclease HBV1-2x.2 It shares at least 90% sequence identity with no. 44.

[0301] Meganuclease that recognizes and cleaves the HBV 5-6 recognition sequence

[0302] The genetically engineered HBV5-6 meganucleases are collectively referred to herein as "HBV5-6 meganucleases." The designed meganucleases (SEQ ID NOs: 22 to 28) contain the HBV5-6 recognition sequence (SEQ ID NO: 1 2) and genotypes A, B, C, D, E, and G ( For example, P proteins of multiple HBV genotypes containing SEQ ID NOs: 3 to 7 and 9, respectively; Each HB is located within the S, preS2 / S, and preS1 / preS2 ORFs. The V5-6 engineered meganuclease utilizes the SV40-derived N-terminal nuclease localization The first meganuclease subunit, the linker sequence, and the second meganuclease subunit are The first subunit of each HBV5-6 meganuclease is , binds to the HBV5 recognition half site of SEQ ID NO: 12, while the second subunit It binds to the half-site recognized by HBV6 (see Figure 2).

[0303] The HBV5 binding subunit and the HBV6 binding subunit are HVR1 and HVR2, respectively. Each HBV-binding subunit contains a 56-base pair hypervariable region called VR2. The HVR1 region is highly conserved. Similarly, the HBV6 binding subunit is also highly conserved. The HBV5 binding regions of SEQ ID NOs: 22 to 28 are highly conserved outside the VR2 region. Each of SEQ ID NOs: 48 to 54 is provided as a meganuclease. SEQ ID NO: 49, which is the HBV5 binding region of HBV5-6×.33 (SEQ ID NO: 22) The HBV6 binding regions of SEQ ID NOs: 22 to 28 share at least 90% sequence identity with , which are provided as SEQ ID NOs: 55 to 61, respectively. The sequence of the HBV5 binding region of meganuclease HBV5-6x.33 (SEQ ID NO: 22) It shares at least 90% sequence identity with sequence no. 55.

[0304] Meganuclease that recognizes and cleaves the HBV 7-8 recognition sequence

[0305] The genetically engineered HBV7-8 meganucleases are collectively referred to herein as "HBV7-8 meganucleases." The designed meganucleases (SEQ ID NOs: 29 to 32) contain the HBV7-8 recognition sequence (SEQ ID NO: 1 4) and genotypes A, B, C, D, F, and G ( For example, P proteins of multiple HBV genotypes containing SEQ ID NOs: 3 to 6, 8, and 9, respectively Each HBV7-8 engineered meganuclease is located within the protein ORF. N-terminal nuclease localization signal from SV40, the first meganuclease subunit Each HBV7-8 meganuclease contains a linker sequence, a second meganuclease subunit, and a linker sequence. The first subunit of the ganucleases recognizes the HBV7 half site of SEQ ID NO: 14. The first subunit binds to the HBV8 recognition half-site, while the second subunit binds to the HBV8 recognition half-site (Figure 2 reference).

[0306] The HBV7 binding subunit and the HBV8 binding subunit are HVR1 and HVR2, respectively. Each HBV-binding subunit contains a 56-base pair hypervariable region called VR2. The HVR1 region is highly conserved. Similarly, the HBV8 binding subunit is also highly conserved. The HBV7 binding regions of SEQ ID NOs: 29 to 32 are highly conserved outside the VR2 region. Each of SEQ ID NOs: 62 to 65 is provided as a meganuclease. SEQ ID NO: 62, which is the HBV7 binding region of HBV7-8x.2 (SEQ ID NO: 29); The HBV8 binding regions of SEQ ID NOs: 29 to 32 share at least 90% sequence identity. They are provided as SEQ ID NOs: 66 to 69, respectively. Each of SEQ ID NOs: 66 to 69 is SEQ ID NO: 29, which is the HBV8 binding region of meganuclease HBV7-8x.2 No. 66 shares at least 90% sequence identity.

[0307] Meganuclease that recognizes and cleaves the HBV 11-12 recognition sequence

[0308] Genes collectively referred to herein as "HBV11-12 meganuclease" The engineered meganucleases (SEQ ID NOs: 33-39) contain the HBV 11-12 recognition sequence ( Genetically engineered to recognize and cleave SEQ ID NO: 16, and genotypes A, B, C, D, and E P proteins of multiple HBV genotypes, including , F, and G (e.g., SEQ ID NOS: 3-9, respectively). Each HBV11-12 engineered meganuclear The enzyme contains an N-terminal nuclease localization signal derived from SV40, the first meganuclease subunit Each HBV meganuclease comprises a second subunit, a linker sequence, and a second meganuclease subunit. The first subunit of the 11-12 meganuclease recognizes HBV11 of SEQ ID NO: 16. The second subunit binds to the half-site recognized by HBV12. (See Figure 2.)

[0309] The HBV11 binding subunit and the HBV12 binding subunit are HVR1 and HVR2, respectively. It contains 56 base pair hypervariable regions called HVR1 and HVR2. The nit is highly conserved outside the HVR1 region. Similarly, the HBV12 binding subunit are also highly conserved outside the HVR2 region. The regions are provided as SEQ ID NOs: 70 to 76, respectively. represents the HBV11 binding region of meganuclease HBV11-12x.26 (SEQ ID NO: 33). SEQ ID NO: 33-39 share at least 90% sequence identity with SEQ ID NO: 70. The HBV12 binding regions are provided as SEQ ID NOs: 77 to 83, respectively. Each of 7 to 83 is a sequence of meganuclease HBV11-12x.26 (SEQ ID NO: 33). It shares at least 90% sequence identity with the HBV12 binding region, SEQ ID NO:77.

[0310] Cleavage of the HBV recognition sequence in a CHO cell reporter assay

[0311] HBV1-2, HBV5-6, HBV7-8, and HBV11-12 meganucleases recognize their respective recognition sequences (SEQ ID NOs: 10, 12, 14, and 16, respectively). Each engineered meganuclear molecule was then used to determine whether it could be cleaved. The enzyme activity was evaluated using a previously described CHO cell reporter assay (International Publication No. (See Patent Publication No. 2012 / 167192, Figure 6). , a non-functional green fluorescent protein (GFP) gene expression cassette integrated into the genome of the cell. We generated a CHO cell reporter strain carrying the nucleotide sequence. Intracellular cleavage of the recognition sequence stimulates a homologous recombination event to generate a functional GFP gene. The GFP gene of each cell line was interrupted by a pair of recognition sequences.

