EA042452B1 - ENGINEERED MEGANUCLASES SPECIFIC TO THE RECOGNITION SEQUENCES IN THE HEPATITIS B VIRUS GENOME - Google Patents

Description

Technical field

The present invention relates to the field of oncology, molecular biology and technology of recombinant nucleic acids. In particular, the present invention provides engineered meganucleases that are specific for a recognition sequence in at least two genotypes of the hepatitis B virus genome. Such engineered meganucleases are useful in methods of treating hepatitis B virus infection and hepatitis B virus hepatocellular carcinoma.

Link to a sequence listing submitted as a text file via efs-web.

This application includes a sequence listing that has been filed in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The specified ASCII copy, created on October 13, 2017, is named P109070018WO00-SEQ-MJT.txt and is 169011 bytes in size.

State of the art

Hepatitis B virus (HBV) is a major health problem worldwide, with more than 350 million people chronically carriers. HBV infection is a serious and common infectious liver disease. Chronic infection is associated with an increased risk of developing severe liver disease, including cirrhosis and hepatocellular carcinoma (HCC), one of the most common forms of human cancer. The estimated risk of HCC in chronic HBV carriers is approximately 100 times higher than in uninfected individuals. Approximately a third of the world's population has been infected at some point in life, including 240 million to 350 million subjects with chronic infections. More than 750,000 people die every year from hepatitis B. Approximately 300,000 of them are from liver cancer. Currently available medicines for HBV have limitations. For example, administration of interferon alfa is associated with severe adverse reactions. Nucleoside analogs are virusostatic and require long-term administration. The level of genetic variability of the HBV genome is estimated as 1.4-3.2x10'5 nucleotide substitutions per site per year. A large number of virus variants arise during replication as a result of nucleotide insertion errors in the absence of any ability of the viral polymerase to correct errors. This variability has led to well-known subtypes of the virus. HBV was classified into well-defined genotypes based on intergroup divergence of 8% or more across the full-length genome sequence, each with a distinct geographic distribution. For example, genotype A is widespread in sub-Saharan Africa, Northern Europe, and West Africa; genotypes B and C are common in Asia; genotype C is mainly observed in Southeast Asia; genotype D is predominant in Africa, Europe, Mediterranean countries and India; genotype G has been described in France, Germany and the United States; and the H genotype is commonly found in Central and South America. Genotype I has recently been reported in Vietnam and Laos. A newly discovered HBV genotype, the J genotype, has been discovered in the Ryukyu Archipelago in Japan.

HBV is an enveloped DNA virus that belongs to the Hepadnaviridae family. It contains a genome of a small partially double-stranded (DC) relaxed circular DNA (rcDNA) that is replicated by reverse transcription of the messenger RNA, pregenomic RNA (pgRNA). The HBV circular DNA genome is unusual because its DNA is not fully double-stranded. One end of the full length chain is linked to the DNA polymerase of the virus. The genome is approximately 3020 - 3320 nucleotides long (for a full chain) and 1700 - 2800 nucleotides (for a short chain). The antisense (non-coding) strand is complementary to the viral mRNA.

There are four known genes encoded by this genome, which are C, X, P, and S. The core protein is encoded by gene C (HBcAg), and its initiation codon is preceded by an upstream in-frame initiation codon, AUG, from which the precore protein is produced. . HBeAg is formed during the proteolytic processing of the precore protein. DNA polymerase is encoded by the P gene. The S gene encodes the surface antigen (HBsAg). The HBsAg gene is one long open reading frame, but contains three initiation (ATG) codons in one reading frame, which divide the gene into three regions: pre-S1, pre-S2 and S. Due to the presence of several initiation codons, polypeptides of three different sizes are produced , which are called large (in order from the surface to the inside: pre-S1/pre-S2/S), medium (pre-S2/S) and small (S). The function of the protein encoded by the X gene is not fully understood, but it is associated with the development of liver cancer. It stimulates genes that cause cells to grow and inactivates molecules that regulate growth.

Viral DNA is found in the nucleus shortly after infection of the cell. Partially double-stranded DNA becomes fully double-stranded by building up the (+) sense strand and removing a protein molecule from the (-) sense strand and a short RNA sequence from the (+) sense strand. The non-coding bases are removed from the ends of the (-) sense strand and the ends are reattached. The HBV life cycle begins when the virus attaches to a host cell and enters. Recent studies have demonstrated that the cotransporting sodium taurocholate polypeptide (NTCP) is a functional receptor in HBV infection. The relaxed virion circular DNA (rcDNA) is delivered to the nucleus, where it undergoes repair to form a covalently closed circular DNA (cccDNA). Episomal cccDNA serves as template for transcription of pre

- 1 042452 genomic RNA (pgRNA) and other mRNA of the virus by the host RNA polymerase II. The resulting transcripts are then exported to the cytoplasm, where translation of viral proteins occurs. Reverse transcriptase (RT) binds to pgRNA and triggers the assembly of core proteins into immature RNA-containing nucleocapsids. The immature nucleocapsids then undergo a maturation process whereby pgRNA is reverse transcribed by RT to form mature rcDNA. A unique feature of hepadnavirus reverse transcription is the RT-primed initiation of DNA minus strand synthesis, which results in covalent binding of RT to the 5' end of the DNA minus strand. Mature rcDNA-containing nucleocapsids are then enveloped by viral surface proteins and secreted as virions (secretion pathway) or alternatively recycled back into the nucleus to further amplify the cccDNA pool (recycling pathway). Preservation of cccDNA in hepatocytes plays a key role in viral persistence, reactivation of viral replication after withdrawal of antiviral therapy, and resistance to therapy.

Homing endonucleases are a group of naturally occurring nucleases that recognize 15 to 40 bp cleavage sites commonly found in the genomes of plants and fungi. They are often associated with parasitic DNA elements such as group 1 autonomously spliced introns and inteins. They typically cause homologous recombination or insertion of a gene at specific positions in the host genome, creating a double-strand break in the chromosome that recruits the cellular DNA repair apparatus (Stoddard (2006), Q. Rev. Biophys. 38: 49-95). Homing endonucleases are often grouped into four families: the LAGLIDADG family (SEQ ID NO: 2), the GIY-YIG family, the His-Cys box family, and the HNH family. These families differ in structural motifs that affect catalytic activity and sequence recognition. For example, members of the LAGLIDADG family (SEQ ID NO: 2) are distinguished by having either one or two copies of the conserved LAGLIDADG motif (SEQ ID NO: 2) (see Chevalier et al. (2001), Nucleic Acids Res. 29( 18): 3757-3774). Homing endonucleases LAGLIDADG (SEQ ID NO: 2) with one copy of the LAGLIDADG motif (SEQ ID NO: 2) form homodimers, while those with two copies of the LAGLIDADG motif (SEQ ID NO: 2) are found as monomers. Methods for producing homing endonucleases are known in the art.

I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG family of homing endonucleases (SEQ ID NO: 2) that recognizes and cleaves a 22 bp recognition sequence in the chromosome of the Chlamydomonas reinhardtii algal chloroplast. Genetic selection techniques were used to change the wild-type I-CreI cleavage site preference (Sussman et al. (2004), J. Mol. Biol. 342: 31-41; Chames et al. (2005), Nucleic Acids Res. 33: e178, Seligman et al. (2002), Nucleic Acids Res. 30: 3870-9, Amould et al. (2006), J. Mol. Biol. 355: 443-58). Methods have been described for the rational design of mono-LAGLIDADG (SEQ ID NO: 2) homing endonucleases that are able to comprehensively redesign I-CreI and other homing endonucleases to target completely different DNA sites, including sites in the genomes of mammals, yeast, plants, bacteria and viruses (WO 2007/047859).

As first described in WO 2009/059195, I-CreI and its engineered derivatives are usually dimeric, but they can be linked into a single polypeptide using a short peptide linker that connects the C-terminus of the first subunit to the N-terminus of the second subunit (Li et al. (2009), Nucleic Acids Res. 37:1650-62; Grizot et al. (2009), Nucleic Acids Res. 37:5405-19). Thus, a functional single-stranded meganuclease can be expressed from a single transcript.

Proposed the use of engineered meganucleases for the treatment of HBV infections. For example, WO2010/136841 proposes the use of engineered meganucleases to cleave the genome of non-genome-integrating viruses. Such meganucleases include I-CreI variants. The '841 application describes a plurality of 22 bp meganuclease-recognized sequences present in the genome of a single strain of HBV. However, the '841 application did not identify any recognition sequence that is present in multiple genotypes of the HBV genome.

Brief description of the invention

The present invention provides, in part, the development of site-specific, rarely cutting endonucleases that are designed to recognize DNA sequences within at least one open reading frame (ORF) of the HBV genome and in at least two different genotypes of the HBV genome. The present inventors have found specific recognition sequences, conserved among genotypes, that can be targeted by recombinant meganucleases to inactivate multiple virus genotypes. The present invention improves on the prior art in several aspects. The present inventors have surprisingly found that recognition sequences can be found within the ORFs of several genotypes of the HBV genome. By targeting multiple virus genotypes, a single pharmaceutical composition can be obtained that can be used against different genotypes present in certain areas of the world. Thus, the methods and compositions described in this application are useful for treating or reducing the proliferation of HBV in infected individuals throughout the world. Suppression or elimination of HBV replication in the liver leads to an improvement in liver pathology and a decrease in progression.

- 2 042452 in cirrhosis of the liver and hepatocellular carcinoma. Accordingly, the present invention satisfies a need in the art for additional gene therapy approaches for the treatment of HBV infections. In addition, the present invention provides a method for treating hepatocellular carcinoma by providing means of targeting insertion by homologous recombination of a suicide gene into the HBV genome in an HCC cell. Said suicide gene may encode a toxin or a pro-apoptotic protein that directly kills the cancer cell, or a cell surface antigen or MHC Class I antigenic polypeptide that directs the subject's own immune system to kill the cancer cell. The present invention provides engineered meganucleases useful in the treatment of hepatitis B virus (HBV) infection. These engineered meganucleases of the present invention recognize and cleave the recognition sequence within the open reading frame (ORF) of the genome of at least two hepatitis B virus genotypes. non-homologous end joining (NGSK) at the cleavage site. NHSV can result in insertions, deletions, or frameshifts, which can interfere with gene expression. Accordingly, by disrupting normal gene expression, HBV infection and proliferation can be reduced or eliminated, in accordance with the methods described in this application. The present invention also provides pharmaceutical compositions and methods for the treatment of HBV that use an engineered meganuclease specific for recognition sequences located within the ORFs of the genome of at least two hepatitis B virus genotypes. The present invention further provides methods for delivering the engineered meganucleases described herein. , a subject infected with HBV, to reduce the level of HBV and/or reduce the symptoms associated with HBV infection.

Thus, in one aspect, the present invention provides an engineered meganuclease that recognizes and cleaves a recognition sequence within the open reading frame (ORF) of the genome of at least two hepatitis B virus genotypes. The engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds with the first recognized half site of the recognition sequence and contains the first hypervariable (HVR1) region, and the second subunit binds to the second recognized half site of the recognition sequence and contains the second hypervariable (HVR2) region.

In some embodiments, the recognition sequence is found within the ORF of at least 3, at least 4, at least 5, or at least 6 hepatitis B virus genotypes. In one such embodiment, the recognition sequence is within the ORF of genotype A (SEQ ID NO: 3) hepatitis B virus. This embodiment includes isolates of genotype A of hepatitis B virus that differ from SEQ ID NO: 3, but contain one or more HBV meganuclease-recognized sequences described in this application. In further such embodiments, the recognition sequence is within the ORF of genotype A and one or more of genotypes B, C, D, E, F, and G (SEQ ID NOs: 4-9, respectively). Such embodiments include genotype A, B, C, D, E, F, and G isolates of hepatitis B virus that differ from SEQ ID NOs: 3-9 but contain one or more of the HBV meganuclease-recognized sequences described herein. In some embodiments, the recognition sequence is within at least one ORF encoding a protein selected from the group consisting of: polymerase protein (P), large surface protein (preS1/preS2/S), medium surface protein (preS2/S), and small surface protein (S). In some embodiments, the recognition sequence may include SEQ ID NO: 10 (i.e., HBV recognition sequence 1-2), SEQ ID NO: 12 (i.e., HBV recognition sequence 5-6), SEQ ID NO: 14 ( ie HBV recognition sequence 7-8) or SEQ ID NO: 16 (ie HBV recognition sequence 11-12).

In some embodiments in which the recognition sequence includes SEQ ID NO: 10, the HVR1 region may include the amino acid sequence for at least 80%, at least 85%, at least 90%, at least 95% or more identical to the amino acid sequence corresponding to residues 24-79 in the sequence of SEQ ID NO: 18 or 19 or residues 215-270 in the sequence of SEQ ID NO: 20 or 21. In some such embodiments, the HVR1 region may include residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75 and 77 in SEQ ID NOs: 18 or 19, or residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 in SEQ ID NO: 20 or 21. In specific embodiments, the HVR1 region may include residues 2479 in SEQ ID NO: 18 or 19 or residues 215-270 in SEQ ID NO: 20 or 21.

In some such embodiments, in which the recognition sequence comprises SEQ ID NO: 10, the HVR2 region may include an amino acid sequence of at least

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80%, at least 85%, at least 90%, at least 95% or more identical to the amino acid sequence corresponding to residues 215-270 in the sequence of SEQ ID NO: 18 or 19 or residues 24-79 in the sequence SEQ ID NO: 20 or 21. In some such embodiments, the HVR2 region may include residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 in SEQ ID NO: 18 or 19, or residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75 and 77 in SEQ ID NO: 20 or 21. In specific embodiments, the HVR2 region may include residues 215-270 in SEQ ID NO: 18 or 19, or residues 24-79 in SEQ ID NO: 20 or 21.

In those embodiments in which the recognition sequence comprises SEQ ID NO: 10, the first subunit may include the amino acid sequence for at least 80%, at least 85%, at least 90%, at least 95% or more identical to residues 7-153 in the sequence of SEQ ID NO: 18 or 19 or residues 198-344 in the sequence of SEQ ID NO: 20 or 21, and the second subunit may include an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95% or more identical to residues 198-344 in the sequence of SEQ ID NO: 18 or 19 or residues 7-153 in the sequence of SEQ ID NO: 20 or 21. In some embodiments implementation, the first subunit may include residues 7-153 of SEQ ID NO: 18 or 19, or residues 198-344 of SEQ ID NO: 20 or 21. Similarly, in some embodiments, the second subunit may include residues 198-344 of sequences of SEQ ID NO: 18 or 19 or residues 7-153 from the sequence of SEQ ID NO: 20 or 21.

In some such embodiments, in which the recognition sequence comprises SEQ ID NO: 10, the engineered meganuclease may contain a linker, said linker covalently connecting the first subunit and the second subunit. In specific embodiments, the engineered meganuclease may include the amino acid sequence shown in any of SEQ ID NOs: 18-21. In other embodiments, in which the recognition sequence includes SEQ ID NO: 12, the HVR1 region may include the amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95% or more identical to the amino acid sequence corresponding to residues 215270 of any of the sequences of SEQ ID NO: 22-24 or residues 24-79 of any of the sequences of SEQ ID NO: 25-28. In some such embodiments, the HVR1 region may include residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any of the sequences of SEQ ID NO: 22-24, or residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any of SEQ ID NOs: 25-28. In particular embodiments, the HVR1 region may include residues 215-270 of any of SEQ ID NOs: 22-24, or residues 24-79 of any of SEQ ID NOs: 25-28.

In some such embodiments, in which the recognition sequence comprises SEQ ID NO: 12, the HVR2 region may include an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95 % or more identical to the amino acid sequence corresponding to residues 24-79 of any of the sequences of SEQ ID NO: 22-24 or residues 215-270 of any of the sequences of SEQ ID NO: 25-28. In some such embodiments, the HVR2 region may include residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any of the sequences of SEQ ID NO: 22 -24, or residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266 and 268 of any of SEQ ID NOs: 25-28. In specific embodiments, the HVR2 region may include residues 24-79 of any of SEQ ID NOs: 22-24, or residues 215-270 of any of SEQ ID NOs: 25-28.

In some such embodiments, in which the recognition sequence comprises SEQ ID NO: 12, the first subunit may include an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95 % or more identical to residues 198-344 of any of the sequences of SEQ ID NO: 22-24 or residues 7-153 of any of the sequences of SEQ ID NO: 25-28, and the second subunit may include an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95% or more identical to residues 7-153 of any of SEQ ID NOs: 22-24 or residues 198-344 of any of SEQ ID NOs: 25- 28. In some embodiments, the first subunit may include residues 198-344 of any of SEQ ID NOs: 22-24 or residues 7-153 of any of SEQ ID NOs: 25-28. Similarly, in some embodiments, the second subunit may include residues 7-153 of any of SEQ ID NOs: 22-24 or residues 198-344 of any of SEQ ID NOs: 25-28.

In some such embodiments, in which the recognition sequence comprises SEQ ID NO: 12, the engineered meganuclease may contain a linker, said linker covalently connecting the first subunit and the second subunit. In specific implementations

- 4 042452 constructed meganuclease may include the amino acid sequence presented in any of the sequences of SEQ ID NO: 22-28. In some embodiments in which the recognition sequence includes SEQ ID NO: 14, the HVR1 region may include the amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95% or more identical to the amino acid sequence corresponding to residues 215-270 of any of the sequences of SEQ ID NO: 29-32. In some such embodiments, the HVR1 region may include residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266 and 268 of any of the sequences of SEQ ID NO: 29 -32. In specific embodiments, the HVR1 region may include residues 215-270 of any of SEQ ID NOs: 29-32. In some such embodiments, in which the recognition sequence comprises SEQ ID NO: 14, the HVR2 region may include an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95 % or more identical to the amino acid sequence corresponding to residues 24-79 of any of the sequences of SEQ ID NO: 29-32. In some such embodiments, the HVR2 region may include residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any of the sequences of SEQ ID NO: 29 -32. In specific embodiments, the HVR2 region may include residues 24-79 of any of SEQ ID NOs: 29-32.

In some such embodiments, in which the recognition sequence comprises SEQ ID NO: 14, the first subunit may include an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95 % or more identical to residues 198-344 of any of the sequences of SEQ ID NOs: 29-32, and the second subunit may include an amino acid sequence of at least 80%, at least 85%, at least 90%, at least at least 95% or more identical to residues 7-153 of any of the sequences of SEQ ID NO: 29-32. In some embodiments, the first subunit may include residues 198-344 of any of SEQ ID NOs: 29-32. Similarly, in some embodiments, the second subunit may include residues 7-153 of any of SEQ ID NOs: 29-32.

In some such embodiments, in which the recognition sequence comprises SEQ ID NO: 14, the engineered meganuclease may contain a linker, said linker covalently connecting the first subunit and the second subunit. In specific embodiments, the engineered meganuclease may include the amino acid sequence shown in any of SEQ ID NOs: 29-32. In some embodiments in which the recognition sequence includes SEQ ID NO: 16, the HVR1 region may include the amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95% or more identical to residues 215-270 of any of the sequences of SEQ ID NOs: 33-39. In some such embodiments, the HVR1 region may include residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266 and 268 of any of the sequences of SEQ ID NO: 33 -39. In specific embodiments, the HVR1 region may include residues 215-270 of any of SEQ ID NOs: 33-39.

In some such embodiments, in which the recognition sequence comprises SEQ ID NO: 16, the HVR2 region may include an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95 % or more identical to residues 24-79 of any of the sequences of SEQ ID NO: 33-39. In some such embodiments, the HVR2 region may include residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any of the sequences of SEQ ID NO: 33 -39. In specific embodiments, the HVR2 region may include residues 24-79 of any of SEQ ID NOs: 33-39.

In those embodiments in which the recognition sequence comprises SEQ ID NO: 16, the first subunit may include the amino acid sequence at least 80%, at least 85%, at least 90%, at least 95% or more identical to residues 198-344 of any of SEQ ID NOs: 33-39, and the second subunit may include an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95% or more identical to residues 7-153 of any of the sequences of SEQ ID NO: 33-39. In some embodiments, the first subunit may include residues 198-344 of any of SEQ ID NOs: 33-39. Similarly, in some embodiments, the second subunit may include residues 7-153 of any of the sequences of SEQ ID NOs: 33-39.

In some embodiments, the engineered meganuclease may contain a linker, wherein said linker covalently links the first subunit and the second subunit. In specific embodiments, the engineered meganuclease may include the amino acid sequence shown in any of SEQ ID NOs: 33-39. In another aspect of the present invention, an isolated polynucleotide is provided, comprising a nucleic acid sequence encoding any of the engineered meganucleases described herein. In some embodiment, the isolated polynucleotide may be an mRNA. In addition

- 5 042452 real embodiments, the mRNA may be a polycistronic mRNA encoding one or more of the engineered meganucleases described in this application. In some embodiments, the polycistronic mRNA of the present invention may be, without limitation, a bicistronic mRNA encoding two engineered meganucleases described herein, a tricistronic mRNA encoding three engineered meganucleases described herein, or a tetracistronic mRNA encoding four engineered meganucleases. described in this application. In such embodiments in which two or more engineered meganucleases are encoded by a polycistronic mRNA, each engineered meganuclease encoded has specificity for a different HBV recognition sequence. Moreover, in such embodiments, the polycistronic mRNA of the present invention may encode any combination of the engineered meganucleases described herein. In some embodiment, the polycistronic mRNA can encode HBV meganuclease 1-2, HBV meganuclease 5-6, HBV meganuclease 7-8, and HBV meganuclease 11-12. In another specific embodiment, the polycistronic mRNA may be a bicistronic mRNA encoding HBV 5-6 meganuclease and HBV 11-12 meganuclease. In further embodiments, the polycistronic mRNA of the present invention may encode one or more of the engineered meganucleases described herein and one or more additional proteins that elicit a therapeutically beneficial effect in an HBV-infected cell and/or in an HBV-infected subject.