[0312] In the CHO reporter cell line developed for this study, The recognition sequence selected is the HBV1-2 recognition sequence (SEQ ID NO: 10), the HBV5-6 recognition sequence (SEQ ID NO: 11), and the HBV6 recognition sequence (SEQ ID NO: 12). (SEQ ID NO: 12), HBV7-8 recognition sequence (SEQ ID NO: 12), and HBV11-12 recognition sequence The second recognition sequence inserted into the GFP gene was CHO- The 23 / 24 recognition sequence was a control meganucleic acid sequence designated "CHO-23 / 24." The HBV1-2 recognition sequence and the CHO-2 recognition sequence are recognized and cleaved by the HBV1-2 recognition sequence and the CHO-2 recognition sequence. CHO reporter cells containing the 3 / 24 recognition sequence are referred to as "HBV1-2 cells." CHO reporter cells containing the BV5-6 recognition sequence and the CHO-23 / 24 recognition sequence were HBV5-6 cells. HBV7-8 recognition sequence and CHO-23 / 24 recognition sequence. CHO reporter cells containing HBV are called "HBV7-8 cells." CHO reporter cells containing the HBV11- and CHO-23 / 24-recognition sequences were used as the "HBV11- It is called "12 cells."

[0313] CHO reporter cells were cultured with their corresponding engineered meganucleases. Plasmid DNA (e.g., HBV1-2 cells) containing HBV1-2 meganuclease (transfected with plasmid DNA encoding the ) or CHO-23 / 34 meganuclear The cells were transfected with plasmid DNA encoding the enzyme. 5 C HO reporter cells were transfected with Lipofectamine 2000 ( ThermoFisher) to measure 50 ng of plasmid DNA in a 96-well plate. 48 hours after transfection, cells were analyzed by flow cytometry. The number of GFP-positive cells was assessed and compared to a non-transfected negative control (HBV bs). As shown in Figures 7A to 7D, all HBV meganucleases in cell lines containing their corresponding recognition sequences at frequencies significantly exceeding those of the negative control. It was found to produce GFP-positive cells.

[0314] In addition, HBV1-2, HBV5-6, HBV7-8, and HBV11-12 meganucleases The efficacy of the meganucleases was assessed after transfection of the meganucleases into CHO reporter cells. In this study, the Bi- oRad Gene Pulser Xcell is used to detect HBV1-2, HBV5- 6, HBV7-8, or HB11-12 cells (1.0 × 10 6 ) at 1 × 10 per cell 6 10 copies of meganuclease mRNA were electroporated. At this time point, cells were assessed by flow cytometry to determine the percentage of GFP-positive cells. CHO-23 / 24 meganuclease was also included as a positive control at each time point.

[0315] As shown in Figures 8A-8D, the %G produced by different HBV meganucleases FP changed over time. HBV5-6 and HBV11-12 meganucleases were The results were essentially consistent over the 12 days of analysis (Fig. 8B and Fig. 8D), whereas the HBV 1-2 and HBV7-8 meganucleases showed high activity at early time points that decreased over the course of the experiment. The resulting cells were GFP-positive at a high level.

[0316] conclusion

[0317] These studies demonstrated that the HBV meganuclease encompassed by the present invention Can the enzyme efficiently target and cleave the recognition sequence of the target protein, and can the effect be consistent over time? , which showed that it may be transient.

[0318] Example 2: HBV-specific nuclease removes plasmid DNA from E. coli

[0319] Bacterial reporter systems using episomal DNA

[0320] The purpose of this experiment was to demonstrate the HBV meganuclease of the present invention in an E. coli reporter system. The ability of the gene to recognize and cleave the recognition sequence in the episomal DNA plasmid is evaluated. That was the case.

[0321] A plasmid called "pARCUS" was used to drive inducible expression of the ARCUS nuclease. pARCUS was engineered to activate the ARCUS nuclease (herein referred to as pARCUS) (Fig. 9A). HBV5-6×, HBV33 or HBV11-12×) was used as an IPTG-inducible promoter. Furthermore, to allow for the selection of transformed bacteria, encodes the ampicillin resistance gene. pARCUS is a low-copy plasmid. Therefore, induced expression of ARCUS nuclease does not result in overexpression.

[0322] An additional plasmid called "pHBVa" was generated (Figure 9A). Contains a large fragment of the HBV genome, including the recognition sequences for HBV5-6 and HBV11-12 The plasmid also encodes the kanamycin resistance gene and the SacB gene. The kanamycin resistance gene allows for the selection of transformed bacteria. The SacB gene is a toxin that is active in the presence of sucrose. The transformed bacteria survive in the presence of kanamycin in the medium, but do not survive in the presence of kanamycin and sucrose. pHBVa does not survive in the presence of cloning agents. pHBVa may contain many copies of the HBV genome. In an attempt to replicate in HBV-infected cells, a high-copy plasmid was designed Importantly, pHBVa utilizes a different origin of replication than pARCUS, This allows for simultaneous transformation of bacteria with both plasmids.

[0323] Bacteria were simultaneously transformed with these plasmids and subjected to various combinations of selective pressure. When grown in media containing (ampicillin, kanamycin, or sucrose), There are several possible outcomes. Bacteria grown in IPT will carry the pARCUS plasmid, but the growth medium will not Without G supplementation, the encoded nuclease is not expressed. Upon induction with IPTG, The encoded nuclease is expressed. Bacteria transformed with pHBVa express kanamycin. It grows in the presence of ampicillin, but not in the presence of kanamycin and sucrose. pARCUS and pHBVa were co-transformed in the presence of erythromycin and / or kanamycin. The bacteria were sensitive to sucrose. In the transformed bacteria, induction of ARCUS nuclease with IPTG resulted in the production of nuclease. This results in the expression of a target enzyme that can then cleave the target site encoded in pHBVa. It was predicted that cleavage at this site would result in linearization of the pHBVa plasmid. However, it should be rapidly degraded by bacterial nucleases. The degradation of sucrose causes the bacteria to lose their resistance to kanamycin, while the degradation of sucrose This would have two consequences: the ability to survive in its presence;

[0324] To verify the results of simultaneous transformation, bacteria were transfected with pARCUS and pHBVa (elements co-transformed (by microporation) and in the presence of ampicillin for 3 hours before plating. In parallel cultures, the simultaneously transformed cells were treated with IPTG (3 h The cells were then incubated for 1 hour at 4°C for 1 hour, at which time the expression of the nuclease was induced, allowing cleavage of pHBVa. Presence of ampicillin, ampicillin and sucrose, or ampicillin and kanamycin The plates were incubated overnight and the colonies were counted to identify the bacteria. Survival was assessed.