In another aspect, the present invention provides a recombinant DNA construct comprising a nucleic acid sequence that encodes any engineered meganuclease of the present invention. In some embodiments, said recombinant DNA construct comprises a cassette containing a promoter and a nucleic acid sequence encoding the engineered meganuclease described herein. In other embodiments, the recombinant DNA construct comprises two or more cassettes, where each cassette includes a promoter and a nucleic acid sequence encoding the engineered meganuclease described herein, and where each engineered meganuclease has specificity for a different HBV recognition sequence described herein. application. In specific embodiments, the recombinant DNA construct may comprise two cassettes, three cassettes, four cassettes, or more cassettes.

In other embodiments, the recombinant DNA construct comprises a cassette containing a promoter and a polycistronic nucleic acid sequence, said promoter driving expression in the target cell of the polycistronic nucleic acid sequence to form the polycistronic mRNA described herein.

In some embodiment, the recombinant DNA construct encodes a viral vector comprising a nucleic acid sequence encoding any engineered meganuclease described herein. In such an embodiment, said viral vector may be a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV) vector. In some embodiment, the viral vector may be a recombinant AAV vector.

In some embodiments, the viral vector contains a cassette containing a promoter and a nucleic acid sequence encoding the engineered meganuclease described in this application. In other embodiments, the viral vector contains two or more cassettes, where each cassette includes a promoter and a nucleic acid sequence encoding an engineered meganuclease described herein, and where each engineered meganuclease has specificity for a different HBV recognition sequence described in this application. . In other embodiments, the viral vector contains a single cassette containing a promoter and a polycistronic nucleic acid sequence, said promoter driving expression in the target cell of the polycistronic nucleic acid sequence to form the polycistronic mRNA described herein.

In another aspect, the present invention provides a viral vector comprising a nucleic acid sequence that encodes any engineered meganuclease of the present invention. In some embodiments, said viral vector may be a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV) vector. In some embodiment, the viral vector may be a recombinant AAV vector. In some embodiments, the viral vector contains a cassette containing a promoter and a nucleic acid sequence encoding the engineered meganuclease described in this application. In other embodiments, the viral vector contains two or more cassettes, where each cassette includes a promoter and a nucleic acid sequence encoding an engineered meganuclease described herein, and where each engineered meganuclease has specificity for a different HBV recognition sequence described in this application. . In additional embodiments, the viral vector contains a single cassette containing a promoter and a polycistronic nucleic acid sequence, with

- 6 042452 this promoter starts the expression in the target cell of the polycistronic nucleic acid sequence with the formation of polycistronic mRNA described in this application.

In another aspect, the present invention provides a pharmaceutical composition for treating a subject with hepatitis B virus (HBV) or suffering from hepatocellular carcinoma caused by HBV, said pharmaceutical composition comprising a pharmaceutically acceptable carrier and: (a) a nucleic acid encoding the engineered meganuclease described herein; or (b) protein engineered meganuclease described in this application; wherein the engineered meganuclease has specificity for recognition sequences within the open reading frame (ORF) of the genome of at least two hepatitis B virus genotypes. In some embodiments, the recognition sequence is within an ORF of at least 3, at least 4, at least 5, or at least 6 hepatitis B virus genotypes. In one such embodiment, the recognition sequence is within the HBV genotype A ORF (SEQ ID NO: 3), or isolates thereof, that contain the recognition sequence, and at least 1, at least 2 , at least 3, at least 4, at least 5, or at least 6 other HBV genotypes. In various embodiments, additional HBV genotypes may include genotype B, C, D, E, F, and/or G (SEQ ID NOs: 4-9, respectively), or isolates thereof, which contain one or more of the HBV recognition sequences described in this application. In some embodiments, the engineered meganuclease from the pharmaceutical composition, or the encoded nucleic acid sequence from the pharmaceutical composition, has specificity for recognition sequences within at least one ORF encoding a protein selected from the group consisting of: polymerase (P) protein, large surface protein (preS1/preS2/S), medium surface protein (preS2/S), and small surface protein (S). In some embodiments, the recognition sequence may include SEQ ID NO: 10, 12, 14, or 16. In one embodiment, the nucleic acid sequence from the pharmaceutical composition encoding the engineered meganuclease described herein may be the mRNA described herein. In some such embodiments, the mRNA may be a polycistronic mRNA as described herein such that two or more of the engineered meganucleases described herein are expressed in the target cell in vivo.

In another embodiment, the pharmaceutical composition contains the recombinant DNA construct described herein, comprising a nucleic acid sequence encoding the engineered meganuclease described herein. In some such embodiments, the recombinant DNA construct comprises a cassette containing a promoter and a nucleic acid sequence encoding the engineered meganuclease of the present invention. In other embodiments, the recombinant DNA construct from the pharmaceutical composition comprises two or more cassettes, where each cassette includes a promoter and a nucleic acid sequence encoding the engineered meganuclease described herein, and where each engineered meganuclease has specificity for a different HBV recognition sequence, described in this application. In specific embodiments, the pharmaceutical composition recombinant DNA construct may comprise two cassettes, three cassettes, four cassettes, or more cassettes.

In other embodiments, a recombinant DNA construct from a pharmaceutical composition comprises a cassette containing a promoter and a polycistronic nucleic acid sequence, said promoter driving the expression of the polycistronic nucleic acid sequence to form the polycistronic mRNA described herein in the target cell in vivo, so that two or more of the engineered meganucleases described in this application are expressed in the target cell.

In another embodiment, the pharmaceutical composition contains a viral vector comprising a nucleic acid sequence encoding the engineered meganuclease described in this application. In one such embodiment, said viral vector may be a retrovirus, lentivirus, adenovirus, or AAV. In some embodiment, the viral vector may be a recombinant AAV vector. In some such embodiments, the viral vector contains a cassette containing a promoter and a nucleic acid sequence encoding the engineered meganuclease described in this application. In other embodiments, the viral vector contains two or more cassettes, where each cassette includes a promoter and a nucleic acid sequence encoding an engineered meganuclease described herein, and where each engineered meganuclease has specificity for a different HBV recognition sequence described in this application. . In other such embodiments, the viral vector contains a single cassette containing a promoter and a polycistronic nucleic acid sequence, said promoter driving the expression of the polycistronic nucleic acid sequence to form the polycistronic mRNA described herein in the target cell in vivo, such that two or more engineered meganucleases described in this application are expressed in the target cell.

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In one such embodiment, the pharmaceutical composition may comprise an engineered meganuclease as described herein (or a nucleic acid encoding the same) that recognizes and cleaves SEQ ID NO: 10. SEQ ID NO: 18-21.

In another embodiment, the pharmaceutical composition may comprise an engineered meganuclease as described herein (or a nucleic acid encoding the same) that recognizes and cleaves SEQ ID NO: 12. In specific embodiments, the engineered meganuclease may include the amino acid sequence shown in any of the sequences of SEQ ID NO: 22-28.

In another embodiment, the pharmaceutical composition may comprise an engineered meganuclease as described herein (or a nucleic acid encoding the same) that recognizes and cleaves SEQ ID NO: 14. In specific embodiments, the engineered meganuclease may include the amino acid sequence shown in any of the sequences of SEQ ID NO: 29-32.

In another embodiment, the pharmaceutical composition may comprise an engineered meganuclease as described herein (or a nucleic acid encoding the same) that recognizes and cleaves SEQ ID NO: 16. In specific embodiments, the engineered meganuclease may include the amino acid sequence shown in any of the sequences of SEQ ID NO: 33-39.

In various embodiments, the pharmaceutical composition may contain two or more engineered meganuclease proteins described herein, wherein said engineered meganucleases have specificity for different HBV recognition sequences described herein. In other embodiments, the pharmaceutical composition may contain two or more nucleic acids encoding the engineered meganucleases described herein, wherein said engineered meganucleases have specificity for various HBV recognition sequences described herein. In such embodiments, the implementation of these two or more nucleic acids can be included in the mRNA described in this application, in the design of recombinant DNA described in this application, and/or in the viral vectors described in this application. In other embodiments, the pharmaceutical composition may contain one or more engineered meganuclease proteins described herein and one or more nucleic acids encoding the engineered meganuclease described herein (i.e., mRNA, recombinant DNA construct, or viral vector), wherein said engineered meganucleases and encoded engineered meganucleases have specificity for the various HBV recognition sequences described herein. In additional embodiments, the pharmaceutical composition may contain a combination of engineered meganuclease proteins described herein, a combination of nucleic acids encoding engineered meganucleases described herein (i.e., mRNA molecules, recombinant DNA constructs, viral vectors), or a combination of engineered proteins. meganucleases and nucleic acids encoding engineered meganuclease proteins, wherein said combination of engineered meganucleases and/or encoded engineered meganucleases in a pharmaceutical composition can recognize and cleave the recognition sequence in each of the HBV genotypes A, B, C, D, E, F, and G ( sequence SEQ ID NO: 3-9), and isolates of each genotype that contain the HBV recognition sequences described in this application.

In some embodiments, the implementation of the pharmaceutical composition may contain one or more mRNA described in this application, encapsulated within the lipid nanoparticles. In specific embodiments, said lipid nanoparticles from a pharmaceutical composition may contain two or more mRNAs described herein, each of which encodes an engineered meganuclease of the present invention having specificity for a distinct HBV recognition sequence described herein. In specific embodiments, said lipid nanoparticles may contain two, three, or four mRNAs described herein, each of which encodes an engineered meganuclease of the present invention having specificity for a different HBV recognition sequence. In other embodiments, these lipid nanoparticles from a pharmaceutical composition may contain one or more polycistronic mRNAs described in this application, with each polycistronic mRNA encoding two or more engineered meganucleases according to the present invention, having specificity for various HBV recognition sequences described in this application. . In specific embodiments, said lipid nanoparticles may contain a polycistronic mRNA encoding two, three, or four of the engineered meganucleases described herein. In other specific embodiments, these lipid nanoparticles may contain two or more polycistronic mRNAs described in this application, each of which encodes two or more designed meganucleases according to the present invention. In some implementations

- 8 042452 said lipid nanoparticles have a composition that enhances delivery and absorption in the liver, and, in particular, inside hepatocytes.

In another aspect of the present invention, a method of treating a subject suffering from HBV is provided. Similarly, the present application provides a method for reducing the level and/or proliferation of HBV, or reducing the symptoms associated with HBV. These methods include delivering to the subject's target cell: (a) a nucleic acid encoding an engineered meganuclease, wherein the engineered meganuclease is expressed in the target cell in vivo; or (b) an engineered meganuclease protein; wherein the engineered meganuclease has specificity for recognition sequences in the ORF of the genome of at least two hepatitis B virus genotypes, and wherein the engineered meganuclease recognizes and cleaves the recognition sequence, thereby cleaving the HBV genome in the target cell. Said method can reduce or eliminate HBV infection and/or proliferation in a subject.

In another aspect of the present invention, a method of treating a subject suffering from HCC caused by HBV is provided. These methods include delivering to the subject's target cell: (1) (a) a nucleic acid encoding an engineered meganuclease, wherein the engineered meganuclease is expressed in the target cell in vivo; or (b) an engineered meganuclease protein; and (2) a nucleic acid comprising a polynucleotide sequence encoding a suicide gene and sequences homologous to sequences flanking a meganuclease cleavage site; wherein the engineered meganuclease has specificity for recognition sequences in the ORF of the genome of at least two hepatitis B virus genotypes, wherein the engineered meganuclease recognizes and cleaves the recognition sequence, thereby cleaving the HBV genome in the target cell; wherein the suicide gene is inserted into the split HBV genome by homologous recombination; and in doing so, the expression of the suicide gene destroys the target cell.

In some embodiments, the suicide gene is directly lethal to the target cell. In some such embodiments, the directly lethal suicide gene encodes for a toxic polypeptide or pro-apoptotic protein. In some embodiments, the suicide gene is indirectly lethal to the target cell and directs the subject's own immune system to kill the target cell. In some such embodiments, the indirectly lethal suicide gene encodes a cell surface protein that is recognized as foreign by the subject's immune system and becomes the target of a humoral or cellular immune response. In other such embodiments, the indirectly lethal suicide gene encodes a polypeptide that presents as a class I MHC molecule, is recognized as foreign by the subject's immune system, and becomes the target of a cytotoxic immune response.

In some embodiments of methods for treating HBV or HCC infection, the recognition sequence is found within the ORF of at least 3, at least 4, at least 5, or at least 6 hepatitis B virus genotypes. In one such embodiment, the recognition sequence is within Hepatitis B virus genotype A (SEQ ID NO: 3) ORF. This embodiment includes hepatitis B virus genotype A isolates that differ from SEQ ID NO: 3 but contain one or more of the HBV meganuclease-recognized sequences described herein. In further such embodiments, the recognition sequence is within the ORF of genotype A and one or more of genotypes B, C, D, E, F, and G (SEQ ID NOs: 4-9, respectively). Such embodiments include genotype A, B, C, D, E, F, and G isolates of hepatitis B virus that differ from SEQ ID NOs: 3-9 but contain one or more of the HBV meganuclease-recognized sequences described herein.

In such embodiments of methods for treating HBV or HCC infection, the recognition sequence may be within at least one ORF encoding a protein selected from the group consisting of: polymerase protein (P), large surface protein (preS1/preS2/S), middle surface protein (preS2/S) and small surface protein (S). In some embodiments, the recognition sequence may include SEQ ID NO: 10 (i.e., HBV recognition sequence 1-2), SEQ ID NO: 12 (i.e., HBV recognition sequence 5-6), SEQ ID NO: 14 ( ie HBV recognition sequence 7-8) or SEQ ID NO: 16 (ie HBV recognition sequence 11-12). In specific embodiments of methods for treating HBV or HCC infection, the engineered meganuclease protein, or the encoded engineered meganuclease, is the engineered meganuclease described herein.

In additional embodiments, methods for treating HBV or HCC infection comprise administering to said subject any pharmaceutical composition of the present invention described herein that contains at least a pharmaceutically acceptable carrier and (a) a nucleic acid encoding the engineered meganuclease described herein. the application, wherein the engineered meganuclease is expressed in the target cell in vivo; or (b) an engineered meganuclease protein as described herein. In some embodiments of methods for treating HBV or HCC infection, the engineered meganuclease or nucleic acid encoding the engineered meganuclease can be delivered to a target hepatocyte. In specific embodiments, an effective amount of an engineered meganuclease or a nucleic acid encoding an engineered meganuclease can be delivered to a target hepatocyte.

In specific embodiments, delivery to the hepatocyte occurs ex vivo, wherein the subject is administered an effective amount of hepatocytes that have received the engineered meganuclease or nucleic acid encoding the engineered meganuclease.

In some embodiments, a hepatotoxic protein, or a nucleic acid or AAB encoding a hepatotoxic protein, is administered together with the pharmaceutical compositions described in this application.

In specific embodiments of these methods, the first recognition sequence may include SEQ ID NO: 10. In certain such embodiments, the engineered meganuclease may be any engineered meganuclease of the present invention that recognizes and cleaves SEQ ID NO: 10. In specific embodiments, the engineered the meganuclease may include the amino acid sequence shown in any of SEQ ID NOs: 18-21.

In another embodiment of these methods, the first recognition sequence may comprise SEQ ID NO: 12. In some such embodiments, the engineered meganuclease may be any engineered meganuclease of the present invention that recognizes and cleaves SEQ ID NO: 12. In specific embodiments, the engineered the meganuclease may include the amino acid sequence shown in any of SEQ ID NOs: 22-28.

In other embodiments of these methods, the first recognition sequence may include SEQ ID NO: 14. In certain such embodiments, the engineered meganuclease may be any engineered meganuclease of the present invention that recognizes and cleaves SEQ ID NO: 14. In specific embodiments, the engineered the meganuclease may include the amino acid sequence shown in any of SEQ ID NOs: 29-32.

In other embodiments of these methods, the first recognition sequence may include SEQ ID NO: 16. In certain such embodiments, the engineered meganuclease may be any engineered meganuclease of the present invention that recognizes and cleaves SEQ ID NO: 16. In specific embodiments, the engineered the meganuclease may include the amino acid sequence shown in any of SEQ ID NOs: 33-39. In specific embodiments of these methods, the subject may be a mammal, such as a human.

In another aspect of the present invention, the engineered meganuclease described herein is provided for use as a drug. The present invention further provides the use of the engineered meganuclease described herein for the manufacture of a medicament for treating HBV, reducing the level or proliferation of HBV, reducing symptoms associated with HBV, or treating HCC. In another aspect of the present invention, there is provided an isolated polynucleotide for use as a drug, characterized in that the isolated polynucleotide comprises a nucleic acid sequence encoding the engineered meganuclease described herein. The present invention further provides the use of an isolated polynucleotide for the manufacture of a medicament for the treatment of HBV, the reduction of the level or proliferation of HBV, the reduction of symptoms associated with HBV, or the treatment of HCC.

In another aspect of the present invention, a recombinant AAV vector is provided for use as a drug, wherein said recombinant AAV vector contains an isolated polynucleotide, and wherein said isolated polynucleotide comprises a nucleic acid sequence encoding the engineered meganuclease described herein. The present invention further provides the use of a recombinant AAV vector for the manufacture of a medicament for the treatment of HBV, the reduction or proliferation of HBV, the reduction of symptoms associated with HBV, or the treatment of HCC, wherein said recombinant AAV vector contains an isolated polynucleotide, and wherein said the selected polynucleotide includes a nucleic acid sequence encoding the engineered meganuclease described in this application.

The above and other aspects and embodiments of the present invention can be more fully understood by reading the following detailed description and claims. Certain features of the present invention, which are described in the context of separate embodiments for clarity, may also be offered in combination in a single embodiment. All combinations of embodiments are expressly included in the scope of the present invention and are described in this application in the same way as if all combinations without exception were separately and explicitly described. Conversely, various features of the present invention, which for brevity are described in the context of one

- 10 042452 implementation options may also be offered separately or in any suitable subcombination. All subcombinations of properties listed in the embodiments are also expressly included in the scope of the present invention and are described in this application in the same way as if all such subcombinations without exception were separately and explicitly described in this application. Embodiments of each aspect of the present invention described in this application apply to every other aspect of the present invention, mutatis mutandis.

Brief description of the figures

In FIG. 1 shows a genomic map of the HBV genome and labels all ORFs.

The viral particles have a partially double-stranded genome (indicated by the dotted line) with a sticky overlap that connects the 5' portion of each strand and is flanked by direct repeat sequences (DR1 and DR2). The S gene encodes the hepatitis B major surface antigen (HBsAg) protein and its glycosylated partner, which are transmembrane proteins within the envelope of the virus. In-frame sequences upstream of the S gene encode pre-S domains that are translated together with S sequences to form pre-S and S polypeptides (medium and large proteins) that contain the viral receptor for infecting hepatocytes. The C gene encodes the hepatitis B core antigen (HBcAg), which forms the nucleocapsid of the virus. The P region encodes the reverse transcriptase of the virus, which also has DNA-dependent DNA polymerase activity and RNase H activity required for viral replication. Although HBV is a DNA virus, it replicates via a pre-genomic RNA messenger. Finally, gene X codes for a small regulatory protein of the virus, hepatitis B antigen x (HBx). HBx is a transactivating protein that stimulates viral gene expression and replication, protects virus-infected cells from immune-mediated destruction, and promotes the development of hepatocellular carcinoma.

Fig. 2. Constructed meganuclease recognition sequences in the HBV genome. A) Each recognition sequence targeted by the engineered meganuclease of the present invention contains two recognizable half sites. Each recognized half-site contains 9 base pairs separated by a central sequence of 4 base pairs. The HBV recognition sequence 1-2 (SEQ ID NO: 10) contains two recognizable half-sites, designated HBV1 and HBV2. The HBV recognition sequence 5-6 (SEQ ID NO: 12) contains two recognizable half-sites, designated HBV5 and HBV6. The HBV recognition sequence 7-8 (SEQ ID NO: 14) contains two recognizable half-sites, designated HBV7 and HBV8. The HBV recognition sequence 11-12 (SEQ ID NO: 16) contains two recognizable half-sites, designated HBV11 and HBV12.

Fig. 3. Illustration of the HBV genotype A genome and the location of the HBV 1-2, HBV 5-6, HBV 7-8 and HBV 11-12 recognition sequences within the genome. Both HBV 1-2 and HBV 5-6 recognition sequences are located within four ORFs of the HBV genome: P, preS1/preS2/S, preS2/S, and S. Each of the HBV 7-8 and HBV 11-12 recognition sequences is located within an ORF, coding polymerase.

Fig. 4. Alignment of HBV recognition sequences in HBV genotypes A-G. The recognition sequences targeted by the present invention are conserved among many HBV genotypes. The HBV recognition sequence 1-2 extends to residues 185-206 of HBV genotype A shown in SEQ ID NO: 3, and this recognition sequence is fully conserved across genotypes B, C, E, F, and G. Genotype D contains a single nucleotide substitution difference G to T at position -4 of the first half site. The HBV recognition sequence 5-6 extends to residues 742-763 of HBV genotype A shown in SEQ ID NO: 3, and this recognition sequence is fully conserved across genotypes B, C, D, E, and G. The F genotype contains a single nucleotide substitution difference G to C at position -3 of the first half site. The HBV recognition sequence 7-8 extends to residues 1183-1204 of the HBV genotype A shown in SEQ ID NO: 3, and this recognition sequence is completely conserved across genotypes B, C, D, F, and G. The E genotype contains a single nucleotide substitution difference G to C at position -1 of the first half site. The HBV recognition sequence 11-12 extends to residues 1259-1280 of HBV genotype A shown in SEQ ID NO: 3 and this recognition sequence is completely conserved across genotypes B, C, D, E, F and G.

Fig. 5. Said engineered meganucleases of the present invention comprise two subunits, wherein said first subunit containing the HVR1 region binds to the first recognized half site (e.g. HBV1, HBV5, HBV7 or HBV11) and the second subunit containing the HVR2 region binds to the second recognized half site. hemisite (eg HBV2, HBV6, HBV8 or HBV12). In embodiments where the engineered meganuclease is a single-stranded meganuclease, the first subunit containing the HVR1 region can be positioned as either an N-terminal or a C-terminal subunit. Similarly, the second subunit containing the HVR2 region can be positioned as either an N-terminal or a C-terminal subunit.