[0325] result

[0326] The number of colonies present on each selection plate indicates whether ARCUS nuclease is a factor in the pHBVa This gave dramatic evidence that the plasmid could be cut. , colony counts from either uninduced or IPTG-induced cultures were measured using pHBVa and The results were comparable for cells cotransformed with either pARCUS plasmid. (Fig. 9B). Bacteria cotransformed with pARCUS encoding HBV5-6x.33. and bacteria cotransformed with pARCUS encoding HBV11-12×.26. There was a difference in the number of colonies between the two, which is thought to reflect the efficiency of transformation.

[0327] In sharp contrast, non-IPTG induction on plates containing both ampicillin and sucrose The number of colonies from the cultures was dramatically reduced, indicating that sucrose was available. This indicates that the SacB gene is effective in cell killing in the case of I (Fig. 9B). PTG-induced cultures grew non-specifically on plates containing both ampicillin and sucrose. The growth was consistently good, indicating that the SacB gene had been removed. Co-transfected with pARCUS encoding BVa and HBV11-12x.26 Colony counts of bacteria induced with IPTG were compared with those of ampicillin control plates. The bacteria transformed with pARCUS5-6x.33 were the same as those in the control group. There was only a slight decrease compared to the control plate.

[0328] Non-IPTG-induced cultures were grown on plates containing both ampicillin and kanamycin. pARCUS encoding HBV 11-12x.26 was able to propagate (Fig. 9B). The number of colonies from bacteria simultaneously transformed with ampicillin was almost the same as that on the control ampicillin-only plate. Almost equally, cells co-transfected with pARCUS HBV5-6×.33 showed a significant increase compared to the control. However, the concentration of the ampicillin- and kanamycin-containing protease inhibitors was only slightly reduced compared to the control. When IPTG-induced cells were grown at this rate, the number of colonies was close to zero, which is This indicated the elimination of the plasmid carrying the synth resistance gene (Fig. 9B ).

[0329] conclusion

[0330] These data indicate that the HBV meganuclease of the present invention, which recognizes a site in the HBV genome, The pHBVa plasmid can be cleaved, resulting in the elimination of the pHBVa plasmid. In IPTG-induced cultures, cells grow in the presence of sucrose. The SacB-containing plasmid was efficiently removed. In IPTG-induced cultures, cells die in the presence of kanamycin, which also This strongly suggests that the pHBVa plasmid was disrupted. In HBV-infected mammalian cells, the HBV meganuclease of the present invention can be used to It is possible that similar results could occur in bacteria. In bacteria, linear DNA is quickly eliminated. Linearized cccDNA in mammalian cells is also thought to be digested by cellular nucleases. Even if the cleavage does not lead to linearization and elimination of cccDNA, the HBV ARC Indel mutations caused by US nuclease disrupt the coding region likely renders cccDNA unable to produce functional HBV proteins. Therefore, the meganuclease of the present invention that targets a site in the HBV genome can inhibit HBV cc It should be an effective method for eliminating or inactivating cDNA.

[0331] Example 3: Targeting the HBV viral genome in cells

[0332] Treatment of AD38 cells expressing the HBV genome

[0333] The primary purpose of these examples is to demonstrate that the meganucleases of the present invention can inhibit the proliferation of HBV-infected mammalian cells. The aim of this study was to evaluate whether the HBV genome could be inactivated and / or eliminated. Ta.

[0334] In the first study, the efficacy of meganucleases was demonstrated by transfecting the HBV genome under the Tet promoter. The virus was evaluated in a stably expressing AD38 cell line. does not produce HBV particles but produces HBV S antigen (HBsAg) that is detectable in cell culture media In this study, cells were transfected with HBV5-6×.33 or HBV11-12×.26. These engineered meganucleases were transfected with DNA plasmids that encode the enzymes On the other hand, overlapping S (HBsAg gene) and P (polymerase gene) reading frames HBV5-6×.33, which targets sequences within the rheme gene, and HBV1, which targets the P gene 1-12×.26 was used to target sequences specific to the HBV genome. 38 cells were transfected with a plasmid encoding the red fluorescent protein (RFP) gene. Cells were seeded and transfected 24 hours later using a liposome-based transfection protocol. 24 hours after transfection, AD38 cells were washed to remove any remaining liposomes. On days 3 and 7 post-transfection, cell supernatants were collected and analyzed by ELI. The presence of HBsAg was assayed by SA.

[0335] result

[0336] ELISA data were collected from RFP-transfected AD38 cells at 3 and 7 days post-transfection. The amount of HBsAg present in the cell supernatant was normalized. Cells transfected with a plasmid encoding 1-12x.26 were transfected with RFP-transfected cells. showed approximately 25% less HBsAg in the supernatant compared with HBV (Fig. 10). Cells transfected with a plasmid encoding 5-6×.33 were significantly more efficient than RFP-transfected cells. The supernatants showed approximately 50% less HBsAg compared to the non-transfected control cells (Figure 10). The cells were essentially the same as those transfected with RFP. The decrease in HBsAg was observed 7 days after transfection. This was even more evident on day 1. AD38 cells transfected with HBV11-12x.26 showed a R showed approximately 50% lower HBsAg levels than cells transfected with FP, and HBV5-6x Cells transfected with .33 showed levels approximately 75% lower than the RFP control (Figure 10). At 7 days post-transfection, cells transfected with RFP had significantly higher levels of H in the supernatant compared to non-transfected cells. There was significantly less BsAg, indicating that the transfection process had a negative effect on the cells' ability to produce HBsAg. Nevertheless, the effect was not observed in the HBV meganuclear The expression of α-glucan in the supernatant was much more pronounced in cells transfected with a plasmid encoding the enzyme. This strongly suggests that the decrease in HBsAg levels in the HBsAg cells was due to the activity of meganucleases. are.

[0337] conclusion

[0338] These data are based on either HBV5-6×.33 or HBV11-12×.26. AD38 cells transfected with a plasmid encoding the RFP receptor were transfected with untransfected cells or The supernatant HBsAg was dramatically reduced compared to cells transfected with the HBsAg gene. The results show that the H of the supernatant in cells transfected with either HBV meganuclease The decrease in BsAg levels was due to the fact that the decrease was related to the meganuclear activity of the HBV genome in AD38 cells. This strongly suggests that this is due to enzyme activity.

[0339] Example 4: Targeting the HBV viral genome in cells

[0340] Treatment of AD38 cells expressing the HBV genome

[0341] The study in Example 3 above investigated the secretion of HBsAg from the HBV genome-expressing AD38 cell line. This study demonstrates the effectiveness of meganucleases in reducing HBsAg secretion. Even greater reductions were achieved using lentiviral delivery instead of plasmid transfection To determine whether HBV11-12x.26 and HBV5-6x.3 This study focused on lentiviral delivery of three meganucleases, either individually or in combination. Repeated this.

[0342] Receptors expressing either RFP, HBV5-6×.33, or HBV11-12×.26 Antivirus was produced and transduced into AD38 cells to induce the megagene for the production of HBsAg. The effect of cleavage on the AD38 cells was assessed. After 24 hours, RFP, HBV5 -6×.33, lentivirus encoding HBV11-12×.26, or HBV megavirus The mice were transduced with either a 1:1 mixture of lentiviruses encoding nucleases. Cells were transduced with a single lentivirus at an MOI of 1, 2, or 4. Cells transduced with a 1:1 mixture of lentiviruses encoding cleavage enzymes were The cells were transduced at a total MOI of 4. The medium of the transduced cells was collected on days 1 and 3 post-transduction. Seven days after transduction, the cell supernatant was collected and the presence of HBsAg was assayed by ELISA. The presence of β-glucan was assayed.

[0343] result

[0344] ELISA data were obtained from supernatants of RFP-transduced AD38 cells 7 days after transfection. Normalized to the amount of HBsAg present. At an MOI of 1, HBV5-6 × 0.33 AD38 cells transduced with RFP-expressing lentivirus were transduced with RFP-expressing lentivirus. Approximately 60% less HBsAg was found in the supernatant than in the cells (Fig. 11). In this case, HBV11-12x.26 showed approximately 40% less HBsAg than RFP. At higher MOIs, the effect of HBV meganuclease was more pronounced. , cells transduced with lentivirus encoding HBV5-6×.33 expressed RFP. At an MOI of 4, the cells showed a reduction of HBsAg by approximately 80% compared to transduced cells. The reduction was approximately 90% in comparison. Similarly, at an MOI of 2, HBV11-12 × . Cells transduced with lentivirus encoding 26 were significantly higher than cells transduced with RFP. At an MOI of 4, the HBsAg levels were approximately 70% lower than those at 100%. Finally, a 1:1 mixture of lentiviruses encoding HBV meganuclease was used. Cells transduced with the product had significantly higher levels of HBsAg in the supernatant compared to RFP-transduced control cells. At a total MOI of 2, the HBV expression level was dramatically reduced (Fig. 11). The antivirus reduced HBsAg expression by approximately 80%, and at a total MOI of 4, expression was reduced by approximately It decreased by 90%.

[0345] conclusion

[0346] These data suggest that HBV expression is significantly increased in comparison to cells transduced with RFP-expressing lentivirus. Coded V 5-6 x .33 or HBV11-12 x .26, or a combination of both AD38 cells transduced with lentiviruses containing HBsAg showed a dramatic decrease in supernatant HBsAg levels. This demonstrates that lentiviruses expressing either or both of the HBV meganucleases The reduction in the supernatant HBsAg levels in cells transduced with HBsAg indicated that the reduction was associated with AD. This strongly suggests that the mutations are due to the activity of meganucleases against the HBV genome in 38 cells. Furthermore, these data suggest that co-expression of HBV meganuclease inhibits HBsAg expression. demonstrated that the co-expressed meganuclease effectively reduced HBV genome expression. This strongly suggests that the nom can be effectively used.

[0347] Example 5: Targeting the HBV viral genome in cells

[0348] Treatment of AD38 cells expressing the HBV genome

[0349] In further studies, the effect of meganuclease treatment on the secretion of HBsAg, cells HBV DNA copies present in the culture medium and HBV cccDNA copies in the cells To observe this, AD38 cells were transfected with HBV5-6 × 0.33 or HBV11-12 × 0.26. They were transduced with a lentivirus encoding the meganuclease.

[0350] As in Example 4 above, RFP (LV212), HBV5-6×.33 (LV224 ), or lentivirus expressing HBV11-12×.26 (LV225) We generated the nucleases and transduced them into AD38 cells to evaluate the effect of the meganucleases on the results of each experiment. AD38 cells were seeded in the presence of tetracycline and, after 24 hours, 4 MOI was administered. I, lentivirus encoding RFP, HBV5-6 x 0.33, or HBV11-12 x 0.26 The medium of the transduced cells was changed 24 hours after transduction. On day 7 after inoculation, cell supernatants were collected and assayed for the presence of HBsAg by ELISA. The number of extracellular HBV DNA copies per 5 μL of cell culture medium was determined by quantitative PCR. Cell lysates were obtained and the HBV cccDNA concentration was determined per 5 μL of cell lysate. The copies in were determined by quantitative PCR.