- 11 042452

Fig. 6. Schematic representation of a reporter assay in CHO cells to evaluate engineered meganucleases targeting the ORF of the genome of at least two hepatitis B virus genotypes. cell genome. The reporter cassette contained, in 5' to 3' order: SV40 early promoter; 5' 2/3 of the GFP gene; a recognition sequence for the engineered meganuclease of the present invention (eg, HBV 1-2 recognition sequence); recognition sequence for meganuclease CHO-23/24 (WO/2012/167192); and 3'2/3 of the GFP gene. Cells stably transfected with this cassette did not express GFP in the absence of a DNA breakage agent. Meganucleases were introduced by transduction of plasmid DNA or mRNA encoding each meganuclease. When a DNA break was caused at any of the sequences recognized by the meganuclease, the duplicated portions of the GFP gene recombined with each other to form a functional GFP gene. The percentage of GFP-expressing cells could then be determined using flow cytometry as a proxy for the frequency of genome cleavage by meganucleases.

Fig. 7. Efficiency of recognition and cleavage by engineered meganucleases of recognition sequences in the ORF of the genome of at least two genotypes of the hepatitis B virus gene in reporter analysis in CHO cells. The engineered meganucleases shown in SEQ ID NOs: 18-39 were designed to target HBV recognition sequence 1-2 (SEQ ID NO: 10), HBV recognition sequence 5-6 (SEQ ID NO: 12), HBV recognition sequence 7-8 (SEQ ID NO: 14) or HBV recognition sequence 11-12 (SEQ ID NO: 16) and screened for their performance in a reporter assay in CHO cells. The results shown represent the percentage of GFP-expressing cells observed in each assay, which corresponds to the efficiency of each meganuclease cleavage of the target recognition sequence or CHO-23/24 recognition sequence. A negative control (bs) was additionally included in each assay. In FIG. 7A shows meganucleases targeting the HBV 1-2 recognition sequence. In FIG. 7B shows meganucleases targeting the HBV recognition sequence 5-6. In FIG. 7C shows meganucleases targeting the HBV recognition sequence 7-8. In FIG. 7D shows meganucleases targeting the HBV recognition sequence 11-12.

Fig. 8. Efficiency of recognition and cleavage by engineered meganucleases of recognition sequences in the ORF of the genome of at least two genotypes of the hepatitis B virus gene in reporter analysis in CHO cells. The engineered meganucleases shown in SEQ ID NOs: 18-39 were designed to target HBV recognition sequence 1-2 (SEQ ID NO: 10), HBV recognition sequence 5-6 (SEQ ID NO: 12), HBV recognition sequence 7-8 (SEQ ID NO: 14) or HBV recognition sequence 11-12 (SEQ ID NO: 16) and screened for their performance in a reporter assay in CHO cells at multiple time points up to 12 days after nucleofection. The results shown represent the percentage of GFP expressing cells observed in each assay over the 12 day assay period, which corresponds to the efficiency of each meganuclease cleavage of the target or CHO-23/24 recognition sequence as a function of time. In FIG. 8A shows meganucleases targeting the HBV 1-2 recognition sequence. In FIG. 8B shows meganucleases targeting the HBV recognition sequence 5-6. In FIG. 8C shows meganucleases targeting the HBV recognition sequence 7-8. In FIG. 8D shows meganucleases targeting the HBV recognition sequence 11-12.

Fig. 9. Evaluation of the ability of HBV meganucleases to recognize and cleave recognition sequences within episomal DNA plasmids in the E. coli reporter system. In FIG. 9A shows plasmid pARCUS and plasmid pHBVa. In FIG. 9B shows the number of colonies present on each selective medium plate, providing evidence for the ability of HBV meganucleases to cleave the pHBVa plasmid.

Fig. 10. Evaluation of HBV meganucleases in AD38 cells. AD38 cells that express the HBV genome and secrete HBsAg were transfected with plasmid DNA encoding HBV 5-6x.33 or HBV 11-12x.26 meganucleases. Plasmid DNA encoding red fluorescent protein was used in this experiment as a control. On days 3 and 7 after transfection, the cell supernatant was collected and analyzed for the presence of HBsAg in it using enzyme-linked immunosorbent assay (ELISA).

Fig. 11. Evaluation of HBV meganucleases in AD38 cells. AD38 cells that express the HBV genome and secrete HBsAg were transduced with a lentivirus encoding HBV meganuclease 5-6x.33 or HBV meganuclease 11-12x.26, or transduced with a combination of these meganucleases. A lentivirus encoding a red fluorescent protein was used as a control in this experiment. Multiplicities of infection (MOIs) of 1, 2, and 4 were examined. On day 7 after transduction, the cell supernatant was collected and analyzed for the presence of HBsAg by ELISA.

Fig. 12. Evaluation of HBV meganucleases in AD38 cells. AD38 cells that express the HBV genome and secrete HBsAg were transduced with a lentivirus encoding HBV meganuclease 5-6x.33 (LV224),

- 12 042452 HBV meganuclease 11-12x.26 (LV225) or red fluorescent protein (LV212). An MW of 4 was examined and, on day 7 post-transduction, the cell supernatant was collected and analyzed for the presence of HBsAg and the copy number of extracellular HBV DNA. In addition, cell lysates were prepared on day 7 post-transduction and analyzed for copy number of intracellular HBV cccDNA. In FIG. 12A shows the concentrations of HBsAg in the culture medium. In FIG. 12B shows the copy number of extracellular HBV DNA in the culture medium. In FIG. 12C shows the copy number of intracellular HBV cccDNA in cell lysates.

Fig. 13. Evaluation of HBV meganucleases in HBV-infected primary human hepatocytes. Lentiviruses were prepared that expressed either red fluorescent protein (RFP) or HBV 56x.33 or HBV 11-12x.26 and HBV-infected primary human hepatocytes were transduced 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 post-infection, cells were washed and 24 hours later (day 2 post-infection) transduced with either lentivirus encoding RFP, HBV 5-6x.33, HBV 11-12x.26, or a 1:1 mixture of lentiviruses encoding HBV meganucleases. As an additional control, infected cells were treated with DMSO. Cell supernatants were harvested and medium changed on days 4, 8, 11 and 13 post-transduction. At each time point, HBsAg and HBeAg were measured in cell supernatants by ELISA. Also measured extracellular DNA in the supernatant on day 13 after infection. To determine whether, in general, lentivirus transduction affected either HBsAg or HBeAg secretion, cell supernatants transduced with the RFP-encoding lentivirus were compared to those treated with DMSO. In FIG. 13A shows that transduction with a lentivirus encoding RFP had little effect on HBsAg expression at an MZ of 1.25, 2.5, or 5. FIG. 13B shows that transduction with an RFP-encoding lentivirus had little effect on HBeAg expression at an MZ of 1.25, 2.5, or 5. FIG. 13C shows that transduction with an RFP-encoding lentivirus had little effect on HBV DNA expression at an MZ of 1.25, 2.5, or 5.

Fig. 14. Evaluation of HBV meganucleases in HBV-infected primary human hepatocytes. Lentiviruses were prepared that expressed either RFP or HBV 5-6x.33 or HBV 11-12x.26 and HBV-infected primary human hepatocytes were transduced to determine the effect of meganucleases on HBsAg and HBeAg production. Primary human hepatocytes were seeded and infected with HBV 24 hours later. Cells were washed 1 day post-infection and 24 hours later (day 2 post-infection) transduced with either lentivirus encoding RFP, HBV 5-6x.33, HBV 11-12x.26, or a 1:1 mixture of lentiviruses encoding HBV meganucleases. As an additional control, infected cells were treated with DMSO. Cell supernatants were harvested and medium changed on days 4, 8, 11 and 13 post-transduction. At each time point, HBsAg and HBeAg were measured in cell supernatants by ELISA. In FIG. 14A shows HBsAg in culture medium after transduction at an MS of 2.5. In FIG. 14B shows HBsAg in culture medium after transduction at an MS of 1.25. In FIG. 14C shows HBeAg in culture medium after transduction at an MS of 2.5. Figure 14D shows HBeAg in culture medium after transduction at an MS of 1.25.

Fig. 15. Delivery of mRNA enclosed in a lipid nanoparticle to the liver. An engineered meganuclease was developed that has specificity for recognition sequences in the mouse CMP-NeuAc (Cmah) hydrolase gene, which is expressed in mouse liver. Capped ARCA mRNA encoding Cmah meganuclease was enclosed in three different commercially available LNP formulations. LNP-encapsulated mRNA was administered to CD-1 mice by intravenous (IV) injection and harvested in the liver 6 days later. Whole liver genomic DNA (gDNA) was isolated and the insertion/deletion (indel) mutation rate in the Cmah gene was determined using T7 endonuclease I (T7E) analysis and deep sequencing.

Brief description of the sequences

SEQ ID NO: 1 shows the amino acid sequence of wild-type I-CreI meganuclease from Chlamydomonas reinhardtii.

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

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

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

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

SEQ ID NO: 6 shows the amino acid sequence of HBV genotype B.

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

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

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

SEQ ID NO: 10 shows the amino acid sequence of the HBV recognition sequence 1-2 (sense).

SEQ ID NO: 11 shows the amino acid sequence of the HBV recognition sequence 1-2 (antisense).

SEQ ID NO: 12 shows the amino acid sequence of the HBV recognition sequence 5-6 (sense).

- 13 042452

SEQ ID NO: 13 shows the amino acid sequence of the HBV recognition sequence 5-6 (antisense).

SEQ ID NO: 14 shows the amino acid sequence of the HBV recognition sequence 7-8 (sense).

SEQ ID NO: 15 shows the amino acid sequence of the HBV recognition sequence 7-8 (antisense).

SEQ ID NO: 16 shows the amino acid sequence of the HBV recognition sequence 11-12 (sense).

SEQ ID NO: 17 shows the amino acid sequence of the HBV recognition sequence 11-12 (antisense).

SEQ ID NO: 18 shows the amino acid sequence of HBV meganuclease 1-2x.2.

SEQ ID NO: 19 shows the amino acid sequence of HBV meganuclease 1-2x.14.

SEQ ID NO: 20 shows the amino acid sequence of HBV meganuclease 1-2x.68.

SEQ ID NO: 21 shows the amino acid sequence of HBV meganuclease 1-2x.93.

SEQ ID NO: 22 shows the amino acid sequence of HBV meganuclease 5-6x.33.

SEQ ID NO: 23 shows the amino acid sequence of HBV meganuclease 5-6x.84.

SEQ ID NO: 24 shows the amino acid sequence of HBV meganuclease 5-6x.90.

SEQ ID NO: 25 shows the amino acid sequence of HBV meganuclease 5-6x.4.

SEQ ID NO: 26 shows the amino acid sequence of HBV meganuclease 5-6x.5.

SEQ ID NO: 27 shows the amino acid sequence of HBV meganuclease 5-6x.68.

SEQ ID NO: 28 shows the amino acid sequence of HBV meganuclease 5-6x.79.

SEQ ID NO: 29 shows the amino acid sequence of HBV meganuclease 7-8x.2.

SEQ ID NO: 30 shows the amino acid sequence of HBV meganuclease 7-8x.9.

SEQ ID NO: 31 shows the amino acid sequence of HBV meganuclease 7-8x.17.

SEQ ID NO: 32 shows the amino acid sequence of HBV meganuclease 7-8x.44.

SEQ ID NO: 33 shows the amino acid sequence of HBV meganuclease 11-12x.26.

SEQ ID NO: 34 shows the amino acid sequence of HBV meganuclease 11-12x.9.

SEQ ID NO: 35 shows the amino acid sequence of HBV meganuclease 11-12x.13.

SEQ ID NO: 36 shows the amino acid sequence of HBV meganuclease 11-12x.16.

SEQ ID NO: 37 shows the amino acid sequence of HBV meganuclease 11-12x.27.

SEQ ID NO: 38 shows the amino acid sequence of HBV meganuclease 11-12x.41.

SEQ ID NO: 39 shows the amino acid sequence of HBV meganuclease 11-12x.48.

SEQ ID NO: 40 shows the amino acid sequence of the HBV1 binding subunit of HBV meganuclease 1-2x.2.

SEQ ID NO: 41 shows the amino acid sequence of the HBV1 binding subunit of HBV meganuclease 1-2x.14.

SEQ ID NO: 42 shows the amino acid sequence of the HBV1 binding subunit of HBV meganuclease 1-2x.68.

SEQ ID NO: 43 shows the amino acid sequence of the HBV1 binding subunit of HBV meganuclease 1-2x.93.

SEQ ID NO: 44 shows the amino acid sequence of the HBV2 binding subunit of HBV meganuclease 1-2x.2.

SEQ ID NO: 45 shows the amino acid sequence of the HBV2 binding subunit of HBV meganuclease 1-2x.14.

SEQ ID NO: 46 shows the amino acid sequence of the HBV2 binding subunit of HBV meganuclease 1-2x.68.

SEQ ID NO: 47 shows the amino acid sequence of the HBV2 binding subunit of HBV meganuclease 1-2x.93.

SEQ ID NO: 48 shows the amino acid sequence of the HBV5 binding subunit of HBV meganuclease 5-6x.33.

SEQ ID NO: 49 shows the amino acid sequence of the HBV5 binding subunit of HBV meganuclease 5-6x.84.

SEQ ID NO: 50 shows the amino acid sequence of the HBV5 binding subunit of HBV meganuclease 5-6x.90.

SEQ ID NO: 51 shows the amino acid sequence of the HBV5 binding subunit of HBV meganuclease 5-6x.4.

SEQ ID NO: 52 shows the amino acid sequence of the HBV5 binding subunit of HBV meganuclease 5-6x.5.

SEQ ID NO: 53 shows the amino acid sequence of the HBV5 binding subunit of HBV meganuclease 5-6x.68.

SEQ ID NO: 54 shows the amino acid sequence of the HBV5 binding subunit of HBV meganuclease 5-6x.79.

- 14 042452

SEQ ID NO: 55 shows the amino acid sequence of the HBV6 binding subunit of HBV meganuclease 5-6x.33.

SEQ ID NO: 56 shows the amino acid sequence of the HBV6 binding subunit of HBV meganuclease 5-6x.84.

SEQ ID NO: 57 shows the amino acid sequence of the HBV6 binding subunit of HBV meganuclease 5-6x.90.

SEQ ID NO: 58 shows the amino acid sequence of the HBV6 binding subunit of HBV meganuclease 5-6x.4.

SEQ ID NO: 59 shows the amino acid sequence of the HBV6 binding subunit of HBV meganuclease 5-6x.5.

SEQ ID NO: 60 shows the amino acid sequence of the HBV6 binding subunit of HBV meganuclease 5-6x.68.

SEQ ID NO: 61 shows the amino acid sequence of the HBV6 binding subunit of HBV meganuclease 5-6x.79.

SEQ ID NO: 62 shows the amino acid sequence of the HBV7 binding subunit of HBV meganuclease 7-8x.2.

SEQ ID NO: 63 shows the amino acid sequence of the HBV7 binding subunit of HBV meganuclease 7-8x.9.

SEQ ID NO: 64 shows the amino acid sequence of the HBV7 binding subunit of HBV meganuclease 7-8x.17.

SEQ ID NO: 65 shows the amino acid sequence of the HBV7 binding subunit of HBV meganuclease 7-8x.44.

SEQ ID NO: 66 shows the amino acid sequence of the HBV8 binding subunit of HBV meganuclease 7-8x.2.

SEQ ID NO: 67 shows the amino acid sequence of the HBV8 binding subunit of HBV meganuclease 7-8x.9.

SEQ ID NO: 68 shows the amino acid sequence of the HBV8 binding subunit of HBV meganuclease 7-8x.17.

SEQ ID NO: 69 shows the amino acid sequence of the HBV8 binding subunit of HBV meganuclease 7-8x.44.

SEQ ID NO: 70 shows the amino acid sequence of the HBV11 binding subunit of HBV meganuclease 11-12x.26.

SEQ ID NO: 71 shows the amino acid sequence of the HBV11 binding subunit of HBV meganuclease 11-12x.9.

SEQ ID NO: 72 shows the amino acid sequence of the HBV11 binding subunit of HBV meganuclease 11-12x.13.

SEQ ID NO: 73 shows the amino acid sequence of the HBV11 binding subunit of HBV meganuclease 11-12x.16.

SEQ ID NO: 74 shows the amino acid sequence of the HBV11 binding subunit of HBV meganuclease 11-12x.27.

SEQ ID NO: 75 shows the amino acid sequence of the HBV11 binding subunit of HBV meganuclease 11-12x.41.

SEQ ID NO: 76 shows the amino acid sequence of the HBV11 binding subunit of HBV meganuclease 11-12x.48.

SEQ ID NO: 77 shows the amino acid sequence of the HBV12 binding subunit of HBV meganuclease 11-12x.26.

SEQ ID NO: 78 shows the amino acid sequence of the HBV12 binding subunit of HBV meganuclease 11-12x.9.

SEQ ID NO: 79 shows the amino acid sequence of the HBV12 binding subunit of HBV meganuclease 11-12x.13.

SEQ ID NO: 80 shows the amino acid sequence of the HBV12 binding subunit of HBV meganuclease 11-12x.16.

SEQ ID NO: 81 shows the amino acid sequence of the HBV12 binding subunit of HBV meganuclease 11-12x.27.

SEQ ID NO: 82 shows the amino acid sequence of the HBV12 binding subunit of HBV meganuclease 11-12x.41.

SEQ ID NO: 83 shows the amino acid sequence of the HBV12 binding subunit of HBV meganuclease 11-12x.48.

SEQ ID NO: 84 shows the nucleic acid sequence of the recognition sequence present in HBV genotype D, which corresponds to the recognition sequence of HBV 1-2 in HBV genotype A with the G replaced by C at position -4 of the first half site.

SEQ ID NO: 85 shows the nucleic acid sequence of the recognition sequence present in HBV genotype F, which corresponds to the recognition sequence of HBV 5-6 in HBV genotype A with G replaced by C at position -3 of the first half site.

SEQ ID NO: 86 shows the nucleic acid sequence of the recognition sequence present in HBV genotype E, which corresponds to the recognition sequence of HBV 7-8 in HBV genotype A with the C replaced with a T at position -1 of the first half site.

Detailed description of the invention

1.1. Contrasted materials and definitions.

The patent and scientific literature referenced in this application indicates the amount of knowledge that is available to those skilled in the art. U.S. published patents, accepted applications, published foreign applications, and references, including sequences from the GenBank database, that are cited in this application are hereby incorporated by reference to the same extent as if each material were specifically and individually indicated as incorporated by reference. The present invention can be implemented in various forms, and should not be construed as limited to the specific implementation options described in this application. Rather, these embodiments are provided so that the present description is comprehensive and complete and fully expresses the scope of the present invention to those skilled in the art. For example, features illustrated with respect to one implementation may be included in other implementations, and features illustrated with respect to a particular implementation may be removed from that implementation. In addition, many changes and additions to the specific implementation options proposed in this application will be obvious to specialists in this field, in light of the present description, which do not deviate from the present invention.

Unless otherwise indicated, all technical and scientific terms used in this application have the same meanings as commonly understood by the average person in the field to which the present invention pertains. The terminology used to describe the present invention in this application is used solely for the purpose of describing specific embodiments and is not intended to limit the present invention.

All publications, patent applications, patents and other opposing materials mentioned in this application are hereby incorporated by reference in their entirety.

In this application, the use of the singular may mean one or more than one. For example, a cell can mean a single cell or a plurality of cells.

In this application, unless specifically stated otherwise, the term or is used in the inclusive sense and/or, but not in the exclusive sense of either/or.

In this application, the terms nuclease and endonuclease are used interchangeably with respect to naturally occurring or engineered enzymes that cleave a phosphodiester bond within a polynucleotide chain.

In this application, the term meganuclease refers to an endonuclease that binds to double-stranded DNA in a recognition sequence that is greater than 12 base pairs. Preferably, said recognition sequence for a meganuclease according to the present invention is 22 base pairs long. The meganuclease may be an endonuclease that is derived from I-CreI and may refer to an engineered variant of I-CreI that has been modified from native I-CreI in terms of, for example, DNA binding specificity, DNA cleavage activity, DNA binding affinity, or ability to dimerize. Methods for preparing such modified I-CreI variants are known in the art (eg WO 2007/047859). The meganuclease proposed in this application binds to double-stranded DNA as a heterodimer. The meganuclease can also be a single stranded meganuclease in which a pair of DNA binding domains are joined into one polypeptide by a peptide linker. The term homingendonuclease is synonymous with the term meganuclease. The meganucleases of the present invention are substantially non-toxic when expressed in cells, do not adversely affect cell viability, and meganuclease cleavage activity is not significantly reduced when measured using the methods described herein.

In this application, the term single-stranded meganuclease refers to a polypeptide containing a pair of nuclease subunits connected by a linker. A single-stranded meganuclease may have the following organization: N-terminal subunit - linker - C-terminal subunit. These two meganuclease subunits will typically not be identical in amino acid sequence and will recognize non-identical DNA sequences. Thus, single-stranded meganucleases typically cleave pseudopalindromic or non-palindromic recognition sequences. A single-stranded meganuclease may be referred to as a single-stranded heterodimer or a single-stranded heterodimeric meganuclease, although it is not actually dimeric. For clarity, unless otherwise indicated, the term meganuclease may refer to a dimeric or single-stranded meganuclease.

In this application, the term linker refers to an exogenous peptide sequence that is used to join two meganuclease subunits into a single polypeptide. The linker sequence may be one that occurs in natural proteins, or may be an artificial sequence that does not occur in any natural protein. The linker may be flexible and have no secondary structure, or may be prone to form a particular three-dimensional structure under physiological conditions. The linker may include, without limitation, linkers included in the scope of US patent No. 8445251 and US patent No. 9434931. In some embodiments, the amino acid sequence of the linker may contain residues 154-195 of any of the sequences of SEQ ID NO: 18-39.

In this application, the term recombinant or engineered with respect to a protein means containing an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids that encode the specified protein, and to cells or organisms that express the specified protein. In relation to a nucleic acid, the term recombinant or engineered means containing an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to: PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis and gene fusion. According to this definition, a protein with an amino acid sequence identical to a naturally occurring protein, but obtained by cloning and expression in a heterologous host, is not considered recombinant.