[0351] result

[0352] Transduced with HBV5-6 × 0.33 and HBV11-12 × 0.26 at an MOI of 4 AD38 cells were transduced with RFP-expressing lentivirus 7 days after transduction. Approximately 58% and 25% less HBsA in the cell culture medium than in AD38 cells, respectively. The copy number of extracellular HBV DNA was also significantly increased in the RFP-expressing lentiviruses (Fig. 12A). Compared with viral transduction, HBV5-6×.33 and HBV11-12× Transduction with .26 meganuclease reduced the levels by approximately 28% and 50%, respectively. (Fig. 12B). Finally, the copy number of HBV cccDNA in cells also correlates with RFP expression. Compared with lentiviral transduction, HBV5-6×.33 and HBV11- Transduction with 12×.26 meganuclease reduced the number of HIV-1 markers by approximately 71% and 60%, respectively. (Figure 12C).

[0353] conclusion

[0354] These data further demonstrate that HBV5-6x.33 or HBV11-12x.26 meganucleases AD38 cells transduced with lentiviruses encoding either RFP or ATPases showed a dramatic reduction in HBsAg in the supernatant compared to cells transduced with lentivirus expressing Furthermore, these data demonstrate that the HBV meganuclease of the present invention It reduces the extracellular HBV DNA copy number and, importantly, HBV cccDNA It has been demonstrated that the intracellular copy number of

[0355] Example 6: Treatment of HBV-infected primary human hepatocytes

[0356] Treatment of HBV-infected primary human hepatocytes

[0357] The study in the above example showed that HBsAg secretion from HBV genome-expressing AD38 cell lines was reduced. The study in this example demonstrates the effectiveness of meganucleases in reducing HBV To determine the efficacy of the HBV meganuclease of the present invention in infected primary human hepatocytes It was held.

[0358] Lentiviruses expressing RFP, HBV5-6×.33, or HBV11-12×.26 We generated a HBV-infected primary human hepatocyte transduction system and investigated the production of HBsAg and HBeAg. The effect of meganucleases on cell viability was determined. Briefly, primary human hepatocytes were seeded and incubated for 2 h. After 4 hours, the cells were infected with HBV. On day 1 post-infection, the cells were washed and 24 hours later (day 2 post-infection), the cells were (eye) lentiviruses encoding RFP, HBV5-6×.33, and HBV11-12×.26 virus, or a 1:1 mixture of lentivirus encoding HBV meganuclease As a further control, infected cells were treated with DMSO. Cell supernatant was collected and the medium was replaced on days 4, 8, 11, and 13 after transduction. At each time point, HBsAg and HBeAg were measured in the cell supernatant by ELISA. In addition, extracellular DNA in the supernatant was measured 13 days after infection.

[0359] result

[0360] In general, lentiviral transduction does not affect the secretion of either HBsAg or HBeAg. To determine whether this effect is due to the presence of RFP, the mice were transduced with a lentivirus encoding RFP. The supernatants obtained from cells were compared with those from cells treated with DMSO (Figure 13). Transduction of HBV-infected primary human hepatocytes with lentivirus encoding HBsAg or had little, if any, effect on HBeAg secretion (Fig. 13A and Fig. 13 B) At an MOI of 5 or 2.5, HBsAg levels were identical to those in DMSO-treated cells. At an MOI of 1.25, there was only a slight decrease (Fig. 13A). The levels of HBeAg in the supernatants of antiviral-transduced cells were significantly higher than those of DMSO-treated cells. When compared, there is only a slight decrease at MOIs of 1.25, 2.5, or 5 ( Furthermore, the amount of extracellular HBV detected in the supernatant of HBV-infected primary human hepatocytes was The results are from DMSO-treated cells and cells transduced with lentivirus encoding RFP. There was little difference between the cells (Fig. 13C).

[0361] Lentiviral transduction generally does not affect HBV function in infected primary human hepatocytes. We demonstrated that lentivirus encoding HBV meganuclease has no adverse effects on the immune system. HBsAg ELISA data were compared with RFP lentivirus. RFP-transduced HBV-infected primary human hepatocytes on days 8, 11, and 13 Normalized to the amount of HBsAg present in the supernatant (Figure 14). In both OIs, cells transduced with lentivirus encoding HBV5-6×.33 , showing a steady decrease in HBsAg over the course of the experiment, when compared to the RFP control. Levels decreased slightly on day 3, decreased by approximately 50-60% by day 8, and decreased by days 11 and 12. By day 13, the HBV11-12x antibody showed a reduction of approximately 90% (Figures 14A and 14B). Infected cells transduced at an MOI of 1.26 showed similar patterns at both MOIs of 2.5 and 1.25. At day 3, there was a more significant decrease (about 50%), but still transduced Approximately 90% reduction was achieved by days 11 and 13 after inoculation (Figures 14A and 14B). Infected cells were transduced with a 1:1 mixture of lentiviruses encoding HBV meganucleases. A similar decrease in HBsAg was observed over time when the cells were transfected with HBsAg. The level decreased by approximately 50% on day 3 after transduction, and the decrease continued until day 11 and day 1 after transduction. By day 3, this reached 90% (Figures 14A and 14B).

[0362] In addition, HBeAg ELISA data were collected on days 3, 8, 11, and 24 post-transduction. and on day 13, the HBeAg present in the supernatant of RFP-transduced HBV-infected primary human hepatocytes was The results were normalized to the amount of HBV5-6 at both MOIs of 2.5 and 1.25 (Figure 14). Cells transduced with lentivirus encoding ×.33 showed no HBV-mediated ... showed a steady decrease in eAg, reaching approximately 25% levels on day 3 when compared to RFP controls reduction, approximately 50% reduction by day 8, and approximately 90% reduction by days 11 and 13 HBV11-12x.26 transduced infected cells showed similar patterns at both MOIs of 2.5 and 1.25, with similar results at day 3. showed a decrease (approximately 30% decrease), yet still showed a decrease by 11 and 13 days post-transduction. Approximately 90% reduction of HBeAg was achieved (Fig. 14C and Fig. 14D). When transduced with a 1:1 mixture of meganuclease-encoding lentiviruses, HBV A similar decrease in eAg was observed over time. At MOIs of 2.5 and 1.25, levels were The number of cells decreased by approximately 50% on day 3 after transduction, and the decrease continued until days 11 and 13 after transduction. By the time the oxidative stress reached 90% (Fig. 14C and Fig. 14D).