In this application, the term wild type refers to the most naturally occurring allele (ie, polynucleotide sequence) in a population of alleles of a single gene type, and the polypeptide encoded by the wild type allele has the original functions. The term wild type also refers to a polypeptide encoded by a wild type allele. Alleles (ie, polynucleotides) and wild-type polypeptides can be distinguished from mutant or variant alleles and polypeptides that contain one or more mutations and/or substitutions compared to wild-type sequence(s). While a wild-type allele or polypeptide can confer a normal phenotype on an organism, a mutant or variant allele or polypeptide can, in some cases, confer an altered phenotype. Wild-type nucleases can be distinguished from recombinant or non-naturally occurring nucleases. The term wild type can also refer to a cell, organism and/or subject that has the wild type allele of a particular gene, or to a cell, organism and/or subject used for comparison purposes.

In this application, the term genetically modified refers to a cell or organism in which, or in the precursor of which, the genomic DNA sequence has been intentionally modified by recombinant technology. In this application, the scope of the term genetically modified includes the term transgenic.

In this application, the term modification in relation to recombinant proteins means any insertion, deletion, or substitution of an amino acid residue in a recombinant sequence relative to the original sequence (eg, wild-type or native sequence).

In this application, the term recognition sequence refers to a DNA sequence that is bound and cleaved by an endonuclease. In the case of a meganuclease, the recognition sequence contains a pair of inverted half-sites of 9 base pairs, which are separated by four base pairs. In the case of a single-stranded meganuclease, the N-terminal domain of the protein contacts the first half-site and the C-terminal domain of the protein contacts the second half-site. Meganuclease cleavage results in a 3' overhang of four base pairs. Overhangs or sticky ends are short single-stranded DNA fragments that can result from the cleavage of a double-stranded DNA sequence by an endonuclease. In the case of meganucleases and single-stranded meganucleases derived from I-CreI, the overhang contains bases 10-13 of the 22 base pair recognition sequence. In this application, the term target site or target sequence refers to a region of the chromosomal DNA of a cell containing a sequence recognized by a nuclease.

In this application, the term DNA binding affinity or binding affinity refers to the propensity of a meganuclease to non-covalently bind to a parent DNA molecule (eg, recognition sequence or random sequence). Binding affinity is measured using the dissociation constant Kd. In this application, a nuclease has an altered binding affinity if the Kd of the nuclease from the original recognition sequence increases or decreases by a statistically significant (p<0.05) value compared to the original nuclease.

In this application, the term specificity means the ability of a meganuclease to recognize and cleave double-stranded DNA molecules only for a certain sequence of base pairs, which is called a recognition sequence, or only for a certain set of recognition sequences. The specified set of recognition sequences will have some common conservative positions or sequence motifs, but they may be degenerate in one or more positions. A highly specific meganuclease is capable of cleaving only one or only a few recognition sequences. Specificity can be determined using any method known in the art. In this application, a meganuclease has an altered specificity if it binds and cleaves a recognition sequence that the parent meganuclease (e.g., wild-type) does not bind or cleave under physiological conditions, or if the cleavage rate of the recognition sequence is increased or decreased by a biologically significant value (for example, at least 2 times or 2-10 times) compared with the original meganuclease.

As used herein, the term homologous recombination or GR refers to the natural cellular process in which a double-stranded DNA break is repaired using a homologous DNA sequence as a repair template (see, for example, Cahill et al. (2006), Front. Biosci. 11 :1958-1976). The homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous nucleic acid that has been delivered to the cell.

As used herein, the term non-homologous end joining or NHSC refers to a natural cellular process in which a double-stranded DNA break is repaired by directly joining two non-homologous DNA fragments (see, e.g., Cahill et al. (2006), Front. Biosci. 11:1958 -1976). DNA repair by non-homologous end joining is error prone and often results in non-template insertion or deletion of DNA sequences at the repair site. In some cases, cleavage at the target recognition sequence results in NGSC at the target recognition site. Nuclease-induced cleavage of a target site in a gene's coding sequence, followed by DNA repair by NHSC, can introduce mutations into the coding sequence, such as frameshift mutations, that impair gene function. Thus, engineered meganucleases can be used for efficient gene knockout in a population of cells. In this application, with respect to both amino acid sequence and nucleic acid sequence, the terms percent identity, sequence identity, percent similarity, sequence similarity, and the like refer to a measure of the degree of similarity between two sequences based on a sequence alignment that maximizes the similarity between aligned amino acid residues or nucleotides, which is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs are available to determine sequence similarity using standard parameters. In this application, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/) and are described, for example, in Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nature Genet. 3:266-272; Madden et al. (1996), Meth. Enzymol, 266:131-141; Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402); Zhang et al. (2000), J. Comput. Biol. 7(1-2):203-14. In this application, the percent similarity of two amino acid sequences is a score based on the following parameters of the BLASTp algorithm: word length=3; gap opening penalty=-11; gap extension penalty=-1; and weight matrix=BLOSUM62. In this application, the percent similarity of two nucleic acid sequences is a score based on the following parameters of the BLASTn algorithm: word length=11; gap opening penalty=-5; gap extension penalty=-2; match reward=1; and mismatch penalty=-3. In this application, the term appropriate in relation to modifications of two proteins or amino acid sequences is used to indicate that a certain modification in the first protein is a replacement of the same amino acid residue as in the modification in the second protein, and that the position of the amino acid modified in the first protein is matches or aligns with the position of the amino acid modified in the second protein when the two proteins are subjected to standard sequence alignment (eg, using the BLASTp program). Thus, modification of an X residue to amino acid A in the first protein will correspond to a modification of a Y residue to amino acid A in the second protein if the X and Y residues match each other in the sequence alignment and despite the fact that X and Y may be different numbers .

In this application, the term recognized half-site, half-site of the recognition sequence or simply half-site means a nucleic acid sequence in a double-stranded DNA molecule that is recognized by a monomer of a homodimeric or heterodimeric meganuclease or by one subunit of a single-stranded meganuclease.

As used herein, the term hypervariable region refers to a localized sequence within a meganuclease monomer or subunit that contains amino acids with relatively high variability. A hypervariable region may contain about 50-60 contiguous residues, about 53-57 contiguous residues, or preferably about 56 residues. In some embodiments, hypervariable region residues may correspond to positions 24-79 or positions 215-270 of any of the sequences of SEQ ID NOs: 18-39. A hypervariable region may contain one or more residues that are in contact with bases.

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DNA in the recognition sequence, and it can be modified to change the preference for the specified monomer or subunit. The hypervariable region may also contain one or more residues that are associated with the DNA backbone when the meganuclease is linked to a recognized sequence in the double stranded DNA. Such residues can be modified to change the binding affinity of the meganuclease to the DNA backbone and to the target recognition sequence. In various embodiments of the present invention, the hypervariable region may contain 1-20 residues that exhibit variability and that can be modified to affect base preference and/or DNA binding affinity. In some embodiments, the variable residues within the hypervariable region correspond to one or more of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 49, 50, 54, 64, 68, 70, 75, and 77 of any of the sequences of SEQ ID NOs: 18-39. In other embodiments, the variable residues within the hypervariable region correspond to one or more of positions 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 240, 241, 245, 255, 259, 261, 266 and 268 of any of the sequences of SEQ ID NOs: 18-39. The terms recombinant DNA construct, recombinant construct, expression cassette, expression construct, chimeric construct, construct and recombinant DNA fragment are used interchangeably in this application and describe nucleic acid fragments. The recombinant construct contains an artificial combination of nucleic acid fragments, including, without limitation, regulatory and coding sequences that do not occur together in nature. For example, a recombinant DNA construct may contain regulatory sequences and coding sequences that are obtained from different sources, or regulatory sequences and coding sequences obtained from the same source but arranged in a manner that is different from that found in nature. Such a construct may be used on its own or may be used in combination with a vector.

As used herein, a vector or recombinant DNA vector may be a construct that contains a replication system and sequences that are capable of transcribing and translating a polypeptide coding sequence in a given host cell. If a vector is used, then the choice of vector depends on the method that will be used to transform the host cells, as is well known to those skilled in the art. Vectors may include, without limitation, plasmid vectors and recombinant AAV vectors, or any other vector known in the art suitable for delivering a gene encoding a meganuclease of the present invention into a target cell. The skilled artisan is well aware of the genetic elements that must be present in a vector in order to successfully transform, select and expand host cells containing any of the isolated nucleotides or nucleic acid sequences of the present invention.

In this application, a vector may also refer to a viral vector.

Viral vectors may include, without limitation, retroviral vectors, lentiviral vectors, adenovirus vectors, and adeno-associated virus (AAV) vectors.

In this application, polycistronic mRNA refers to a single messenger RNA that contains two or more coding sequences (ie cistrons) and codes for more than one protein. The polycistronic mRNA may contain any element known in the art that allows translation of two or more genes from the same mRNA molecule, including but not limited to: the IRES element, the T2A element, the P2A element, the E2A element, and the F2A element.

In this application, the control or control cell refers to a cell that is a benchmark for measuring changes in the genotype or phenotype of a genetically modified cell. The control cell may include, for example: (a) a wild-type cell, ie. a cell of the same genotype as the source material for the genetic change that results in the genetically modified cell; (b) a cell of the same genotype as the genetically modified cell, but which has been transformed with an empty construct (ie, a construct that has no known effect on the trait of interest); or (c) a cell that is genetically identical to the genetically modified cell, but that has not been subjected to conditions or stimuli or additional genetic modifications that would cause the expression of the altered genotype or phenotype. In this application, the term appropriate in relation to modifications of two proteins or amino acid sequences is used to indicate that a certain modification in the first protein is a replacement of the same amino acid residue as in the modification in the second protein, and that the position of the amino acid modified in the first protein is matches or aligns with the position of the amino acid modified in the second protein when the two proteins are subjected to standard sequence alignment (eg, using the BLASTp program). Thus, modification of an X residue to amino acid A in the first protein will correspond to a modification of a Y residue to amino acid A in the second protein if the X and Y residues match each other in the sequence alignment and despite the fact that X and Y may be different numbers .

In this application, the terms treatment or treatment of a subject refer to the administration of an engineered meganuclease of the present invention or a nucleic acid encoding a construct

- 19 042452 ruianized meganuclease according to the present invention, a subject infected with HBV, in order to slow down the rate or stop the proliferation of the HBV virus by splitting the genome of at least one HBV particle. Such treatment reduces or prevents transfection and replication of HBV in the subject and either partially or completely alleviates one or more symptoms of HBV in the subject. Means of assessing symptomatic relief of HBV infection may include measuring liver function by measuring alanine aminotransferase (ALT) enzyme levels or by measuring serum conversion, ie HbeAg clearance. In addition, relief or reduction in HBV symptoms can be determined by examining liver biopsies and measuring the level of tissue fibrosis using methods well known in the art. The number of circulating viral particles can be determined, for example, by measuring levels of HBV DNA using PCR, or by detecting levels of HBsAg in the blood. The terms treatment or treatment of a subject may further refer to administering a cell (e.g., a hepatocyte) containing a nucleic acid encoding an engineered meganuclease, said cell being delivered to a target tissue (e.g., liver), where it produces the engineered meganuclease in an amount sufficient for treatment. HBV infection in a subject, thereby resulting in either partial or complete relief of one or more symptoms of HBV. In some aspects, an engineered meganuclease according to the present invention, a nucleic acid encoding it, or a genetically modified cell according to the present invention is administered during treatment in the form of a pharmaceutical composition according to the present invention.

The term hepatitis B virus infection refers to any condition associated with or resulting from hepatitis B virus infection, such as chronic liver diseases/disorders, inflammation, fibrotic conditions, and proliferative disorders such as liver cancers. Chronic resistant HBV infection can cause fatigue, liver damage, cirrhosis of the liver, and hepatocellular carcinoma, the primary cancer of the liver.

The term proliferating and proliferation in this application refers to HBV cells that actively divide and infect human cells. Thus, reduction in proliferation refers to any reduction in HBV proliferation, including a reduction of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95% or 100%, compared to a suitable control, which did not enter the specified engineered meganuclease or nucleic acid encoding the engineered meganuclease described in this application. Throughout this application, the term proliferative disorder refers to any disease/disorder characterized by unwanted or aberrant tissue proliferation. In this application, the term proliferative disorder also refers to conditions in which uncontrolled and/or abnormal cell growth may lead to the development of an undesirable condition or disease, which may or may not be cancerous.

The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce beneficial or desirable biological and/or clinical results. A therapeutically effective amount will vary depending on the formulation or composition of the meganuclease, the particular disease and its severity, and the age, body weight, physical condition, and responsiveness to treatment of the subject being treated. In specific embodiments, an effective amount of the engineered meganuclease or pharmaceutical compositions described herein reduces the level or proliferation of HBV or reduces at least one symptom of HBV in a subject with an HBV infection.

The term lipid nanoparticle refers to a lipid composition typically having a spherical structure with an average diameter of 10 to 1000 nanometers. In some formulations, the lipid nanoparticles may contain at least one cationic lipid, at least one non-cationic lipid, and at least one conjugated lipid. Lipid nanoparticles known in the art that are suitable for encapsulating nucleic acids such as mRNA are provided for use in the present invention.

In this application, it is contemplated that listing a numeric range for a variable expresses that the present invention can be practiced with a variable equal to any of the values within that range. Thus, for a variable that is essentially discrete, the specified variable can be equal to any integer value within the specified numeric range, including the endpoints of the range. Likewise, for a variable that is essentially continuous, the specified variable can be equal to any real value within the specified numeric range, including the endpoints of the range. By way of example, and without limitation, a variable that is described as having values between 0 and 2 may take on the values 0, 1, or 2 if said variable is substantially discrete, and may take on the values 0.0, 0.1, 0, 01, 0.001, or any other real value, and 12 if the specified variable is essentially continuous.

2.1. The principle of the present invention.

The present invention is based in part on the hypothesis that engineered meganucleases can

- 20 042452 but used to reduce the level of HBV or slow down the proliferation of HBV by splitting the ORF within the HBV genome. In particular, meganucleases can be engineered to recognize and cleave the recognition sequence present within the genome of multiple HBV genotypes. Thus, a single engineered meganuclease can be used to reduce the level or proliferation of HBV, or reduce the symptoms of HBV infection, of multiple hepatitis B virus genotypes. Thus, engineered meganucleases that recognize and cleave a recognition sequence within an ORF of at least 2 genotypes of the HBV genome. Also within the scope of the present invention are methods for using such engineered meganucleases in pharmaceutical compositions and in methods for treating HBV infection. Also within the scope of the present invention are pharmaceutical compositions containing engineered meganuclease proteins or nucleic acids encoding engineered meganucleases and the use of such compositions for the treatment of HBV infection.

2.2. Meganucleases for recognition and cleavage of recognition sequences within the HBV genome.

It is known in the art that a site-specific nuclease can be used to create a DNA break in the genome of a virus and that such a DNA break can result in permanent modification of the genome by NHSC such that the HBV virion can no longer divide or infect human cells. Thus, in one embodiment, the present invention can be carried out using engineered recombinant meganucleases.

In preferred embodiments, the nucleases used to practice the present invention are single-stranded meganucleases. A single chain meganuclease contains an N-terminal subunit and a C-terminal subunit connected by a linker peptide. Each of these two domains recognizes half of the recognition sequence (ie, the recognized half site), and the DNA cleavage site is in the middle of the recognition sequence near the boundary between the two subunits. DNA strand breaks are displaced by four base pairs, so that DNA cleavage by meganuclease results in a four-base pair of 3'-single-stranded overhangs. In some examples, engineered meganucleases of the present invention were engineered to recognize and cleave the HBV 1-2 recognition sequence (SEQ ID NO: 10). The HBV 1-2 recognition sequence is located within the P, S, preS2/S and preS1/preS2 protein ORFs of multiple HBV genotypes. The HBV recognition sequence 1-2 can be found in at least the genome of a variety of HBV genotypes, including genotype A, B, C, E, F, and G (eg, sequences SEQ ID NOs: 3-5 and 7-9, respectively). Such engineered meganucleases are collectively referred to herein as HBV 1-2 meganucleases. Examples of HBV meganucleases 1-2 are provided in SEQ ID NOs: 18-21.

In additional examples, engineered meganucleases of the present invention were engineered to recognize and cleave the HBV recognition sequence 5-6 (SEQ ID NO: 12). The HBV 5-6 recognition sequence is located within the P, S, preS2/S and preS1/preS2 protein ORFs of multiple HBV genotypes. The HBV recognition sequence 5-6 can be found in at least the genome of a variety of HBV genotypes, including genotype A, B, C, D, E, and G (eg, SEQ ID NOs: 3-7 and 9, respectively). Such engineered meganucleases are collectively referred to herein as HBV 5-6 meganucleases. Examples of HBV meganucleases 5-6 are provided in SEQ ID NOs: 22-28.

In additional examples, engineered meganucleases of the present invention were engineered to recognize and cleave the HBV recognition sequence 7-8 (SEQ ID NO: 14). The HBV 7-8 recognition sequence is located within the P ORF protein of many HBV genotypes. The HBV recognition sequence 7-8 can be found in at least the genome of a variety of HBV genotypes, including genotype A, B, C, D, F, and G (eg, SEQ ID NOs: 3-6, 8, and 9, respectively). Such engineered meganucleases are collectively referred to herein as HBV 7-8 meganucleases. Examples of HBV meganucleases 7-8 are provided in SEQ ID NOs: 29-32. In additional examples, engineered meganucleases of the present invention were engineered to recognize and cleave the HBV recognition sequence 11-12 (SEQ ID NO: 16). The HBV recognition sequence 11-12 is located within the P ORF protein of many HBV genotypes. The HBV recognition sequence 11-12 can be found in at least the genome of a variety of HBV genotypes, including genotype A, B, C, D, E, F, and G (eg, sequences SEQ ID NOs: 3-9, respectively). Such engineered meganucleases are collectively referred to herein as HBV 11-12 meganucleases. Examples of HBV meganucleases 11-12 are provided in SEQ ID NOs: 34-40.

The engineered meganucleases of the present invention comprise a first subunit containing a first hypervariable (HVR1) region and a second subunit containing a second hypervariable (HVR2) region. In addition, the first subunit binds to the first recognized half-site in the recognition sequence (e.g., the HBV1, HBV5, HBV7, or HBV11 half-site) and the second subunit binds to the second recognized half-site after

- 21 042452 recognition distance (for example, half-site HBV2, HBV6, HBV8 or HBV12). In embodiments where the engineered meganuclease is a single-stranded meganuclease, the first and second subunits can be oriented such that the first subunit, which contains the HVR1 region and binds the first half site, is positioned as the N-terminal subunit, and the second subunit, which contains the HVR2 region and links the second half-site, located as the C-terminal subunit. In alternative implementations, the first and second subunits can be oriented such that the first subunit, which contains the HVR1 region and binds the first half site, is located as a C-terminal subunit, and the second subunit, which contains the HVR2 region and binds the second half site, is located as N- terminal subunit. Examples of HBV meganucleases 1-2 according to the present invention are proposed in table. 1. Examples of HBV 56 meganucleases according to the present invention are proposed in table. 2. Examples of HBV meganucleases 7-8 according to the present invention are proposed in table. 3. Examples of HBV meganucleases 11-12 according to the present invention are proposed in table. 4.

Table 1. Examples of engineered meganucleases that are engineered to recognize and cleave the HBV 1-2 recognition sequence (SEQ ID NO: 10)

Meganuclease AK SEQ ID Residues of the HBV1 subunit SEQ ID subunits HBV1 *% HBV1 subunit Residues of the HBV2 subunit SEQ ID HBV2 subunits *% HBV2 subunit VGV 1 - 2x.2 18 7-153 40 100 198 - 344 44 100 VGV 1 -2x.14 19 7-153 41 93.2 198 - 344 45 91.16 VGV 1 - 2x.68 20 198 -344 42 93.2 7-153 46 91.16 VGV 1 - 2x.93 21 198 -344 43 100 7-153 47 95.92

* % HBV1 subunit and % HBV2 subunit denote amino acid sequence identity between HBV1-binding and HBV2-binding subunit regions of each meganuclease and HBV1-binding and HBV2-binding subunit regions, respectively, of HBV meganuclease 1-2x.2.

Table 2. Examples of engineered meganucleases that are engineered to recognize and cleave the HBV 5-6 recognition sequence (SEQ ID NO: 12)

Meganuclease AK SEQ ID Remains of the HBV5 subunit SEQ ID HBV5 subunit *% HBV5 subunit Remains of the HBV6 subunit SEQ ID HBV6 subunits *% HBV6 subunits VGV 5 - bx.33 22 198 -344 48 100 7-153 55 100 VGV 5 - bh.84 23 198 -344 49 93.2 7-153 56 92.52 VGV 5 - 6x.9O 24 198 -344 50 91.84 7-153 57 93.2 VGV 5 - 6x 4 25 7-153 51 92.52 198 -344 58 95.92 VGV 5 - 6x.5 26 7-153 52 92.52 198 -344 59 93.88 VGV 5 - 6x.68 27 7-153 53 91.16 198 -344 60 93.88 VGV 5 - 6x.79 28 7-153 54 93.2 198 -344 61 93.88

* % HBV5 subunit and % HBV6 subunit denote amino acid sequence identity between HBV5-binding and HBV6-binding subunit regions of each meganuclease and HBV5-binding and HBV6-binding subunit regions, respectively, of HBV meganuclease 5-6x.33.

Table 3. Examples of engineered meganucleases that are engineered to recognize and cleave the HBV 7-8 recognition sequence (SEQ ID NO: 14)

Meganuclease AK SEQ ID Residues of the HBV7 subunit SEQ ID subunits HBV7 *% HBV7 subunit Residues of the HBV8 subunit SEQ ID subunits HBV8 *% HBV8 subunit VGV 7 - 8x.2 29 198 -344 62 100 7-153 66 100 VGV 7 - 8x.9 thirty 198 -344 63 98.64 7-153 67 99.32 VGV 7-8x. 17 31 198 -344 64 98.64 7-153 68 99.32 VGV 7 - 8x.44 32 198 -344 65 96.6 7-153 69 99.32

* % HBV7 subunit and % HBV8 subunit denote amino acid sequence identity between the HBV7-binding and HBV8-binding subunit regions of each meganuclease and the HBV7-binding and HBV8-binding subunit regions, respectively, of HBV meganuclease 7-8x.2.

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Table 4. Examples of engineered meganucleases that are engineered to recognize and cleave the HBV recognition sequence 11-12 (SEQ ID NO: 16)

Meganu cleaza AK SEQ ID Residues of the HBV11 subunit SEQ ID HBV11 subunits *% HBV11 subunit Remains of the HBV12 subunit SEQ ID subunits HBV12 *% HBV12 subunit VGV 11 - 12x.26 33 198 -344 70 100 7-153 77 100 VGV 11 - 12x.9 34 198 -344 71 93.2 7-153 78 93.88 VGV 11 - 12x.13 35 198 -344 72 93.88 7-153 79 93.88 VGV 11 - 12x.16 36 198 -344 73 95.92 7-153 80 95.92 VGV 11 - 12x.27 37 198 -344 74 100 7-153 81 93.2 VGV 11 - 12x.41 38 198 -344 75 95.92 7-153 82 93.2 VGV 11 - 12x.48 39 198 -344 76 93.88 7-153 83 93.2

* % HBV11 subunit and % HBV12 subunit denote amino acid sequence identity between HBV11-binding and HBV12-binding subunit regions of each meganuclease and HBV11-binding and HBV12-binding subunit regions, respectively, of HBV 11-12x meganuclease.26.

2.3. Methods for delivery and expression of endonucleases.

This application describes methods of treating an HBV or HCC infection in a subject. Similarly, methods are provided to reduce the symptoms of HBV infection and reduce the amount of HBV, reduce the rate of HBV proliferation, or treat HCC in a subject, comprising administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an engineered meganuclease described herein (or a nucleic acid encoding an engineered meganuclease) . In the methods of the present invention, the engineered meganuclease described herein can be delivered and/or expressed from DNA/RNA in target cells, allowing the engineered meganuclease to be provided with the HBV gene. The engineered meganucleases described herein can be delivered to the cell as a protein or, preferably, as a nucleic acid encoding the engineered meganuclease. Such a nucleic acid may be DNA (eg, circular or linearized plasmid DNA or PCR products) or RNA (eg, mRNA). In embodiments in which the engineered meganuclease coding sequence is delivered as DNA, it must be operably linked to a promoter to cause transcription of the nuclease gene. Mammalian promoters suitable for the present invention include constitutive promoters such as the cytomegalovirus (CMV) early promoter (Thomsen et al. (1984), Proc Natl Acad Sci USA. 81(3):659-63) or the SV40 early promoter (Benoist and Chambon (1981), Nature. 290(5804):304-10) as well as inducible promoters such as the tetracycline inducible promoter (Dingermann et al. (1992) Mol Cell Biol. 12(9):4038-45) . The engineered meganuclease of the present invention can also be operably linked to a synthetic promoter. Synthetic promoters may include, without limitation, the JeT promoter (WO 2002/012514). In specific embodiments, the nucleic acid sequence encoding the engineered meganuclease described herein may be operably linked to a liver-specific promoter. Examples of liver-specific promoters include, without limitation, the human alpha-1 antitrypsin promoter and the apolipoprotein A-II promoter.

In specific embodiments, a nucleic acid sequence encoding at least one engineered meganuclease is delivered as part of a recombinant DNA construct or expression cassette. For example, a recombinant DNA construct may comprise an expression cassette (ie, cassette) containing a promoter and a nucleic acid sequence encoding the engineered meganuclease described herein. In other embodiments, the recombinant DNA construct comprises two or more cassettes, where each cassette contains a promoter and a nucleic acid sequence encoding the engineered meganuclease described herein, and where each engineered meganuclease has specificity for a different HBV recognition sequence described herein. application. In specific embodiments, the recombinant DNA construct may comprise two cassettes, three cassettes, four cassettes, or more cassettes. For example, a cassette or combination of cassettes can encode any number or combination of HBV meganuclease 1-2, HBV meganuclease 5-6, HBV meganuclease 78, and HBV meganuclease 11-12. In some embodiments, a single cassette may encode HBV meganuclease 1-2, HBV meganuclease 5-6, HBV meganuclease 7-8, and HBV meganuclease 11-12. In another specific embodiment, the cassette or combination of cassettes may encode HBV 56 meganuclease and HBV 11-12 meganuclease. In other embodiments, the recombinant DNA construct comprises a cassette containing a promoter and a polycistronic nucleic acid sequence, said promoter driving expression in the target cell of the polycistronic nucleic acid sequence to form the polycistronic mRNA described herein.

In some embodiments, mRNA encoding the engineered meganuclease is delivered to the cell, as this reduces the likelihood that the gene encoding the engineered meganuclease will be inserted into the cell's genome. Such mRNA encoding an engineered meganuclease can be obtained using methods known in the art, such as in vitro transcription. In some embodiments, the mRNA is capped using 7-methylguanosine. In some embodiments, the mRNA may be polyadenylated. In specific embodiments, the mRNA encoding the engineered nuclease of the present invention may be a polycistronic mRNA encoding two or more nucleases that are simultaneously expressed in the cell. In some embodiments, the polycistronic mRNA may encode two or more of the meganucleases described herein that target different recognition sequences in the HBV genome such that the HBV genome is cut at multiple sites. In some embodiments, the polycistronic mRNA may encode two or more of the meganucleases described herein and at least one additional protein that elicits a therapeutically beneficial effect in a cell. The polycistronic mRNA of the present invention may contain any element known in the art to allow translation of two or more genes from the same mRNA molecule, including but not limited to: the IRES element, the T2A element, the P2A element, the E2A element, and the F2A element . In specific embodiments, the polycistronic mRNA is a bicistronic mRNA encoding two meganucleases described in this application, a tricistronic mRNA encoding three meganucleases described in this application, or a tetracistronic mRNA encoding four meganucleases described in this application, while the nucleases encoded each mRNA have specificity for different recognition sequences in the HBV genome. For example, a polycistronic mRNA can encode any number or combination of HBV meganuclease 1-2, HBV meganuclease 5-6, HBV meganuclease 7-8, and HBV meganuclease 11-12. In some embodiments, the polycistronic mRNA can encode for HBV meganuclease 1-2, HBV meganuclease 5-6, HBV meganuclease 7-8, and HBV meganuclease 11-12. In another specific embodiment, the polycistronic mRNA may be a bicistronic mRNA encoding HBV 5-6 meganuclease and HBV 11-12 meganuclease.

In another specific embodiment, a nucleic acid encoding an endonuclease of the present invention can be introduced into a cell using a single-stranded DNA template. Said single stranded DNA may further comprise a 5' and/or 3' inverted terminal repeat (ITR) of AAB upstream and/or downstream of the engineered meganuclease coding sequence. In other embodiments, the single-stranded DNA may further comprise a 5' and/or 3' homology arm upstream and/or downstream from the engineered meganuclease coding sequence.

In another specific embodiment, the genes encoding the endonuclease of the present invention can be introduced into a cell using a linearized DNA template. In some examples, plasmid DNA encoding an endonuclease can be cleaved with one or more restriction enzymes such that the circular plasmid DNA is linearized before being introduced into a cell.

Purified nuclease proteins can be delivered to cells for cleavage of genomic DNA using a wide variety of mechanisms known in the art, including mechanisms further detailed below in this application.

Target tissue(s) for delivery of the engineered meganucleases of the present invention include(s) without limitation: liver cells such as a hepatocyte or preferably a primary hepatocyte, more preferably a human hepatocyte or a primary human hepatocyte, a HepG2.2.15 cell or HepG2-hNTCP. As already discussed, the meganucleases of the present invention can be delivered as a purified protein or as RNA or DNA encoding the meganuclease. In one embodiment, meganuclease proteins, or mRNA or DNA vectors encoding endonucleases, are delivered to target cells (eg, liver cells) by injection directly into the target tissue. Alternatively, the endonuclease protein, mRNA or DNA can be delivered systemically through the bloodstream. In some embodiments, endonuclease proteins, or DNA/mRNA encoding endonucleases, are formulated for systemic administration or administration to target tissues in a pharmaceutically acceptable carrier in accordance with known techniques. See, for example, Remington, The Science and Practice of Pharmacy (21st ed., 2005). In the manufacture of a pharmaceutical composition according to the present invention, proteins/RNA/mRNA are usually mixed with a pharmaceutically acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with all other ingredients of the formulation and must not be harmful to the patient. The carrier may be solid or liquid, or solid and liquid, and may be included in a single dose formulation with said compound.

In some embodiments, endonuclease proteins or DNA/mRNA encoding an endonuclease are coupled to a cell-penetrating peptide or targeting ligand to promote cellular uptake. Examples of cell-penetrating peptides known in the art include polyarginine (Jearawiriyapaisarn, et al. (2008) Mol Ther. 16:1624-9), the TAT peptide from the HIV virus (Hudecz et al. (2005), Med. Res. Rev. 25:679-736), MPG (Simeoni, et al. (2003) Nucleic Acids Res. 31:27172724), Pep-1 (Deshayes et al. (2004) Biochemistry 43:7698-7706) and HSV-1 VP-22 (Deshayes et al. (2005) Cell

- 24 042452

Mol Life Sci. 62:1839-49). In an alternative embodiment, endonuclease proteins, or DNA/mRNA encoding an endonuclease, are linked covalently or non-covalently to an antibody that recognizes a specific cell surface receptor expressed on target cells such that the endonuclease protein/DNA/mRNA binds to it and enters the cells. -targets. Alternatively, the endonuclease protein/DNA/mRNA can be coupled covalently or non-covalently to a natural ligand (or part of a natural ligand) for such a cell surface receptor. (McCall, et al. (2014) Tissue Barriers. 2(4):e944449; Dinda, et al. (2013) Curr Pharm Biotechnol. 14:1264-74; Kang, et al. (2014) Curr Pharm Biotechnol. 15 (3):220-30; Qian et al. (2014) Expert Opin Drug Metab Toxicol 10(11):1491-508).

In some embodiments, endonuclease proteins, or DNA/mRNA encoding endonucleases, are embedded (encapsulated) in biodegradable hydrogels for injection or implantation within a desired region of the liver (e.g., near hepatic sinusoidal capillary endothelial cells or hematopoietic endothelial or progenitor cells, which are differentiated in them). Hydrogels can provide a sustained and controlled release of a therapeutic load to the desired target tissue area without the need for frequent injections, and stimulus-sensitive materials (e.g., temperature and pH sensitive hydrogels) can be designed to release a load in response to environmental cues or external signals (Kang Derwent et al. (2008) Trans Am Ophthalmol Soc. 106:206-214). In some embodiments, endonuclease proteins, or DNA/mRNA encoding endonucleases, are covalently or preferably non-covalently attached to, or encapsulated within, a nanoparticle using methods known in the art (Sharma, et al. (2014) Biomed Res Int. 2014). The nanoparticle is a nanoscale delivery system, the linear scale of which is <1 μm, preferably <100 nm. Such nanoparticles can be designed using a core composed of a metal, lipid, polymer, or biological macromolecule, and multiple copies of proteins, mRNA, or endonuclease DNA can be attached to or encapsulated in said nanoparticle core. This increases the number of protein/mRNA/DNA copies that are delivered to each cell and therefore increases the intracellular expression of each endonuclease to maximize the likelihood that target recognition sequences will be cut. The surface of such a nanoparticle can be further modified with polymers or lipids (e.g., chitosan, cationic polymers, or cationic lipids) to produce a core-shell nanoparticle whose surface contains additional functional groups to improve cell delivery and load absorption (Jian et al. (2012) Biomaterials 33(30):7621-30). The nanoparticles can additionally be successfully coupled with targeting molecules to target the nanoparticle to the appropriate cell type and/or increase the likelihood of uptake by the cell. Examples of such targeting molecules include antibodies specific for cell surface receptors and natural ligands (or portions of natural ligands) for cell surface receptors.

In some embodiments, endonuclease proteins or DNA/mRNA encoding endonucleases are encapsulated in liposomes or complexed with cationic lipids (e.g., Lipofectamine transfection reagent, Life Technologies Corp., Carlsbad, CA; Zuris et al. (2015) Nat Biotechnol 33:73-80; Mishra et al. (2011) J Drug Deliv 2011:863734). The liposome and lipoplex formulations can protect the load from degradation, increase uptake and retention in the target area, and promote cellular uptake and delivery efficiency through fusion with and/or rupture of cell membranes of target cells.

In some embodiments, endonuclease proteins, or DNA/mRNA encoding endonucleases, are encapsulated in polymeric scaffolds (e.g., lactic acid glycolic acid copolymer (PLGA)) or complexed with cationic polymers (e.g., polyethyleneimine (PEI), poly-L-lysine (PLL)) (Tamboli et al. (2011) Ther Deliv. 2(4):523-536). Polymer carriers can be designed to allow controlled drug release rates by controlling polymer degradation and drug diffusion, and high drug encapsulation efficiencies can provide protection of the therapeutic load until intracellular delivery to the desired target cell population. In some embodiments, endonuclease proteins, or DNA/mRNA encoding engineered meganucleases, are combined with amphiphilic molecules that self-assemble into micelles (Tong et al. (2007) J Gene Med. 9(11):956-66). The polymer micelles may contain a micellar shell formed together with a hydrophilic polymer (eg, polyethylene glycol) which can prevent aggregation, shield charge interactions, and reduce non-specific interactions.

In some embodiments, the endonuclease proteins, or DNA/mRNA encoding the endonuclease, are in the form of an emulsion or nanoemulsion (ie, <1 nm mean particle diameter) for administration and/or delivery to a target cell. The term emulsion refers, without limitation, to any oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil dispersions or droplets containing lipid structures that can be formed by hydrophobic forces,

- 25 042452 which lead non-polar residues (eg long hydrocarbon chains) away from the water and polar heads towards the water when the water-immiscible phase is mixed with the aqueous phase. These other lipid structures include, but are not limited to, monolamellar, oligolamellar, and multilamellar lipid vesicles, micelles, and lamellar phases. Emulsions consist of an aqueous phase and a lipophilic phase (usually containing an oil and an organic solvent). Emulsions also often contain one or more surfactants. Nanoemulsion formulations are well known, such as those described in US Patent Applications Nos. 2002/0045667 and 2004/0043041 and US Pat.

In some embodiments, endonuclease proteins, or DNA/mRNA encoding endonucleases, are covalently attached or non-covalently linked to multifunctional polymeric conjugates, DNA dendrimers, and polymeric dendrimers (Mastorakos et al. (2015) Nanoscale. 7(9):3845-56; Cheng et al (2008) J Pharm Sci 97(1):123-43). The formation of a dendrimer can control the capacity and size of the load and can provide high drug loading capacity. Moreover, multiple surface group display can be manipulated to improve stability, reduce non-specific interactions, and increase specific cell targeting and drug release.

In some embodiments, the genes encoding the endonuclease are delivered using a viral vector. Such vectors are known in the art and include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated virus (AAV) vectors (discussed in Vannucci, et al. (2013 New Microbiol. 36:1-22). In some embodiments, viral vectors are administered by injection directly into target tissues (e.g., liver tissue) In alternative embodiments, viral vectors are delivered systemically via the bloodstream It is known in the art that different AAV vectors tend to localize in different tissues In target liver tissues, efficient transduction of hepatocytes has been shown, for example, by AAV serotypes 2, 8, and 9 (Sands (2011) Methods Mol. Biol. 807:141-157) AAV vectors can also be self-complementary, so that they do not require second-strand DNA synthesis in the host cell (McCarty, et al. (2001) Gene Ther. 8:1248-54).

In one embodiment, the viral vector that is used to deliver the endonuclease gene is a self-limiting viral vector. A self-limiting viral vector may have limited persistence in a cell or organism due to the presence of a recognition sequence for the engineered meganuclease within the vector. Thus, a self-limiting viral vector can be designed to encode a promoter, an endonuclease as described herein, and an endonuclease recognition site within the ITR. The self-limiting viral vector delivers an endonuclease gene into a cell, tissue, or organism such that the endonuclease is expressed and is able to cut the cell's genome at an endogenous recognition sequence within the genome. The delivered endonuclease will also find the target site within the self-limiting viral vector itself and cut the vector at the target site. After cutting, the 5' and 3' ends of the viral genome will be exposed and degraded by exonucleases, thus destroying the virus and stopping the production of the endonuclease. If the endonuclease genes are delivered as DNA (eg, plasmids) and/or via a viral vector (eg, AAB), then they must be operably linked to a promoter. In some embodiments, this may be a viral promoter, such as endogenous promoters from a viral vector (eg, LTR from a lentiviral vector) or well-known cytomegalovirus or SV40 virus early promoters. In a preferred embodiment, the meganuclease genes are operably linked to a promoter that drives gene expression preferentially in target cells. Examples of liver-specific promoters include, without limitation, the human alpha-1 antitrypsin promoter and the apolipoprotein A-II promoter.

In specific embodiments, the viral vector contains a cassette containing a promoter and a nucleic acid sequence encoding the engineered meganuclease described in this application. The specified viral vector may also contain two or more cassettes, with each cassette containing a promoter and a nucleic acid sequence encoding an engineered meganuclease described in this application, and each engineered meganuclease has specificity for a different HBV recognition sequence described in this application. In some embodiments, the viral vector contains a single cassette containing a promoter and a polycistronic nucleic acid sequence, said promoter driving the expression of the polycistronic nucleic acid sequence to form a polycistronic mRNA, such as a polycistronic mRNA encoding the engineered meganuclease described herein, in the target cell. .

Methods and compositions are provided for delivering the meganuclease described herein to the liver of a subject infected with HBV. In one embodiment, native hepatocytes that have been removed from a mammal can be transduced with a vector that encodes an engineered meganuclease. Alternatively, native hepatocytes from an HBV-infected subject

- 26 042452 can be transduced ex vivo with an adenoviral vector that encodes an engineered meganuclease and/or a molecule that stimulates liver regeneration, such as hepatotoxin. Preferably, the hepatotoxin is a urokinase-type plasminogen activator (uPA) and has been modified to inhibit its secretion from the hepatocyte upon expression by a viral vector. In another embodiment, said vector encodes a tissue-type plasminogen activator (tPA) that can stimulate de novo regeneration of hepatocytes. Transduced hepatocytes that have been removed from the mammal can then be returned to the mammal, which has conditions that are favorable for expression of the engineered meganuclease. Typically, the transduced hepatocytes can be returned to the patient by infusion through the spleen or portal vasculature, and single or multiple administrations can be made over a period of 1 to 5 or more days. In the in vivo aspect of the methods of the present invention, a retroviral, pseudotype, or adenovirus-associated vector is constructed that encodes the engineered meganuclease and administered to a subject. The introduction of a vector encoding an engineered meganuclease can be carried out together with the introduction of an adenoviral vector that encodes a non-secretory hepatotoxin or encodes a tPA that stimulates hepatocyte regeneration without acting as a hepatotoxin.

Appropriate doses will depend, among other factors, on the characteristics of each selected AAV vector (e.g., serotype, etc.), on the route of administration, on the subject being treated (i.e., age, body weight, sex, and general condition subject), and route of administration. Thus, a suitable dosage may vary from patient to patient. A suitable effective amount can be readily determined by one skilled in the art. The dosing schedule may be a single dose schedule or a multiple dose schedule. Moreover, as many doses as needed can be administered to a subject. A person skilled in the art can easily determine the appropriate number of doses. It may be necessary to change the dosage to take into account an alternative route of administration or to balance the therapeutic benefit and any side effects.

2.4. pharmaceutical compositions.

In some embodiments, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an engineered meganuclease of the present invention, or a pharmaceutically acceptable carrier and an isolated polynucleotide comprising a nucleic acid encoding an engineered meganuclease of the present invention. In other embodiments, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a cell of the present invention that can be delivered to a target tissue, wherein said cell expresses an engineered meganuclease as described herein. Pharmaceutical compositions of the present invention may be useful for treating a subject suffering from HBV, reducing the level or proliferation of HBV, reducing at least one symptom of HBV, or treating HCC.

Pharmaceutical compositions can be designed or selected according to the genotype of the target HBV strain. As described in detail in this application, the meganucleases of the present invention were designed to recognize and cleave recognition sequences in specific HBV genotypes. For example, HBV 1-2 meganucleases (e.g., sequences of SEQ ID NOs: 18-21) recognize and cleave the HBV recognition sequence 1-2, which is found in the genome of at least HBV genotypes A, B, C, E, F, and G. (for example, the sequences of SEQ ID NO: 35 and 7-9, respectively). In addition, the recognition sequences of these engineered meganucleases described in this application can be found in isolates of HBV genotypes A, B, C, D, E, F and G, which are not 100% identical to the sequences of the corresponding examples of genotypes presented in the SEQ ID sequences. NO: 3-9. In this application, the sequences of HBV isolates can be 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 identical to the corresponding genotype example shown in any of SEQ ID NOs: 3-9. In some embodiments, the pharmaceutical compositions described herein may be administered to a subject containing any HBV genotype containing the recognition sequence shown in SEQ ID NO: 10, 12, 14, or 16.

Such pharmaceutical compositions can be obtained in accordance with known methods. See, for example, Remington, The Science and Practice of Pharmacy (21st ed., 2005). In the manufacture of a pharmaceutical composition according to the present invention, the endonuclease polypeptides (or DNA/RNA encoding them) are usually mixed with a pharmaceutically acceptable carrier and the resulting composition is administered to the subject. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be harmful to the subject. In some embodiments, the pharmaceutical compositions of the present invention may further comprise one or more additional agents or biological molecules useful in the treatment of a disease in a subject. Likewise, additional agent(s) and/or biological

- 27 042452 geic(s) molecule(s) can be administered together as a separate composition.

In specific embodiments, the pharmaceutical compositions of the present invention may contain combinations of the engineered meganucleases described herein (or nucleic acids encoding the engineered meganucleases), each engineered meganuclease having specificity for a different HBV recognition sequence such that a single pharmaceutical composition is useful for treating a wide range of HBV genotypes and/or isolates of genotypes in a subject. Similarly, in other embodiments, pharmaceutical compositions of the present invention may contain polycistronic mRNAs (or recombinant DNA constructs or viral vectors containing cassettes that, when expressed, produce polycistronic mRNAs) that encode for a variety of engineered meganucleases described herein. , which are specific to different HBV recognition sequences. Such pharmaceutical compositions are also useful for treating a wide range of HBV genotypes and/or isolates of genotypes in a subject. In any case, such pharmaceutical compositions may be useful as monotherapy when the particular HBV genotype or isolate in the subject is known or unknown.