[0363] conclusion

[0364] These data suggest that HBV expression is significantly increased in comparison to cells transduced with RFP-expressing lentivirus. V 5-6 x .33 or HBV 11-12 x .26, or a combination of the two HBV-infected primary human hepatocytes transduced with lentivirus encoding HBsA in the supernatant The results demonstrate that the HBsAg and HBeAg levels in the supernatants of infected cells are dramatically reduced. The decrease in the levels of HBeAg and HBeAg was due to the use of either or both of the HBV meganucleases. The cells were transduced with lentivirus expressing the α-glucanase (α-glucanase) and the reduction in the α-glucanase activity was confirmed by the meganuclease activity against infectious virus. This strongly suggests that this is due to the activity of lyase.

[0365] Example 7: Delivery of LNP-encapsulated nuclease mRNA to the liver

[0366] Encapsulation of mRNA in lipid nanoparticles and delivery to mice

[0367] The aim of this study was to develop lipid nanoparticles (LNPs) encoding genetically engineered meganucleases. NP)-encapsulated mRNA can be produced and administered in vivo, resulting in gene editing in the liver, The purpose is to demonstrate that it can be observed.

[0368] The mouse CMP-NeuAc hydrolase (Cmah) gene is expressed in mouse liver. We expressed a genetically engineered meganuclease with specificity for the recognition sequence of C ARCA-capped mRNA encoding the mah meganuclease was transduced into TriLink Bio oTechnologies LLC (San Diego, California) and are encapsulated in different commercially available LNP formulations, each containing various ratios of ionic to cationic lipids. PEG lipids and cholesterol. LNP-encapsulated mRNA was 1.0 mg / kg. The dose was administered to CD-1 mice by IV injection. Livers were harvested 6 days after administration. Before organ collection, animals were perfused with saline. Total liver genomic DNA (gDNA) was isolated and analyzed by C The frequency of insertion / deletion (indel) mutations in the mah gene was determined using T7 endonuclease The T7E assay was performed using the T7E enzyme I (T7E) assay and deep sequencing. In the first study, the region of the genome containing the Cmah recognition sequence was amplified by PCR, and the results were compared between wild-type and mutant strains. A heterogeneous mixture of mutant amplified alleles was obtained. The PCR products were denatured and slowly re-aliquoted. annealing to allow heteroduplex formation between the wild-type and mutant alleles Such heteroduplexes are susceptible to cleavage by T7 endonuclease I, which cleaves at mismatches. Visualization of T7E cleavage products on a gel revealed that T7E cleavage of heteroduplexes is highly susceptible to cleavage. Due to this, multiple bands are present in the sample, whereas the wild-type sample has a single band. The ratio of cleaved to uncleaved products is a measure of the level of nuclease cleavage activity. Provides degrees.

[0369] result

[0370] The results of this study are shown in Figure 15. As shown in lanes 1 and 2, luciferase was co-transfected with gDNA obtained from control mice administered LNP-encapsulated mRNA containing Cmah No evidence of cleavage at the recognition sequence was observed in luciferase mRNA-treated animals. The ratio of cleaved to uncleaved mRNA was comparable to that observed in animals not administered mRNA. In contrast, lanes 3 to 7 and 9 to 18 each contained multiple The animals were treated with Cmah meganuclease, as indicated by the presence of a band of The figure shows the alterations in the Cmah recognition sequence upon administration of LNP-encapsulated mRNA encoding Cm To confirm and quantify the proportion of genetic modifications in the ah recognition sequence, Deep sequencing was also performed on the gDNA. The resulting percentages are shown below each lane as %KO in Figure 15. Lanes 3 to 7 are , ranging from 3.41% to 21.49% alterations in replicate animals administered LNP formulation #1. Lanes 9-13 show a decrease from 18.4% to 2% in replicate animals administered LNP formulation #2. Lanes 14-18 show a range of alterations of 3.2%. The alterations range from 51.1% to 64.8%.

[0371] conclusion

[0372] This study demonstrates that the use of genetically engineered meganucleases such as the HBV-specific meganuclease of the present invention The mRNA encoding the cleavage enzyme is encapsulated in LNPs and then delivered to target hepatocytes in vivo (i.e. , hepatocytes) and induce gene editing at targeted recognition sequences in the genome. This clearly shows that:

Claims

1. The open reading frames of the genomes of at least two genotypes of hepatitis B virus It is a genetically engineered meganuclease that recognizes and cleaves a recognition sequence within the ORF. So, comprising a first subunit and a second subunit; The first subunit binds to a first recognition half site of the recognition sequence, and the first comprising a hypervariable (HVR1) region, The second subunit binds to a second recognition half site of the recognition sequence, A genetically engineered meganuclease comprising the hypervariable (HVR2) region of

2. The recognition sequence is a sequence of genotype A (SEQ ID NO: 3) and a sequence of genotype B (SEQ ID NO: 4), Genotype C (SEQ ID NO: 5), genotype D (SEQ ID NO: 6), genotype E (SEQ ID NO: 7), genotype 1, wherein the genotype is within one or more ORFs of genotype F (SEQ ID NO: 8), and genotype G (SEQ ID NO: 9).

2. A genetically engineered meganuclease according to claim 1.

3. The recognition sequence is located in the polymerase (P) protein, the major surface (preS1 / preS2 / S) proteins, middle surface (preS2 / S) proteins, and small surface (S) proteins A claim within at least one ORF encoding a protein selected from the group consisting of:

3. The genetically engineered meganuclease of claim 1 or 2.

4. The genetic manipulation of any one of claims 1 to 3, wherein the recognition sequence comprises SEQ ID NO:

12. Meganucleases.

5. the HVR1 region is selected from residues 215 to 270 of any one of SEQ ID NOs: 22 to 24, or At least one amino acid sequence corresponding to residues 24-79 of any one of SEQ ID NOs. 25-28 5. The genetically engineered polypeptide of claim 4, comprising an amino acid sequence having at least 80% sequence identity. Meganucleases.

6. the HVR1 region is selected from residues 215, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 2 ９、２２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６ Residues corresponding to 1, 266, and 268, or residues of any one of SEQ ID NOs: 25-28 ２４、２６、２８、３０、３２、３３、３８、４０、４２、４４、４６、６８、７０、７ 6. The genetically engineered meganucleic acid of claim 4 or 5, comprising residues corresponding to 5, 7, and 77. Rease.

7. the HVR1 region is selected from residues 215 to 270 of any one of SEQ ID NOs: 22 to 24, or Any one of claims 4 to 6, comprising residues 24 to 79 of any one of sequences 25 to 28 2. A genetically engineered meganuclease according to claim 1.

8. the HVR2 region is selected from residues 24 to 79 of any one of SEQ ID NOs: 22 to 24 or At least one amino acid sequence corresponding to residues 215-270 of any one of Nos. 25-28 8. The method according to claim 4, wherein the amino acid sequence has at least 80% sequence identity with the 1. The described engineered meganuclease.

9. the HVR2 region is selected from residues 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, Corresponding to 0, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 or residues 215, 217, 219, 221 of any one of SEQ ID NOs: 25-28 、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６１、２６６ 10. The genetically engineered protein of claim 4, comprising a residue corresponding to 268, Meganucleases.

10. the HVR2 region comprises residues 24 to 79 of any one of SEQ ID NOs: 22 to 24, or Any of claims 4 to 9, comprising residues 215 to 270 of any one of sequences 25 to 28 2. The genetically engineered meganuclease of claim 1.

11. the first subunit is selected from residues 198 to 344 of any one of SEQ ID NOs: 22 to 24 or a sequence that is at least 80% identical to residues 7-153 of any one of SEQ ID NOs: 25-28. and the second subunit comprises an amino acid sequence having sequence identity with SEQ ID NOs: 22 to 24. or residues 7 to 153 of any one of SEQ ID NOs:25 to 28 344, comprising an amino acid sequence having at least 80% sequence identity with 11. The genetically engineered meganuclease of any one of claims 10.

12. the first subunit is selected from residues 198 to 344 of any one of SEQ ID NOs: 22 to 24 or any one of claims 4 to 11, comprising residues 7 to 153 of any one of SEQ ID NOs: 25 to 28. The genetically engineered meganuclease of any one of claims 1 to 4.

13. the second subunit is selected from residues 7 to 153 of any one of SEQ ID NOs: 22 to 24; or Any of claims 4 to 12, comprising residues 198 to 344 of any one of SEQ ID NOs: 25 to 28. The genetically engineered meganuclease of any one of claims 1 to 4.

14. a linker, said linker connecting said first subunit and said second subunit; The genetically engineered megakaryon according to any one of claims 4 to 13, wherein Nuclease.

15. Any one of claims 4 to 14, comprising the amino acid sequence of any one of SEQ ID NOs: 22 to 28.

2. The genetically engineered meganuclease of claim 1.

16. The genetic manipulation of any one of claims 1 to 3, wherein the recognition sequence comprises SEQ ID NO:

16. Meganucleases.

17. the HVR1 region is substituted for residues 215-270 of any one of SEQ ID NOs: 33-39 17. The gene of claim 16, comprising an amino acid sequence having at least 80% sequence identity with Engineered meganucleases.

18. the HVR1 region comprising residues 215, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239 ９、２２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６ 18. The genetic manipulation of claim 16 or 17, comprising residues corresponding to 1, 266, and 268. Meganucleases.

19. the HVR1 region comprises residues 215 to 270 of any one of SEQ ID NOs: 33 to 39 19. The genetically engineered meganuclease according to any one of claims 16 to 18.

20. the HVR2 region is at least 5'-5' with respect to residues 24-79 of any one of SEQ ID NOs: 33-39; Any of claims 16 to 19, comprising an amino acid sequence having at least 80% sequence identity.

2. The genetically engineered meganuclease of claim 1.

21. the HVR2 region is selected from residues 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, Corresponding to 0, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 The genetically engineered meganuclea of ​​any one of claims 16 to 20, comprising a residue -ze.

22. the HVR2 region comprises residues 24 to 79 of any one of SEQ ID NOs: 33 to 39.

22. The genetically engineered meganuclease of any one of claims 16 to 21.

23. the first subunit is selected from residues 198 to 344 of any one of SEQ ID NOs: 33 to 39 and wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to said The unit has at least 80% affinity to residues 7-153 of any one of SEQ ID NOs: 33-39. % sequence identity of the amino acid sequence of any one of claims 16 to 22 Genetically engineered meganucleases.

24. the first subunit is selected from residues 198 to 344 of any one of SEQ ID NOs: 33 to 39 24. The engineered meganuclease of any one of claims 16 to 23, comprising:

25. The second subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 33 to 39. The genetically engineered meganuclease of any one of claims 16 to 24.

26. a linker, said linker connecting said first subunit and said second subunit; The genetically engineered membrane according to any one of claims 16 to 25, wherein Ganuclease.

27. Any one of claims 16 to 26, comprising an amino acid sequence of any one of SEQ ID NOs: 33 to 39.

1. A genetically engineered meganuclease according to claim 1.

28. A gene encoding the engineered meganuclease of any one of claims 1 to 27. A polynucleotide comprising a nucleic acid sequence comprising:

29. 29. The polynucleotide of claim 28, wherein the polynucleotide is mRNA.

30. The mRNA is one or more of the engineered meganucleases of claims 1 to 27.

30. The polynucleotide of claim 29, which is a polycistronic mRNA encoding 。

31. The polycistronic mRNA is (a) a genetically engineered meganuclease that recognizes and cleaves a recognition sequence comprising SEQ ID NO: 12; and (b) an engineered meganuclease that recognizes and cleaves a recognition sequence comprising SEQ ID NO: 16; 31. The polynucleotide of claim 30, encoding

32. The polycistronic mRNA is (a) an engineered meganuclease according to any one of claims 4 to 15; and (b) a genetically engineered meganuclease according to any one of claims 16 to 27.

32. The polynucleotide of claim 30 or 31, encoding

33. The polycistronic mRNA is a genetically engineered mRNA comprising the amino acid sequence of SEQ ID NO:

22. A genetically engineered gene encoding a meganuclease comprising the amino acid sequence of SEQ ID NO:

33. The polynucleotide according to any one of claims 30 to 32, encoding a meganuclease Reotide.

34. A gene encoding the engineered meganuclease of any one of claims 1 to 27. A recombinant DNA construct comprising a nucleic acid sequence comprising:

35. A promoter and a genetically engineered meganuclear according to any one of claims 1 to 27. and a nucleic acid sequence encoding the α-glucanase. thing.