For example, pharmaceutical compositions containing many different recombinant meganucleases described in this application, or containing nucleic molecules encoding many different recombinant meganucleases described in this application, can be administered to a patient infected with multiple HBV genotypes or infected with unknown HBV genotypes. Accordingly, the provision of pharmaceutical compositions with a variety of different recombinant meganucleases, or containing nucleic molecules encoding a variety of different recombinant meganucleases, allows for flexible choice for the treatment and control of HBV infection when resources do not allow accurate HBV genotyping and when rapid, broad-spectrum treatments are desired.

In specific embodiments of the present invention, the pharmaceutical composition may contain one or more mRNA described in this application, encapsulated within lipid nanoparticles, which are described elsewhere in this application. In particular embodiments, the lipid nanoparticles may comprise two or more mRNAs described herein, each of which encodes an engineered meganuclease of the present invention having specificity for the distinct HBV recognition sequence described herein. In specific embodiments, the lipid nanoparticles may contain two, three, or four mRNAs described herein, each of which encodes an engineered meganuclease of the present invention having specificity for a different HBV recognition sequence. In other embodiments, the lipid nanoparticles may contain one or more polycistronic mRNAs described herein, with each polycistronic mRNA encoding two or more engineered meganucleases of the present invention having specificity for the various HBV recognition sequences described herein. In specific embodiments, the lipid nanoparticles may contain a polycistronic mRNA encoding two, three, or four of the engineered meganucleases described herein. In other specific embodiments, the implementation of lipid nanoparticles may contain two or more polycistronic mRNA described in this application, each of which encodes two or more designed meganucleases according to the present invention. Some lipid nanoparticles for use in the present invention contain at least one cationic lipid, at least one non-cationic lipid, and at least one conjugated lipid. In more specific examples, the lipid nanoparticles may contain from about 50 mole % to about 85 mole % cationic lipid, from about 13 mole % to about 49.5 mole % non-cationic lipid, and from about 0.5 mole % to about 10 mole % lipid conjugate. and be made in such a way that they have a non-lamellar (i.e., non-bilayer) structure. In other specific examples, the lipid nanoparticles may contain from about 40 mole % to about 85 mole % cationic lipid, from about 13 mole % to about 49.5 mole % non-cationic lipid, and from about 0.5 mole % to about 10 mole % lipid conjugate. and be made in such a way that they have a non-lamellar (i.e., non-bilayer) structure.

Cationic lipids may include, for example, one or more of the following: palmitoyl-oleoyl-norarginine (PONA), MPDACA, GUADACA, (6Z, 9Z, 28Z, 31Z)-heptatriaconta-6, 9, 28, 31-tetraen-19yl-4- (dimethylamino)butanoate) (MC3), LenMC3, CP-LenMC3, y-LenMC3, CP-y-LenMC3, MC3MC, MC2MC, MC3 ether, MC4 ether, MC3 amide, Pan-MC3, Rap-MC4 and Rap-MC5, 1,2-dilinoleyloxy-N,Ndimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin- K-C2-DMA; XTC2), 2,2-dilinoleyl-4-(3dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl )[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6DMA), 2,2-dilinoleyl-4-N -methylpepyrazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4- 28 042452 dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy -3 dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLinDAC), 1,2-dilinoleyl hydroxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3dimethylaminopropane (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-(M,M-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-duoleulαmino)-1,2-pronαnduol (DOAP ), 1,2-dilinoleyloxo-3-(2-N,Ndimethylamino)ethoxypropane (DLin-EG-DMA), N,N-diodeide-N,N-dimethide ammonium chloride (DODAC), 1,2-dioleyloxy-N, N-dimethylaminopropane (DODMA), 1,2-distearidoxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N ,N-distearideN,N-dimethylammonium bromide (DDAB), N-(1-(2,3-diodeoidoxy)propide)-N,N,N-trimethidammonium chloride (DOTAP), 3-(N-(N',N '-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(1,2dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydrox andethylammonium bromide (DMRIE), 2,3-dioleyloxy-N[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propaneammonium trifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-( cholest-5-en-3-beta-hydroxybutane-4-oxy)-1-(cis,cis-9,12octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta- hydroxy)-3'-osapentoxy)-3-dimethyl-1(cis,cis-9', 1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1 ,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP) or mixtures thereof. The cationic lipid may also be DLinDMA, DLin-K-C2-DMA (XTC2), MC3, LenMC3, CP-LenMC3, y-LenMC3, CP-y-LenMC3, MC3MC, MC2MC, ether MC3, ether MC4, amide MC3, Pan-MC3, Pan-MC4, Pan MC5 or mixtures thereof.

In various embodiments, the cationic lipid can be from about 50 mole % to about 90 mole %, from about 50 mole % to about 85 mole %, from about 50 mole % to about 80 mole %, from about 50 mole % to about 75 mole % %, from about 50 mole% to about 70 mole%, from about 50 mole% to about 65 mole%, or from about 50 mole% to about 60 mole% of the total amount of lipids present in the specified particle.

In other embodiments, the cationic lipid may be from about 40 mole % to about 90 mole %, from about 40 mole % to about 85 mole %, from about 40 mole % to about 80 mole %, from about 40 mole % to about 75 mole % %, from about 40 mole% to about 70 mole%, from about 40 mole% to about 65 mole%, or from about 40 mole% to about 60 mole% of the total amount of lipids present in the specified particle.

The non-cationic lipid may include, for example, one or more anionic lipids and/or neutral lipids. In preferred embodiments, the non-cationic lipid comprises one of the following neutral lipid components: (1) cholesterol or a derivative thereof; (2) a phospholipid; or (3) a mixture of a phospholipid and cholesterol, or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenon, coprostanol, cholesteryl 2'-hydroxyethyl ether, cholesteryl 4'-hydroxybutyl ether, and mixtures thereof. The phospholipid may be a neutral lipid including, but not limited to: dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylglycerol (POPG), dipalmitoylphosphatidylethanolamine (DPPE) DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), and mixtures thereof. In some preferred embodiments, the phospholipid is DPPC, DSPC, or mixtures thereof. In some embodiments, the non-cationic lipid (e.g., one or more of the phospholipids and/or cholesterol) can be from about 10 mole % to about 60 mole %, from about 15 mole % to about 60 mole %, from about 20 mole % to about 60 mole %, from about 25 mole % to about 60 mole %, from about 30 mole % to about 60 mole %, from about 10 mole % to about 55 mole %, from about 15 mole % to about 55 mole %, from about 20 mole% to about 55 mole%, about 25 mole% to about 55 mole%, about 30 mole% to about 55 mole%, about 13 mole% to about 50 mole%, about 15 mole% to about 50 mole % or from about 20 mole % to about

- 29 042452 mole % of the total amount of lipids present in the specified particle. If the non-cationic lipid is a mixture of a phospholipid and cholesterol or a cholesterol derivative, then said mixture may contain up to about 40, 50, or 60 mole % of the total lipid present in said particle.

The conjugated lipid that inhibits particle aggregation may include, for example, one or more of the following lipids: polyethylene glycol (PEG)-lipid conjugate, polyamide (ATTA)-lipid conjugate, cationic polymer-lipid (CPL) conjugates, or mixtures thereof. In one preferred embodiment, the nucleic acid-lipid particles comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate. In some embodiments, a PEG-lipid conjugate or an ATTAlipid conjugate is used in conjunction with the CPL. The conjugated lipid that inhibits particle aggregation may include a PEG lipid, including, for example, PEG diacylglycerol (DAT), PEG dialkyloxypropyl (DAA), PEG phospholipid, PEG ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEGdipalmityloxypropyl (C16), PEG-distearyloxypropyl (C18) or mixtures thereof.

Additional PEG-lipid conjugates suitable for use in the present invention include, but are not limited to, m-PEG-2000-1,2-di-O-αkyl-sn3-carbomoylglyceride (PEG-CDOMG). The synthesis of PEG-C-DOMG is described in PCT Application No. PCT/US08/88676. Still further PEG-lipid conjugates suitable for use in the present invention include, without limitation: methyl polyethylene glycol (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in US Pat. No. 7,404,969.

In some instances, a conjugated lipid that inhibits particle aggregation (e.g., a PEG-lipid conjugate) may be from about 0.1 mole % to about 2 mole %, from about 0.5 mole % to about 2 mole %, from about 1 mole % to about 2 mole %, from about 0.6 mole % to about 1.9 mole %, from about 0.7 mole % to about 1.8 mole %, from about 0.8 mole % to about 1.7 mole%, from about 1 mole% to about 1.8 mole%, from about 1.2 mole% to about 1.8 mole%, from about 1.2 mole% to about 1.7 mole%, from about 1, 3 mole% to about 1.6 mole%, about 1.4 mole% to about 1.5 mole%, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1 .6, 1.7, 1.8, 1.9, or 2 mole % (or any fraction or range thereof) of total lipids present in the given particle. Typically, in such cases, the average molecular weight of the PEG molecule is approximately 2000 daltons. In other instances, a conjugated lipid that inhibits particle aggregation (e.g., a PEG-lipid conjugate) may be from about 5.0 mole % to about 10 mole %, from about 5 mole % to about 9 mole %, from about 5 mole % to about 8 mole%, from about 6 mole% to about 9 mole%, from about 6 mole% to about 8 mole%, or about 5 mole%, 6 mole%, 7 mole%, 8 mole%, 9 mole%, or 10 mole % (or any fractional part or range thereof) of the total lipids present in said particle. Typically, in such cases, the average molecular weight of the PEG molecule is approximately 750 daltons.

In other embodiments, the composition may contain amphoteric liposomes that contain at least one positively charged carrier and at least one negatively charged carrier that is different from the positively charged carrier, the isoelectric point of said liposomes is between 4 and 8. This purpose achieved due to the fact that the liposomes are prepared with a pH-dependent, changing charge.

Liposomal structures with desirable properties are formed, for example, when the amount of membrane-forming or membrane-based carriers with a cationic charge exceeds the number of carriers with an anionic charge at low pH, and this ratio is reversed at higher pH. This happens whenever the pKa value of the ionizable components is between 4 and 9. As the pH of the medium decreases, all cationic charged carriers become more charged and all anionic charged carriers lose their charge. Cationic compounds suitable for amphoteric liposomes include those cationic compounds that have been described above in this application. Without limitation, highly cationic compounds may include, for example: DC-Choi 3-P-[N-(N',N'-dimethylmethane)carbamoyl]cholesterol, TC-Chol 3-P-[N-(N',N' ,N'trimethylaminoethane)carbamoyl]cholesterol, BGSC bis-guanidinium-spermidine-cholesterol, BGTC (bisguanidinium-tren-cholesterol), DOTAP (1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride, DOSPER (1,3 -dioleoyloxy-2-(6-carboxyspermyl)-propylamide, DOTMA (1,2-dioleoyloxypropyl)N,N,N-trimethylamronium chloride) (Lipofectin®), DORIE ((1,2-dioleoyloxypropyl)-3dimethylhydroxyethylammonium bromide), DOSC (1,2-dioleoyl-3-succinyl-sn-glycerylcholine ester), DOGSDSO (1,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyldisulfidornithine), DDAB (dimethyldioctadecylammonium bromide), DOGS ((C18) 2GlySper3+) N,N-dioctadecylamidoglycol- 30 042452 spermine (Transfectam®) (C18)2Gly+ (N,N-dioctadecylamidoglycine), CTAB (cetyltrimethylammonium bromide), CpyC (cetylpyridinium chloride), DOEPC (1,2-dioleoyl-sn-glycero -3-ethylphosphocholine) or other O-alkylphosphatidylcholine or e tanolamines, amides from lysine, arginine or ornithine, and phosphatidylethanolamine.

Examples of weakly cationic compounds include, without limitation: His-Chol (histaminelcholesterol hemisuccinate), Mo-Chol (morpholine-N-ethylaminocholesterol hemisuccinate), or histidinyl-PE.

Examples of neutral compounds include, without limitation: cholesterol, ceramides, phosphatidylcholines, phosphatidylethanolamines, tetraether lipids, or diacylglycerols.

Anionic compounds suitable for amphoteric liposomes include those non-cationic compounds that have been previously described in this application. Without limitation, examples of weakly anionic compounds may include: CHEMS (cholesterol hemisuccinate), alkyl carboxylic acids with 8-25 carbon atoms, or diacylglycerol hemisuccinate. Additional weakly anionic compounds may include aspartic acid or glutamic acid amides and PE, as well as PS and its amides with glycine, alanine, glutamine, asparagine, serine, cysteine, threonine, tyrosine, glutamic acid, aspartic acid, or other amino acids or amino dicarboxylic acids. According to the same principle, esters of hydroxycarboxylic acids or hydroxydicarboxylic acids and PS are also weakly anionic compounds.

In some embodiments, amphoteric liposomes may contain a conjugated lipid, such as those described above in this application. Specific examples of useful conjugated lipids include, without limitation, PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropan-3-amines. PEG-modified diacylglycerols and dialkylglycerols are particularly preferred. In some embodiments, neutral lipids can comprise from about 10 mole % to about 60 mole %, from about 15 mole % to about 60 mole %, from about 20 mole % to about 60 mole %, from about 25 mole % to about 60 mole % %, from about 30 mole % to about 60 mole %, from about 10 mole % to about 55 mole %, from about 15 mole % to about 55 mole %, from about 20 mole % to about 55 mole %, from about 25 mole % % to about 55 mole %, from about 30 mole % to about 55 mole %, from about 13 mole % to about 50 mole %, from about 15 mole % to about 50 mole %, or from about 20 mole % to about 50 mole % from the total amount of lipids present in the specified particle.

In some instances, a conjugated lipid that inhibits particle aggregation (e.g., a PEG-lipid conjugate) may be from about 0.1 mole % to about 2 mole %, from about 0.5 mole % to about 2 mole %, from about 1 mole % to about 2 mole %, from about 0.6 mole % to about 1.9 mole %, from about 0.7 mole % to about 1.8 mole %, from about 0.8 mole % to about 1.7 mole%, from about 1 mole% to about 1.8 mole%, from about 1.2 mole% to about 1.8 mole%, from about 1.2 mole% to about 1.7 mole%, from about 1, 3 mole% to about 1.6 mole%, about 1.4 mole% to about 1.5 mole%, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1 .6, 1.7, 1.8, 1.9, or 2 mole % (or any fraction or range thereof) of total lipids present in the given particle. Typically, in such cases, the average molecular weight of the PEG molecule is approximately 2000 daltons. In other instances, a conjugated lipid that inhibits particle aggregation (e.g., a PEG-lipid conjugate) may be from about 5.0 mole % to about 10 mole %, from about 5 mole % to about 9 mole %, from about 5 mole % to about 8 mole%, from about 6 mole% to about 9 mole%, from about 6 mole% to about 8 mole%, or about 5 mole%, 6 mole%, 7 mole%, 8 mole%, 9 mole%, or 10 mole % (or any fractional part or range thereof) of the total lipids present in said particle. Typically, in such cases, the average molecular weight of the PEG molecule is approximately 750 daltons.

Given the total amount of neutral and conjugated lipids, the remaining balance of the amphoteric liposome may be a mixture of cationic compounds and anionic compounds, composed in various ratios. The ratio of cationic to anionic lipids can be selected to achieve the desired nucleic acid encapsulation properties, zeta potential, pKa, or other physicochemical property that is at least in part dependent on the presence of charged lipid components.

In some embodiments, said lipid nanoparticles have a composition that specifically enhances delivery and absorption in the liver and, in particular, within hepatocytes.

2.5. Methods for obtaining recombinant AAV vectors.

- 31 042452

In some embodiments of the present invention, recombinant AAV vectors are provided for use in the methods of the present invention. Recombinant AAV vectors are usually produced in mammalian cell lines such as HEK-293. Since the viral cap and rep genes are removed from the vector to prevent it from self-replicating and to make room for the therapeutic gene(s) to be delivered (e.g. the endonuclease gene), this must be done in trans in the packaging line. cells. In addition, ancillary (eg, adenoviral) components needed to support replication must be provided (Cots D, Bosch A, Chillon M (2013) Curr. Gene Ther. 13(5): 370-81). Often, recombinant AAV vectors are generated using a triple transfection in which a cell line is transfected with a first plasmid encoding accessory components, a second plasmid containing the cap and rep genes, and a third plasmid containing the viral ITRs, between which is the DNA sequence to be packaged into the virus. Viral particles containing the genome (ITR and intervening gene(s) of interest) enclosed in a capsid are then isolated from cells by freeze-thaw cycles, sonication, detergent, or other means known to in this area. The particles are then purified using cesium chloride density gradient centrifugation or affinity chromatography, and then the gene(s) of interest are delivered to cells, tissues, or an organism, such as a human patient.

Since recombinant AAV particles are usually produced (produced) in cells, precautions must be taken in the practice of the present invention to ensure that the site-specific endonuclease is not expressed in the packaging cells. Since the viral genomes of the present invention contain an endonuclease-recognizable sequence, any endonuclease expressed in a packaging cell line will be able to cleave the viral genome before it can be packaged into viral particles. This will lead to reduced packaging efficiency and/or packaging of fragmented genomes. Several approaches can be used to prevent endonuclease expression in packaging cells, including those described below.

The endonuclease can be placed under the control of a tissue-specific promoter that is inactive in packaging cells. For example, if the viral vector is designed to deliver the endonuclease gene(s) to muscle tissue, then a muscle-specific promoter can be used. Examples of muscle-specific promoters include C5-12 (Liu, et al. (2004) Hum Gene Ther. 15:783-92), muscle-specific creatine kinase (MCK) promoter (Yuasa, et al. (2002) Gene Ther. 9 :1576-88) or smooth muscle promoter 22 (SM22) (Haase, et al. (2013) BMC Biotechnol. 13:49-54). Examples of CNS (neuronal) specific promoters include the NSE, synapsin, and MeCP2 promoters (Lentz, et al. (2012) Neurobiol Dis. 48:179-88). Examples of liver-specific promoters include those for albumin (such as Palb), human a1-antitrypsin (such as Pal AT) and hemopexin (such as Phpx) (Kramer, MG et al., (2003) Mol. Therapy 7:375-85 ). Examples of eye-specific promoters include the opsin promoter and the corneal epithelium-specific K12 promoter (Martin KRG, Klein RL, and Quigley HA (2002) Methods (28): 267-75) (Tong Y, et al., (2007) J Gene Med 9:956-66). These promoters, or other tissue-specific promoters known in the art, are not highly active in HEK-293 cells and therefore would not be expected to give significant levels of endonuclease gene expression in packaging cells when inserted into the viral vectors of the present invention. Similarly, the use of other cell lines with viral vectors of the present invention using incompatible tissue-specific promoters (ie, the well-known HeLa (human epithelial cell) cell line and use of the liver-specific hemopexin promoter) is contemplated. Other examples of tissue-specific promoters include: synovial sarcomas PDZD4 (cerebellum), C6 (liver), ASB5 (muscle), PPP1R12B (heart), SLC5A12 (kidney), cholesterol regulation AROM (liver), ADPRHL1 (heart), and monogenic congenital malformation syndromes TP73L (muscle). (Jacox E, et al., (2010) PLoS One vol 5(8):e12274). Alternatively, the vector can be packaged in cells from a distinct species in which the endonuclease is unlikely to be expressed. For example, viral particles can be produced in microbial, insect, or plant cells using mammalian promoters, such as the well-known cytomegalovirus or SV40 early promoters, which are inactive in non-mammalian packaging cells. In a preferred embodiment, viral particles are generated in insect cells using a baculovirus system as described by Gao, et al. (Gao, H., et al. (2007) J. Biotechnol. 131 (2): 138-43). An endonuclease under the control of a mammalian promoter is unlikely to be expressed in these cells (Airenne, KJ, et al. (2013) Mol. Ther. 21(4):739-49). Moreover, insect cells use different mRNA splicing motifs from those in mammalian cells. Therefore, a mammalian intron, such as the human growth hormone (HGH) intron or the SV40 large T antigen intron, can be included in the endonuclease coding sequence. Since these introns are not able to efficiently splice from pre-mRNA transcripts in insect cells, insect cells will not express a functional endonuclease and will package a full-length genome. Conversely, mammalian cells delivered with the resulting recombinant AAV particles will properly splice the pre-mRNA and express a functional endonuclease protein.

- 32 042452

Haifeng Chen described the use of HGH introns and the SV40 large T antigen to reduce the expression of toxic barnase proteins and diphtheria toxin fragment A in insect packaging cells, allowing the production of recombinant AAB vectors carrying genes for these toxins (Chen, H (2012) Mol Ther Nucleic Acids .1(11): e57). An endonuclease gene can be operably linked to an inducible promoter such that a small molecular weight inducer is required for expression of the endonuclease. Examples of inducible promoters include the Tet-On system (Clontech; Chen H., et al., (2015) BMC Biotechnol. 15(1):4)) and the RheoSwitch system (Intrexon; Sowa G., et al., (2011) Spine, 36(10): E623-8). Both systems, as well as similar systems known in the art, use ligand-inducible transcription factors (Tet repressor variants and ecdysone receptors, respectively) that activate transcription in response to a small molecule activator (doxycycline or ecdysone, respectively). The implementation of the present invention using such ligand-inducible transcription activators includes: 1) placing the endonuclease gene under the control of a promoter that is responsive to the appropriate transcription factor, said endonuclease gene contains the binding site(s) for said transcription factor; and 2) incorporating a gene encoding said transcription factor into the packaged viral genome. The last step is necessary because the endonuclease will not be expressed in target cells or tissues after delivery of recombinant AAV unless a transcription activator is also introduced into the same cells. The transcription activator then induces expression of the endonuclease gene only in cells or tissues that have been treated with the appropriate small molecule activator. This approach is preferable, since it allows spatiotemporal regulation of endonuclease gene expression by choosing when and to which tissues to deliver the low molecular weight inducer. However, the need to include the inducer in the viral genome, which is significantly limited in capacity, is a disadvantage of this approach.

In another preferred embodiment, recombinant AAV particles are produced in a mammalian cell line that expresses a transcriptional repressor that prevents endonuclease expression. Transcriptional repressors are known in the art and include the Tet repressor, the Lac repressor, the Cro repressor, and the Lambda repressor. Many nuclear hormone receptors, such as ecdysone receptors, also act as transcriptional repressors in the absence of a recognizable hormone ligand. To implement the present invention, packaging cells are transfected/transduced with a vector encoding a transcriptional repressor and an endonuclease gene in the viral genome (packaging vector) is operably linked to a promoter that is modified to contain repressor binding sites such that the repressor turns off the promoter. A gene encoding a transcriptional repressor can be placed in various positions. It can be encoded on a separate vector; it can be included in the packing vector outside of the ITR sequences; it can be included in a cap/rep vector or in an adenoviral helper vector; or, most preferably, it can be stably inserted into the genome of the packaging cell so that it is constitutively expressed. Methods for modifying widely distributed mammalian promoters to contain transcriptional repressor sites are known in the art. For example, Chang and Roninson modified the strong constitutive promoters of CMV and RSV to contain operators for the Lac repressor and showed that gene expression from the modified promoters was greatly reduced in cells expressing the repressor (Chang BD, and Roninson IB (1996) Gene 183:137-42). The use of a non-human transcriptional repressor ensures that transcription of the endonuclease gene is only repressed in packaging cells expressing the repressor, and not in target cells or tissues transduced with the resulting recombinant AAV vector.

2.6. Variants of engineered meganucleases.

Embodiments of the present invention include the engineered meganucleases described herein and variants thereof. Additional embodiments of the present invention include isolated polynucleotides comprising a nucleic acid sequence encoding the endonucleases described herein and variants of such polynucleotides.

In this application, the term variants is intended to mean substantially similar sequences. A variant polypeptide is meant to mean a polypeptide derived from a native polypeptide by deletion or insertion of one or more amino acids at one or more internal sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native polypeptide. In this application, the native polynucleotide or polypeptide includes the original sequence from which these variants are derived. The polypeptide variants included in the embodiments are biologically active. That is, they continue to have the desired biological activity of the native protein; those. the ability to recognize and cleave a recognition sequence within the genome ORF of at least two hepatitis B virus genotypes, e.g., HBV recognition sequence 1-2 (SEQ ID NO: 10), HBV recognition sequence 56 (SEQ ID NO: 12), HBV recognition sequence 7 -8 (SEQ ID NO: 14) or HBV recognition sequence 11-12 (SEQ ID NO: 16). Such variants can be obtained as a result of, for example, human processing. The sequences of biologically active variants of the native

- 33 042452 polypeptide according to embodiments (for example, the sequence of SEQ ID NO: 18-39) or biologically active subunit variants that bind the recognized half-site described in this application will be at least about 40%, about 45%, about 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% amino acid sequence identity of the native polypeptide or native subunit, as determined by using programs and parameters for aligning sequences described elsewhere in this application. A biologically active variant polypeptide or subunit according to embodiments may differ from said polypeptide or subunit by as little as about 1-40 amino acid residues, as little as about 1-20, as little as about 1-10, as little as about 5, as little as 4, 3, 2 or even 1 amino acid residue.

The polypeptides of the embodiments can be altered 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 obtained by mutating DNA. Methods for mutagenesis and alteration of polynucleotides are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; US patent number 4873192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and references cited in references cited. Guidance on suitable amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model by Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), included in this application by reference. Conservative substitutions, such as replacing one amino acid with another having similar properties, may be optimal.

In some embodiments, engineered meganucleases of the present invention may contain variants of the HVR1 and HVR2 regions described herein. HVR parent regions may include, for example, residues 24-79 or residues 215-270 of exemplary engineered meganucleases. Thus, HVR variants may include an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or more identical to the amino acid sequence corresponding to residues 24-79 or residues 215-270 of said engineered meganucleases exemplified in this application, such that said HVR region variants retain the biological activity of the engineered meganuclease (ie, binding and cleavage of the recognition sequence). In addition, in some embodiments of the present invention, a variant HVR1 region or a variant HVR2 region may include residues corresponding to amino acid residues located at certain positions within the original HVR. In this context, relevant means that the amino acid residue in the HVR variant is the same amino acid residue (i.e., a single identical residue) that is present in the original HVR sequence in the same relative position (i.e., relative to the rest of the amino acids in the original sequence). ). As an example, if the original HVR sequence contains a serine residue at position 26, then the HVR variant that contains the residue corresponding to residue 26 will also contain the serine at the position that is compared to the original position 26. A significant number of amino acid modifications have previously been identified in the domain DNA recognition of wild-type I-CreI meganuclease (e.g., US Pat. No. 8,021,867) which, alone or in combination, result in engineered meganucleases with specificities altered at single bases within the half-site of the DNA recognition sequence such that rationally designed meganucleases have specificities half-sites are different from those of the wild-type enzyme. In table. 5 shows potential substitutions that can be made in the engineered meganuclease monomer or subunit to increase specificity based on the base present at each position (-1 to -9) of the recognition half site.

- 34 042452

Table 5

Preferred base in the sense strand According to lies A With G T A/T A/C A/G S/T G/T A/G/T A/C/G/T -1 Y75 L75* C75* Y139* С46* A46* R70* H75* R75* H46* K46* R46* K70 E70* E75* E46* D46* Q70* C70 L70 Y75* Q75* H75* H139 Q46* H46* T46* G70 A70 S70 G46* -2 Q70 T44* A44* V44* 144* L44* N44* E70 D70 K44* R44* H70 D44* E44* Q44* С44* -3 Q68 C24* 124* E68 F68 K24* R24* R68 M68 C68 L68 F68 H68 Y68 K68 -4 A26* Q77 E77 K26* R77 E26* S77 Q26* S26* -5 E42 R42 K28* C28* Q42 M66 K66 -6 Q40 C28* E40 R28* R40 С40 140 V40 C79 179 V79 Q28* A40 A79 A28* H28* S40 S28* -7 N30* Q38 E38 KZO* R30* KZ 8 R38 EZO* 138 L38 C38 H38 N38 Q30* -8 F33 Y33 E3Z D33 F33 NZZ L33 V33 133 F33 szz R32* R33 -9 E32 R32 K32 L32 V32 D32 132 S32 N32

Preferred base in the sense strand A32 C32 H32 Q32 T32

Residues in bold are wild-type contact residues and are not modifications described in this application. The asterisk indicates that the residue is in contact with a base on the antisense strand.

- 35 042452

For polynucleotides, the variant includes the deletion and/or insertion of one or more nucleotides at one or more sites within the native polynucleotide. One skilled in the art will recognize that the nucleic acid variants of the embodiments will be designed to retain an open reading frame. For polynucleotides, conservative variants include those sequences that, due to the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the embodiments. Polynucleotide variants include synthetically produced polynucleotides such as those obtained, for example, using site-directed mutagenesis, but which still encode an engineered meganuclease according to the embodiments. Typically, the sequences of variants of a particular polynucleotide according to embodiments will be at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, about 98%, about 99% or more identical to this particular polynucleotide, as determined by the programs and parameters for aligning sequences described elsewhere in this application. Variants of a particular polynucleotide according to embodiments (ie, the parent polynucleotide) can also be assessed by comparing the percent sequence identity between the polypeptide encoded by the polynucleotide variant and the polypeptide encoded by the parent polynucleotide. It is not expected that deletions, insertions and substitutions of protein sequences included in the scope of the present description, cause radical changes in the properties of the specified polypeptide. However, in cases where it is difficult to predict in advance the exact effect of a substitution, deletion, or insertion, it will be apparent to one skilled in the art that the effect will be assessed by screening for the ability of the polypeptide to preferentially recognize and cleave the recognition sequence within the ORF of the genome of at least two genotypes. hepatitis b virus.

Examples

The present invention is further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will identify, or be able to ascertain using no more than routine research, many equivalents to the specific substances and procedures described in this application. Such equivalents are intended to be included within the scope of the claims, which appear after the examples below.

Example 1

Characteristics of meganucleases that recognize and cleave HBV recognition sequences.

Meganucleases that recognize and cleave the HBV 1-2 recognition sequence.

The engineered meganucleases (SEQ ID NOs: 18-21), collectively referred to herein as HBV 1-2 meganucleases, were engineered to recognize and cleave the HBV 1-2 recognition sequence (SEQ ID NO: 10) that is located within the ORF of the P protein , S, preS2/S, and preS1/preS2 of multiple HBV genotypes, including genotype A, B, C, E, F, and G (eg, SEQ ID NOs: 3-5 and 7-9, respectively). Each engineered HBV 1-2 meganuclease contains an N-terminal nuclease localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit in each HBV 1-2 meganuclease binds to the HBV1 recognition half-site of SEQ ID NO: 10, while the second subunit binds to the HBV2 recognition half-site (see FIG. 2).

The HBV1 binding subunits and the HBV2 binding subunits each contain a 56 bp hypervariable region, designated HVR1 and HVR2, respectively. The HBV1 binding subunits are highly conserved beyond the HVR1 region. Similarly, the HBV2 binding subunits are also highly conserved beyond the HVR2 region. HBV1 binding sites with SEQ ID NOs: 18-21 are shown in SEQ ID NOs: 40-43, respectively. Each of SEQ ID NOs: 40-43 is at least 90% identical to SEQ ID NO: 40, which is the HBV1 binding site of HBV meganuclease 1-2x.2 (SEQ ID NO: 18). HBV2 binding sites with SEQ ID NOs: 18-21 are shown in SEQ ID NOs: 44-47, respectively. Each of SEQ ID NOs: 44-47 is at least 90% identical to SEQ ID NO: 44, which is the HBV2 binding site of HBV meganuclease 1-2x.2 (SEQ ID NO: 18).

Meganucleases that recognize and cleave the HBV 5-6 recognition sequence.

The engineered meganucleases (SEQ ID NOs: 22-28), collectively referred to herein as HBV 5-6 meganucleases, were engineered to recognize and cleave the HBV 5-6 recognition sequence (SEQ ID NO: 12) that is located within the ORF of the P protein , S, preS2/S, and preS1/preS2 of multiple HBV genotypes, including genotype A, B, C, D, E, and G (eg, SEQ ID NOs: 3-7 and 9, respectively). Each engineered HBV 5-6 meganuclease contains an N-terminal nuclease localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit in each HBV 5-6 meganuclease binds to a recognized HBV 5 half-site with the sequence SEQ

ID NO: 12, while the second subunit binds to the recognized HBV6 half-site (see FIG. 2).

The HBV5 binding subunits and the HBV6 binding subunits each contain a 56 bp hypervariable region designated HVR1 and HVR2, respectively. HBV5 binding subunits are highly conserved beyond the HVR1 region. Similarly, the HBV6 binding subunits are also highly conserved beyond the HVR2 region. HBV5 binding sites with SEQ ID NOs: 22-28 are shown in SEQ ID NOs: 48-54, respectively. Each of SEQ ID NOs: 48-54 is at least 90% identical to SEQ ID NO: 49, which is the HBV5 binding site of HBV meganuclease 5-6x.33 (SEQ ID NO: 22). HBV6 binding sites with SEQ ID NOs: 22-28 are shown in SEQ ID NOs: 55-61, respectively. Each of SEQ ID NOs: 55-61 is at least 90% identical to SEQ ID NO: 55, which is the HBV5 binding site of HBV meganuclease 5-6x.33 (SEQ ID NO: 22).

Meganucleases that recognize and cleave the HBV 7-8 recognition sequence.

The engineered meganucleases (SEQ ID NOs: 29-32), collectively referred to herein as HBV 7-8 meganucleases, were engineered to recognize and cleave the HBV 7-8 recognition sequence (SEQ ID NO: 14) which is located within the ORF of the P protein multiple HBV genotypes, including genotype A, B, C, D, F, and G (eg, SEQ ID NOs: 3-6, 8, and 9, respectively). Each engineered HBV 7-8 meganuclease contains an N-terminal nuclease localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit in each HBV 7-8 meganuclease binds to the HBV7 recognition half-site of SEQ ID NO: 14, while the second subunit binds to the HBV8 recognition half-site (see FIG. 2). The HBV7 binding subunits and the HBV8 binding subunits each contain a 56 bp hypervariable region designated HVR1 and HVR2, respectively. HBV7 binding subunits are highly conserved beyond the HVR1 region. Similarly, the HBV8 binding subunits are also highly conserved beyond the HVR2 region. HBV7 binding sites with SEQ ID NOs: 29-32 are shown in SEQ ID NOs: 62-65, respectively. Each of SEQ ID NOs: 62-65 is at least 90% identical to SEQ ID NO: 62, which is the HBV7 binding site of HBV meganuclease 7-8x.2 (SEQ ID NO: 29). HBV8 binding sites with SEQ ID NOs: 29-32 are shown in SEQ ID NOs: 66-69, respectively. Each of SEQ ID NOs: 66-69 is at least 90% identical to SEQ ID NO: 66, which is the HBV8 binding site of HBV meganuclease 7-8x.2 (SEQ ID NO: 29).

Meganucleases that recognize and cleave the recognition sequence of HBV 11-12.

The engineered meganucleases (SEQ ID NOs: 33-39), collectively referred to herein as HBV 11-12 meganucleases, were engineered to recognize and cleave the HBV recognition sequence 11-12 (SEQ ID NO: 16) which is located within the ORF of the P protein multiple HBV genotypes including genotype A, B, C, D, E, F, and G (eg, SEQ ID NOs: 3-9, respectively). Each engineered HBV 11-12 meganuclease contains an N-terminal nuclease localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit in each HBV 11-12 meganuclease binds to the HBV11 recognition half-site of SEQ ID NO: 16, while the second subunit binds to the HBV 12 recognition half-site (see FIG. 2). The HBV 11 binding subunits and the HBV 12 binding subunits each contain a 56 bp hypervariable region, designated HVR1 and HVR2, respectively. The HBV 11 binding subunits are highly conserved beyond the HVR1 region. Similarly, the HBV 12 binding subunits are also highly conserved beyond the HVR2 region. HBV 11 binding sites with SEQ ID NOs: 33-39 are shown in SEQ ID NOs: 70-76, respectively. Each of SEQ ID NOs: 70-76 is at least 90% identical to SEQ ID NO: 70, which is the HBV11 binding site of HBV meganuclease 11-12x.26 (SEQ ID NO: 33). HBV 12 binding sites with sequences of SEQ ID NOs: 33-39 are shown in sequences of SEQ ID NOs: 77-83, respectively. Each of SEQ ID NOs: 77-83 is at least 90% identical to SEQ ID NO: 77, which is the HBV 12 binding site of HBV meganuclease 11-12x.26 (SEQ ID NO:33).

Cleavage of HBV recognition sequences in reporter analysis in CHO cells.

In order to determine whether the HBV 1-2, HBV 5-6, HBV 7-8, and HBV 11-12 meganucleases can recognize and cleave the respective recognition sequences (SEQ ID NOs: 10, 12, 14, and 16, respectively), each engineered meganuclease was evaluated using

- 37 042452 reporter assay in CHO cells as previously described (see WO/2012/167192 and FIG. 6). For these assays, CHO reporter cell lines were generated that carried a non-functional green fluorescent protein (GFP) gene expression cassette inserted into the genome of these cells. The GFP gene in each cell line was interrupted by a pair of recognition sequences such that intracellular cleavage of both recognition sequences by the meganuclease would stimulate a homologous recombination event resulting in the production of a functional GFP gene. In the CHO reporter cell lines developed for this study, one recognition sequence inserted into the GFP gene was HBV recognition sequence 1-2 (SEQ ID NO: 10), HBV recognition sequence 5-6 (SEQ ID NO: 12), an HBV 78 recognition sequence (SEQ ID NO: 12); and an HBV11-12 recognition sequence (SEQ ID NO: 16). The second recognition sequence inserted into the GFP gene was the CHO-23/24 recognition sequence, which is recognized and cleaved by a control meganuclease named CHO-23/24. CHO reporter cells containing the HBV 1-2 recognition sequence and the CHO-23/24 recognition sequence are referred to as HBV 1-2 cells. CHO reporter cells containing the HBV 5-6 recognition sequence and the CHO-23/24 recognition sequence are referred to as HBV 5-6 cells. CHO reporter cells containing the HBV 7-8 recognition sequence and the CHO-23/24 recognition sequence are referred to as HBV 7-8 cells. CHO reporter cells containing the HBV 11-12 recognition sequence and the CHO-23/24 recognition sequence are referred to as HBV 11-12 cells. CHO reporter cells were transfected with plasmid DNA encoding the appropriate engineered meganucleases (eg, HBV 1-2 cells were transfected with plasmid DNA encoding HBV 1-2 meganucleases) or encoding CHO-23/34 meganuclease. In each assay, 4e ⁵ CHO reporter cells were transfected with 50 ng of plasmid DNA in a 96-well plate using Lipofectamine 2000 (ThermoFisher) according to the manufacturer's instructions. 48 hours post-transfection, cells were assessed by flow cytometry to determine the percentage of GFP-positive cells compared to a non-transfected negative control (HBV bs). As shown in Figures 7A-7D, all HBV meganucleases were found to produce GFP-positive cells in cell lines containing the appropriate recognition sequence, and their occurrence significantly exceeded that of the negative control.

The efficacy of HBV 1-2, HBV 5-6, HBV 7-8, and HBV 11-12 meganucleases was also determined in a time-dependent manner at 2, 5, 7, 9, and 12 days after introduction of the meganucleases into CHO reporter cells. In this study, HBV 1-2, HBV 5-6, HBV 7-8, or HBV 11-12 (1.0x106) cells were electroporated with 1x106 copies of meganuclease mRNA per cell using a BioRad Gene Pulser Xcell following the manufacturer's instructions. At the indicated time points after transfection, cells were assessed by flow cytometry to determine the percentage of GFP-positive cells. Meganuclease CHO-23/24 was also included as a positive control at each time point.

Figures 8A-8D show that %GFP generated by various HBV meganucleases changed over time. HBV 5-6 and HBV 11-12 meganucleases were essentially constant over the 12-day assay period (Figures 8B and 8D), while HBV 1-2 and HBV 7-8 meganucleases produced high levels of GFP-positive cells in more than early time points, which decreased over time during the experiment.

Conclusions.

These studies demonstrate that the HBV meganucleases within the scope of the present invention can effectively target and cleave appropriate recognition sequences in cells, and that this effect can either be permanent or change over time.

Example 2

HBV-specific nucleases destroy plasmid DNA in E. coli.

Bacterial reporter system using episomal DNA.

The purpose of this experiment was to evaluate the ability of the HBV meganucleases of the present invention to recognize and cleave recognition sequences within episomal DNA plasmids in the E. coli reporter system. A plasmid named pARCUS was generated to drive inducible expression of the ARCUS nuclease (FIG. 9A). In pARCUS, the ARCUS nuclease (here either HBV 5-6x.33 or HBV 11-12x) was placed under the control of an IPTG inducible promoter. In addition, to allow selection of transformed bacteria, pARCUS encodes an ampicillin resistance gene. pARCUS is a low copy plasmid such that induced expression of the ARCUS nuclease does not result in overexpression.

Received additional plasmid, named pHBVa (Fig. 9A).

pHBVa carries a large fragment of the HBV genome, including the HBV 5-6 and HBV 11-12 recognition sequences. This plasmid also encodes the kanamycin resistance gene and the SacB gene. The kanamycin resistance gene allows the selection of transformed bacteria, and the SacB gene is a

- 38 042452 fighting a toxin that is active in the presence of sucrose. Thus, bacteria transformed with pHBVa will survive in the presence of kanamycin in the medium, but will not survive in the presence of kanamycin and sucrose. pHBVa was designed as a high copy number plasmid in an attempt to replicate an HBV-infected cell that can carry multiple copies of the HBV genome. Importantly, pHBVa uses a different origin of replication from that of pARCUS, which allows co-transformation of bacteria by both plasmids.

When bacteria are co-transformed with these plasmids and grown in media containing various combinations of selective pressure (ampicillin, kanamycin, or sucrose), several possible outcomes result. Bacteria that have transformed pARCUS and grown in the presence of ampicillin will carry the pARCUS plasmid but will not express the encoded nuclease unless the growth medium is supplemented with IPTG. After IPTG induction, the encoded nuclease will be expressed. Bacteria transformed with pHBVa will grow in the presence of kanamycin, but not in the presence of kanamycin and sucrose. Bacteria co-transformed with pARCUS and pHBVa in the presence of ampicillin and/or kanamycin will be sensitive to sucrose. In bacteria co-transformed with pARCUS and pHBV, induction of the ARCUS IPTG nuclease was predicted to result in the expression of the nuclease, which would then be able to cleave the target site encoded by pHBVa. Cleavage at this site was expected to result in linearization of the pHBVa plasmid, which should then be rapidly degraded by bacterial nucleases. Degradation of the pHBV plasmid will have two results: the bacteria will lose resistance to kanamycin but will be able to survive in the presence of sucrose.

In order to test the results of co-transformation, the bacteria were co-transformed (by electroporation) with pARCUS and pHBVa plasmids and cultured in the presence of ampicillin for 3 hours before seeding. In parallel cultures, co-transformed cells were treated with IPTG (3 hours) to induce nuclease expression and allow cleavage of pHBVa. Cells were then seeded on agar plates in the presence of ampicillin, ampicillin and sucrose or ampicillin and kanamycin. The plates were incubated overnight and colonies were counted to assess bacterial survival.

Results.

The number of colonies present on each plate of selective medium provided significant evidence for the ability of ARCUS nucleases to cleave the pHBVa plasmid. On the control plates with ampicillin, the number of colonies from both non-induced and IPTG-induced cultures was equal for cells co-transformed with pHBVa and any pARCUS plasmid (Fig. 9B). There was a difference in colony count between bacteria co-transformed with pARCUS encoding HBV 5-6x.33 and bacteria co-transformed with pARCUS encoding HBV 11-12x.26, but this probably reflects transformation efficiency.