36. The method according to claim 1, further comprising the step of: a nucleic acid sequence encoding the engineered meganuclease of any one of claims 1 to 4, Each of the engineered meganucleases has specificity for a different HBV recognition sequence.

35. The recombinant DNA construct of claim 34, comprising:

37. A promoter and a genetically engineered meganucleus according to any one of claims 1 to 27. and a polycistronic nucleic acid sequence encoding one or more of the enzymes, A promoter drives expression of the polycistronic nucleic acid sequence to produce the polycistronic nucleic acid sequence in a target cell.

35. The recombinant DNA construct of claim 34, which produces a lycistronic mRNA.

38. The polycistronic mRNA is a polycistronic mRNA according to any one of claims 30 to 33.

37. The recombinant DNA construct of claim 36, which is a stronic mRNA.

39. A gene encoding the engineered meganuclease of any one of claims 1 to 27.

35. The recombinant DNA of claim 34, encoding a viral vector comprising said nucleic acid sequence Construction.

40. The recombinant DNA of claim 39, wherein the viral vector is a recombinant AAV vector. A construct.

41. A gene encoding the engineered meganuclease of any one of claims 1 to 27. A viral vector comprising a nucleic acid sequence comprising:

42. The viral vector of claim 41, wherein the viral vector is a recombinant AAV vector. Kutar.

43. The viral vector contains a promoter and the gene according to any one of claims 1 to 27. and a nucleic acid sequence encoding the engineered meganuclease. The viral vector described in

44. The method according to claim 1, further comprising the step of: a nucleic acid sequence encoding the engineered meganuclease of any one of claims 1 to 4; Each of the engineered meganucleases is specific for a different HBV recognition sequence. The viral vector of claim 41 .

45. A promoter and a genetically engineered meganucleus according to any one of claims 1 to 27. and a polycistronic nucleic acid sequence encoding one or more of the enzymes, A promoter drives expression of the polycistronic nucleic acid sequence to produce the polycistronic nucleic acid sequence in a target cell. The viral vector of claim 41, which produces a lysistronic mRNA.

46. The polycistronic mRNA is the polycistronic mRNA of any one of claims 30 to 33. The viral vector of claim 45, which is a lysistronic mRNA.

47. Treating subjects with hepatitis B virus (HBV) or hepatocellular carcinoma caused by HBV A pharmaceutical composition for the treatment of a rheumatoid arthritis, comprising a pharmaceutically acceptable carrier, and (a) a method for producing a recombinant meganuclease comprising: or (b) a genetically engineered meganuclease according to any one of claims 1 to 27; A pharmaceutical composition comprising:

48. The nucleic acid encoding the engineered meganuclease is selected from the group consisting of:

48. The pharmaceutical composition of claim 47, wherein the mRNA is any one of the mRNAs.

49. A method according to claim 47, comprising the recombinant DNA construct according to any one of claims 34 to 40. The pharmaceutical composition described above.

50. The virus vector according to claim 47, comprising the virus vector according to any one of claims 41 to 46. A pharmaceutical composition comprising:

51. A method of producing a recombinant human meganuclease comprising the genetically engineered meganuclease of any one of claims 1 to 27.

48. The pharmaceutical composition of claim 47.

52. Two or more of the engineered meganucleases according to any one of claims 1 to 27 wherein the engineered meganucleases are specific for different HBV recognition sequences.

48. The pharmaceutical composition of claim 47, having the formula:

53. Two or more of the engineered meganucleases according to any one of claims 1 to 27 and wherein the engineered meganuclease comprises two or more nucleic acids encoding different HBs.

48. The pharmaceutical composition of claim 47, having specificity for a V recognition sequence.

54. The mRNA of any one of claims 29 to 33, encapsulated in a lipid nanoparticle.

48. The pharmaceutical composition of claim 47, comprising one or more of A.

55. 1. A method of treating a subject with HBV, comprising administering to a target cell of said subject: (a) encoding a genetically engineered meganuclease, and in vivo in said target cell; the nucleic acid to be expressed; or (b) genetically engineered meganucleases; delivering The engineered meganuclease binds to at least two genes of the hepatitis B virus. The genetically engineered megagene has specificity for a recognition sequence in the ORF of the mutant genome. The cleavage enzyme recognizes and cleaves the recognition sequence in the target cell, resulting in the production of HBV in the subject. The method of claim 1, wherein the infection and / or proliferation of B. difficile is reduced or eliminated.

56. The recognition sequence is selected from genotype A, genotype B (SEQ ID NO: 4), genotype C (SEQ ID NO: No. 5), genotype D (SEQ ID NO: 6), genotype E (SEQ ID NO: 7), genotype F (SEQ ID NO: 8), and genotype G (SEQ ID NO: 9). Law.

57. The recognition sequences are selected from at least three, at least four, at least five, or 57. The method of claim 55 or 56, wherein the ORFs are found in one or at least six genotypes. How to do it.

58. The recognition sequence is located in the polymerase (P) protein, the major surface (preS1 / preS2 / S) proteins, middle surface (preS2 / S) proteins, and small surface (S) proteins A claim within at least one ORF encoding a protein selected from the group consisting of:

58. The method according to any one of claims 55 to 57.

59. The recognition sequence comprises SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:10, or SEQ ID NO:14 59. The method according to any one of claims 55 to 58.

60. 60. The method of any one of claims 55 to 59, wherein the recognition sequence comprises SEQ ID NO:

12.

61. The engineered meganuclease is a gene according to any one of claims 4 to 15. The method of any one of claims 55 to 59, wherein the meganuclease is a genetically engineered meganuclease. 。

62. 60. The method of any one of claims 55 to 59, wherein the recognition sequence comprises SEQ ID NO:

16.

63. The engineered meganuclease of any one of claims 16 to 27 63. The method of claim 62, wherein the meganuclease is a genetically engineered meganuclease.

64. administering to said subject a pharmaceutical composition according to any one of claims 47 to 54.

64. The method according to any one of claims 55 to 63.

65. The genetically engineered meganuclease or the genetically engineered meganuclease 65. The method of claim 55, wherein the nucleic acid encoding the How to post.

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