Conversely, the number of colonies in non-IPTG-induced cultures on plates containing both ampicillin and sucrose was greatly reduced, indicating that the SacB gene allowed effective cell killing when sucrose was available (Fig. 9B). However, cultures that induced IPTG grew quite well on plates containing both ampicillin and sucrose, indicating deletion of the SacB gene. Colony numbers of bacteria co-transformed (but not induced by IPTG) with pHBVa and pARCUS encoding HBV 11-12x.26 were the same as on control ampicillin plates, and colony numbers of bacteria co-transformed with pARCUS 5-6x.33 were only slightly less than the control plates.

Non-induced IPTG cultures were able to grow on plates containing both ampicillin and kanamycin (FIG. 9B). The colony numbers of bacteria co-transformed with pARCUS encoding HBV 11-12x.26 were approximately equal to those on control plates containing only ampicillin, and the numbers of cells co-transformed with pARCUS HBV 5-6x.33 were only slightly lower compared to controls. . However, when IPTG-induced cells were grown on plates containing ampicillin and kanamycin, colony numbers were close to zero, indicating the elimination of plasmids carrying the kanamycin resistance gene (FIG. 9B).

Conclusions.

These results clearly demonstrate that the HBV meganucleases of the present invention, which recognize sites in the HBV genome, are able to cut the pHBVa plasmid, resulting in the removal of the pHBVa plasmid. In IPTG-induced cultures, cells grew in the presence of sucrose, indicating efficient removal of the SacB-containing plasmid. Similarly, in IPTG-induced cultures, cells died in the presence of kanamycin, also strongly indicating destruction of the pHBVa plasmid. It is possible that similar results can be obtained in HBV-infected mammalian cells by using the HBV meganucleases of the present invention to cleave HBV cccDNA. In bacteria, linear DNA is rapidly destroyed. It can be assumed that linearized cccDNA in mammalian cells will also be cleaved by cellular nucleases. Even if cleavage does not linearize and eliminate cccDNA, it is likely that insertion/deletion mutations caused by HBV ARCUS nuclease will disrupt coding regions and make

- 39 042452 cccDNA unable to produce functional HBV proteins. Thus, the meganucleases of the present invention, targeting sites in the HBV genome, should be an efficient way to kill or inactivate HBV cccDNA.

Example 3

Targeting HBV viral genomes in cells.

Treatment of AD38 cells expressing the HBV genome.

The primary purpose of these examples was to evaluate whether the meganucleases of the present invention are capable of inactivating and/or eliminating HBV genomes from HBV-infected mammalian cells.

In the first study, the efficacy of meganuclease was evaluated in the AD38 cell line, which stably expresses the HBV genome under the control of the Tet promoter. This AD38 cell line does not produce active viral particles, but produces HBV S antigen (HBsAg), which can be detected in cell-conditioned media. In this study, cells were transfected with plasmid DNA encoding either HBV 5-6x.33 or HBV 11-12x.26. Both of these engineered meganucleases target a sequence specific to the HBV genome, with HBV 5-6x.33 targeting the sequence within the overlapping reading frames S (HBsAg gene) and P (polymerase gene) and HBV 1112x.26 targeting the P gene. As a control, AD38 cells were transfected with a plasmid encoding the red fluorescent protein (RFP) gene. Cells were seeded and 24 hours later transfected with plasmids using a liposome-based transfection protocol. 24 hours after transfection, AD38 cells were washed to remove remaining liposome complexes. On days 3 and 7 after transfection, the cell supernatant was collected and analyzed for the presence of HBsAg by ELISA.

Results.

ELISA results were normalized to the amount of HBsAg present in the supernatant of RFP-transfected AD38 cells on day 3 and day 7 post-transfection. On day 3 post-transfection, cells transfected with the plasmid encoding HBV 11-12x.26 showed approximately 25% less HBsAg in the supernatant compared to RFP-transfected cells (FIG. 10). At the same time point, cells transfected with the plasmid encoding HBV 5-6x.33 showed approximately 50% less HBsAg in the supernatant compared to RFP-transfected cells (FIG. 10). Non-transfected control cells were essentially the same as RFP-transfected cells. The decrease in HBsAg was even more pronounced on day 7 post-transfection. AD38 cells transfected with HBV 11-12x.26 showed HBsAg levels approximately 50% lower than cells transfected with RFP, and cells transfected with HBV 5-6x.33 levels were approximately 75% lower than in RFP control (Fig. 10). At day 7 post-transfection, cells transfected with RFP had significantly less HBsAg in the supernatant compared to non-transfected cells, suggesting that the transfection process had a negative effect on the ability of these cells to produce HBsAg. However, the effect was much more pronounced in cells transfected with plasmids encoding HBV meganucleases, strongly suggesting that the decrease in HBsAg levels in the supernatant was due to meganuclease activity.

Conclusions.

These results demonstrate that AD38 cells transfected with plasmids encoding either HBV 5-6x.33 or HBV 11-12x.26 show a significant reduction in HBsAg levels in the supernatant compared to either non-transfected cells or cells transfected with the reporter gene. RFP. The decrease in HBsAg levels in the supernatant conditioned by cells transfected with any of the HBV meganucleases strongly suggests that this decrease is due to the activity of the meganuclease against the HBV genome in AD38 cells.

Example 4

Targeting HBV viral genomes in cells.

Treatment of AD38 cells expressing the HBV genome.

The studies in Example 3 above demonstrate the efficacy of the meganuclease in reducing HBsAg secretion from the HBV genome-expressing AD38 cell line. To determine whether a large reduction in HBsAg secretion could be achieved using lentiviral delivery instead of plasmid transfection, this study was repeated with lentiviral delivery using HBV 11-12x.26 and HBV 5-6x.33 meganucleases alone or in combination.

Lentiviruses were prepared that expressed either RFP, HBV 5-6x.33 or HBV 11-12x.26, and AD38 cells were transduced to determine the effect of meganucleases on HBsAg production. AD38 cells were seeded and 24 hours later transduced with either lentivirus encoding RFP, HBV 5-6x.33, HBV 11-12x.26, or a 1:1 mixture of lentiviruses encoding HBV meganucleases. Cells were transduced with individual lentiviruses at MVs of 1, 2, or 4. Cells that were transduced with a 1:1 mixture of HBV meganuclease-encoding lentiviruses were transduced at MVs of 2 and 4. Transduced cell media were replaced on day 1 and day 3 post-transduction. On day 7 after transduction, cell supernatants were collected and analyzed for the presence of HBsAg in them using ELISA.

- 40 042452

Results.

ELISA results were normalized to the amount of HBsAg present in the supernatant of RFP-transduced AD38 cells on day 7 post-transfection. At an MZ of 1, AD38 cells transduced with HBV 5-6x33 detected approximately 60% less HBsAg in the supernatant than AD38 cells transduced with RFP-expressing lentivirus (FIG. 11). Also, at an MZ of 1, HBV 11-12x.26 detected approximately 40% less HBsAg than RFP. At higher MS, the action of HBV meganucleases was more pronounced. At an MZ of 2, cells transduced with lentivirus encoding HBV 5-6x.33 showed a decrease in HBsAg of approximately 80% compared to transduced RFP cells, and at an MZ of 4, the reduction was approximately 90% compared with transduced cells. RFP cells. Similarly, at an MR of 2, cells transduced with lentivirus encoding HBV 11-12x.26 had approximately 70% less HBsAg than RFP-transduced cells, and at an MR of 4, HBsAg levels were approximately 80% lower. . Finally, cells transduced with a 1:1 mixture of lentiviruses encoding HBV meganucleases also showed a dramatic decrease in supernatant HBsAg levels compared to RFP-transduced control cells (FIG. 11). At a total MR of 2, HBV-expressing lentiviruses reduced HBsAg expression by approximately 80%, and at a total MR of 4, expression was reduced by approximately 90%.

Conclusions.

These results demonstrate that AD38 cells transduced with lentiviruses encoding either HBV 5-6x.33 or HBV 11-12x.26 or a combination of both showed a dramatic decrease in HBsAg levels in the supernatant compared to cells transduced with RFP-expressing lentivirus. . The decrease in HBsAg levels in the supernatant of cells transduced with lentiviruses expressing either or both HBV meganucleases strongly suggests that this decrease is due to meganuclease activity against the HBV genome in AD38 cells. In addition, the results demonstrate that co-expression of HBV meganucleases effectively reduces HBsAg expression, suggesting that co-expressed meganucleases can effectively degrade the HBV genome.

Example 5

Targeting HBV genomes in cells.

Treatment of AD38 cells expressing the HBV genome.

In an additional study, AD38 cells were transduced with lentiviruses encoding HBV 5-6x.33 or HBV 11-12x.26 meganucleases to observe the effect of meganuclease treatment on HBsAg secretion, copies of HBV DNA present in the culture medium, and copies of intracellular HBV cccDNA. As in Example 4 above, lentiviruses were prepared that expressed either RFP (LV212) or HBV 5-6x.33 (LV224) or HBV 11-12x.26 (LV225) and AD38 cells were transduced to determine the effect of meganucleases on each experimental result. AD38 cells were seeded in the presence of tetracycline and transduced 24 hours later with lentivirus encoding RFP, HBV 56x.33 or HBV 11-12x.26 at an MZ of 4. The media of the transduced cells were replaced 24 hours after transduction. On day 7 after transduction, cell supernatants were collected and analyzed for the presence of HBsAg in them using ELISA. Copies of extracellular HBV DNA per 5 μl of cell culture were determined using quantitative PCR. Cell lysates were prepared and the copy number of intracellular HBV cccDNA per 5 μl of cell lysate was determined by quantitative PCR.

Results.

At an MZ of 4, AD38 cells transduced with HBV 5-6x.33 and HBV 11-12x.26 showed approximately 58% and 25% less HBsAg in culture media, respectively, 7 days post-transduction than AD38 cells. transduced with an RFP expressing lentivirus (FIG. 12A). The copy number of extracellular HBV DNA after transduction with HBV 5-6x.33 and HBV 1112x.26 meganucleases was also reduced by approximately 28% and 50%, respectively, compared to transduction with RFP-expressing lentivirus (Fig. 12B). Finally, the copy number of intracellular HBV cccDNA was also reduced after transduction with HBV 5-6x.33 and HBV 11-12x.26 meganucleases by approximately 71% and 60%, respectively, compared to transduction with RFP-expressing lentivirus (Fig. 12C).

Conclusions.

These results further demonstrate that AD38 cells transduced with lentiviruses encoding either of the HBV 5-6x.33 or HBV 11-12x.26 meganucleases showed a dramatic decrease in supernatant HBsAg levels compared to cells transduced with RFP-expressing lentivirus. In addition, the results demonstrate that the HBV meganucleases of the present invention also reduced the copy number of extracellular HBV DNA and, importantly, significantly reduced the copy number of intracellular HBV cccDNA.

Example 6

Treatment of HBV-infected primary human hepatocytes.

The studies in the examples above demonstrate the efficacy of meganuclease in reducing HBsAg secretion from an HBV genome-expressing AD38 cell line. Studies described in

- 41 042452 of the present example, was carried out to determine the effectiveness of HBV meganucleases according to the present invention in HBV-infected primary human hepatocytes.

Lentiviruses were prepared that expressed either RFP or HBV 5-6x.33 or HBV 1112x.26 and HBV-infected primary human hepatocytes were transduced to determine the effect of meganucleases on HBsAg and HBeAg production. Briefly, primary human hepatocytes were seeded and infected with HBV 24 hours later. On day 1 post-infection, cells were washed and 24 hours later (day 2 post-infection) transduced with either lentivirus encoding RFP, HBV 5-6x.33, HBV 11-12x.26, or a 1:1 mixture of lentiviruses encoding HBV meganucleases. As an additional control, infected cells were treated with DMSO. Cell supernatants were harvested and medium changed on days 4, 8, 11 and 13 post-transduction. At each time point, HBsAg and HBeAg were measured in cell supernatants by ELISA. Also measured extracellular DNA in the supernatant on day 13 after infection.

Results.

To determine whether lentivirus transduction generally affected either HBsAg or HBeAg secretion, supernatants of cells transduced with RFP-encoding lentivirus were compared to supernatants of cells treated with DMSO (FIG. 13). Transduction of HBV-infected primary human hepatocytes with RFP-encoding lentivirus had little, if any, effect on HBsAg or HBeAg secretion (FIGS. 13A and 13B). At an MV of 5 or 2.5, HBsAg levels were identical to those in DMSO-treated cells, and at an MV of 1.25 only a modest decrease was observed (FIG. 13A). Similarly, only modest reductions in HBeAg levels were shown in supernatants of RFP lentivirus-transduced cells at MW of 1.25, 2.5, or 5 compared to DMSO-treated cells (FIG. 13B). In addition, there was no significant difference between the amount of extracellular HBV found in the supernatant of HBV-infected primary human hepatocytes, DMSO-treated cells, and cells transduced with RFP-encoding lentivirus (Fig. 13C).

Showing that transduction with lentivirus did not generally affect function.

HBV in infected primary human hepatocytes, lentiviruses encoding HBV meganucleases were compared to lentivirus RFP. HBsAg ELISA results were normalized to the amount of HBsAg present in the supernatant of RFP-transduced HBV-infected primary human hepatocytes at days 3, 8, 11, and 13 post-transduction (FIG. 14). At both MVs of 2.5 and 1.25, cells transduced with lentivirus encoding HBV 5-6x.33 showed a uniform decrease in HBsAg levels over the duration of the experiment: slightly reduced levels on day 3, a decrease of approximately 50-60 % by day 8 and a decrease of approximately 90% by days 11 and 13 compared to the RFP control (FIGS. 14A and 14B). Infected cells transduced with HBV 11-12x.26 showed a similar pattern at both MHs of 2.5 and 1.25, with a more marked decrease seen at time point day 3 (approximately 50% decrease), and a decrease also reached approximately 90% by days 11 and 13 post-transduction (FIGS. 14A and 14B). When infected cells were transduced with a 1:1 mixture of lentiviruses encoding HBV meganucleases, a similar decrease in HBsAg levels was observed over time. At an MH of 2.5, levels decreased by approximately 50% on day 3 post-transduction and the decline continued, reaching 90% by days 11 and 13 post-transduction (FIGS. 14A and 14B).

HBeAg ELISA results were also normalized to the amount of HBeAg present in the supernatant of RFP-transduced HBV-infected primary human hepatocytes on days 3, 8, 11, and 13 post-transduction (FIG. 14). At both MVs of 2.5 and 1.25, cells transduced with lentivirus encoding HBV 5-6x. 50% by day 8 and decreased by approximately 90% by days 11 and 13 compared to the RFP control (FIGS. 14C and 14D). Infected cells transduced with HBV 11-12x.26 showed a similar pattern at both MHs of 2.5 and 1.25 with a similar decrease at time point day 3 (approximately 30% decrease), and the decrease in HBeAg levels also reached approximately 90% by days 11 and 13 post-transduction (FIGS. 14C and 14D). When infected cells were transduced with a 1:1 mixture of lentiviruses encoding HBV meganucleases, a similar decrease in HBeAg levels was observed over time. At MVs of 2.5 and 1.25, levels decreased by approximately 50% on day 3 post-transduction and the decline continued, reaching 90% by days 11 and 13 post-transduction (FIGS. 14C and 14D).

Conclusions.

These results demonstrate that HBV-infected primary human hepatocytes transduced with lentiviruses encoding either HBV 5-6x.33 or HBV 11-12x.26 or a combination of both showed a dramatic decrease in HBsAg and HBeAg levels in the supernatant compared to cells transduced with a lentivirus expressing RFP. The reduction in HBsAg and HBeAg levels in the supernatant of infected cells transduced with lentiviruses expressing either or both HBV meganucleases strongly suggests that the reduction is due to the activity of the meganucleases against the infectious virus.

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Claims (17)

Example 7 Delivery of LNP-encapsulated nuclease mRNA to the liver. Encapsulation of mRNA into lipid nanoparticles and delivery to mice. The aim of this study was to demonstrate that a lipid nanoparticle (LNP) encapsulated mRNA encoding an engineered meganuclease can be prepared and administered in vivo, and that gene editing occurs and can be observed in the liver. An engineered meganuclease was developed that has specificity for recognition sequences in the mouse CMP-NeuAc (Cmah) hydrolase gene, which is expressed in mouse liver. Capped ARCA mRNA encoding Cmah meganuclease was obtained from TriLink BioTechnologies LLC (San Diego, CA) and encapsulated in three different commercially available LNP formulations, each containing different ratios of ionizable cationic lipid, PEG lipid and cholesterol. LNP-encapsulated mRNA was administered to CD-1 mice by intravenous injection at a dose of 1.0 mg/kg. The livers were harvested 6 days after administration. Animals were perfused with saline prior to organ harvesting. Whole liver genomic DNA (gDNA) was isolated and the insertion/deletion (indel) mutation rate in the Cmah gene was determined using IT7 endonuclease (T7E) analysis and deep sequencing. In the T7E assay, a region of the genome containing the Cmah recognition sequence was amplified by PCR, resulting in a heterogeneous mixture of amplified wild-type and mutant alleles. The PCR product was denatured and allowed to slowly hybridize again, allowing the formation of heteroduplexes between the wild-type and mutant alleles. Such heteroduplexes are susceptible to cleavage by the IT7 endonuclease, which cuts mismatches. When the cleaved T7E products were visualized on the gel, multiple bands were present due to cleavage of the T7E heteroduplexes, while a single band was present in the wild-type samples. The ratio of cleaved to uncleaved product is a measure of the level of nuclease cleavage activity. Results. The results of this study are shown in Fig. 15. Lane 1 and 2 show no evidence of cleavage in the Cmah recognition sequence of gDNA derived from control mice injected with LNP-encapsulated mRNA encoding luciferase. The ratio of cleaved:uncleaved gDNA observed in animals injected with luciferase mRNA was compared with the ratio observed in animals not injected with mRNA (lane 8). In contrast, lanes 3-7 and 9-18 each show a modification in the Cmah recognition sequence when animals are injected with LNP-encapsulated mRNA encoding Cmah meganuclease, as evidenced by the presence of multiple bands in each lane. Deep sequencing of the gDNA obtained from each liver was also performed to confirm and determine the percentage of gene modification in the Cmah recognition sequence. The percentages resulting from deep sequencing are shown in FIG. 15 under each lane as % knockout (% ON). Lanes 3-7 show modifications ranging from 3.41 to 21.49% in duplicate animals treated with LNP formulation #1. Lanes 9-13 show modifications ranging from 18.4% to 23.2% in duplicate animals treated with LNP formulation #2. Lanes 14-18 show modifications ranging from 51.1 to 64.8% in duplicate animals treated with formulation #3 LNP. Conclusions. This study clearly demonstrates that mRNA encoding an engineered meganuclease, such as the HBV-specific meganucleases of the present invention, can be encapsulated in LNP, delivered to target liver cells (i.e., hepatocytes) in vivo, and can induce gene editing in the target recognition sequences in the genome. CLAIM 1. An engineered meganuclease that recognizes and cleaves a recognition sequence consisting of SEQ ID NO: 16, wherein said engineered meganuclease contains a first subunit and a second subunit, wherein said first subunit binds to the first recognized half site of said recognition sequence and contains the first hypervariable ( HVR1) region, and wherein said second subunit binds to the second recognized half-site of said recognition sequence and contains a second hypervariable (HVR2) region, and said engineered meganuclease contains an amino acid sequence that is at least 96% identical to SEQ ID NO: 33. 2. The constructed meganuclease according to claim 1, characterized in that said first subunit comprises an amino acid sequence at least 96% identical to residues 198-344 of SEQ ID NO: 33, and wherein said second subunit comprises an amino acid sequence of at least at least 96% identical to residues 7-153 of SEQ ID NO: 33. 3. A constructed meganuclease according to any one of claims 1 or 2, characterized in that said first subunit comprises residues 198-344 of SEQ ID NO: 33. - 43 042452 4. Constructed meganuclease according to any one of claims 1-3, characterized in that said second subunit comprises residues 7-153 of SEQ ID NO: 33. 5. An engineered meganuclease according to any one of claims 1 to 4, characterized in that said engineered meganuclease comprises the amino acid sequence shown in the sequence of SEQ ID NO: 33. 6. A polynucleotide for expressing an engineered meganuclease according to any one of claims 1 to 5, comprising a nucleic acid sequence encoding said engineered meganuclease according to any one of claims 1 to 5. 7. A polynucleotide according to claim 6, characterized in that said polynucleotide is an mRNA. 8. The design of recombinant DNA for the expression of the engineered meganuclease according to any one of claims 1 to 5, containing a nucleic acid sequence encoding the specified engineered meganuclease according to any one of paragraphs. 1-5. 9. A viral vector for expressing an engineered meganuclease according to any one of claims 1 to 5, containing a nucleic acid sequence encoding said engineered meganuclease according to any one of claims 1 to 5. 10. Viral vector according to claim 9, characterized in that said viral vector is a recombinant AAV vector. 11. A pharmaceutical composition for the treatment of a subject with hepatitis B virus (HBV) or hepatocellular carcinoma caused by HBV, said pharmaceutical composition contains a pharmaceutically acceptable carrier and an effective amount of: (a) a nucleic acid encoding said engineered meganuclease according to any one of claims 1 to 5; or (b) said engineered meganuclease according to any one of claims 1-5. 12. Pharmaceutical composition according to claim 11, characterized in that said nucleic acid encoding said engineered meganuclease is said mRNA according to claim 7. 13. Pharmaceutical composition according to claim 12, characterized in that said mRNA is encapsulated in lipid nanoparticles. 14. Pharmaceutical composition according to claim 11, characterized in that said pharmaceutical composition contains said viral vector according to claim 9 or 10. 15. Use of an engineered meganuclease according to any one of claims 1-5 or a nucleic acid encoding said engineered meganuclease according to any one of claims 1-5 to treat a subject with hepatitis B virus (HBV) or hepatocellular carcinoma caused by HBV. 16. A method of treating a subject with hepatitis B virus (HBV) or hepatocellular carcinoma caused by HBV, including delivery to the target cell of the subject: (a) a nucleic acid encoding an engineered meganuclease according to any one of claims 1 to 5, wherein said engineered meganuclease is expressed in said target cell in vivo; or (b) an engineered meganuclease according to any one of claims 1-5. 17. The method of claim 16, wherein said target cell is a hepatocyte. -