Transcription activator-like effector nucleases (talens) targeting hbv

Abstract

This invention describes nucleic acid molecules encoding transcription activator effector nucleases (TALENs) targeting the hepatitis B virus (HBV) genomic body. It also describes compositions, host cells, lipids, and pharmaceutical compositions containing such TALENs. Furthermore, it describes methods for treating hepatitis infections, particularly in individuals with chronic HBV infection, using the pharmaceutical compositions of this invention.

Description

Technical field

[0001] The present invention relates generally to the fields of molecular biology and genetic engineering, including the use of transcription activator-like effector nuclease (TALEN) sequences for gene targeting and regulation of gene expression.

Prior Technology

[0002] Hepatitis B virus (HBV) is a 3.2-kb hepatotropic small DNA virus encoding four open reading frames and seven proteins. About two billion people are infected with HBV, and approximately 240 million people have chronic hepatitis B infection (chronic HBV), which is characterized by persistent viral and subviral particles in the blood lasting more than 6 months (Cohen et al. J . Viral Hepat. (2011) 18(6), 377-83). Persistent HBV infection leads to T-cell depletion of circulating and intrahepatic HBV-specific CD4+ and CD8+ T cells through chronic stimulation of HBV-specific T-cell receptors by viral peptides and circulating antigens. Consequently, T cell pluripotency is diminished (ie, reduced levels of IL-2, tumor necrosis factor (TNF)-α, IFN-γ and lack of proliferation).

[0003] Chronic HBV (CHB) is currently treated with IFN-α and nucleoside or nucleotide analogues, but due to the persistence in infected hepatocytes of a protein called covalent Intracellular viral replication intermediates of closed circular DNA (cccDNA) and the resulting new virions are therefore ultimately incurable. The induced virus-specific T-cell and B-cell responses are thought to effectively eliminate cccDNA-carrying hepatocytes. Current therapies targeting HBV polymerase suppress viremia but provide limited effects on production of cccDNA and associated circulating antigens present in the nucleus. The most rigorous form of cure may be the elimination of the organism's HBV cccDNA, which is neither observed as a result of natural occurrence nor as a result of any therapeutic intervention. However, HBV surface antigen (HBsAg) reduction is clinically credible equivalent to cure, since disease relapse is only likely to occur in severe immunosuppressed situations, which can then be prevented by prophylactic therapy. Thus, at least from a clinical point of view, HBsAg reduction correlates with the most stringent form of immune reconstitution against HBV.

[0004] Hepatitis D virus (HDV) infection occurs only in cases of superinfection with HBV because it requires the presence of HBsAg for HDV to form infectious virions. Superinfection is associated with earlier development of cirrhosis, increased risk of hepatocellular carcinoma (HCC) development, and increased liver-related and overall mortality.

[0005] Accordingly, it would be useful to have compositions and methods that enable targeted HBV gene body lysis in chronically infected patients with concomitant high efficiency and reduced cytotoxicity.

Content of invention

[0006] Accordingly, there is an unmet medical need for gene-edited hepatitis, especially hepatitis B virus (HBV) and more particularly chronic HBV therapy, for targeting HBV cccDNA, in particular HBsAg. The present invention meets this need by providing compositions and methods for inducing directed cleavage of specific DNA sequences.

[0007] In a general aspect, provided herein is a method of treating a hepatitis infection in an individual in need thereof, the method comprising administering to the individual a combination of mRNA molecules comprising: (1) a first mRNA molecule comprising an encoding A polynucleotide sequence of a first type of transcription activator effector nuclease (TALEN) monomer, the first TALEN monomer comprising a first TALE DNA binding domain and a first nuclease catalytic domain, wherein the first TALE DNA binds The domain can be combined to the first half-site sequence of the target nucleic acid sequence in the HBV gene body; and (2) a second mRNA molecule, which comprises a polynucleotide sequence encoding a second TALEN monomer, and the second TALEN monomer comprises a second TALE DNA binding domain and a second nuclease catalytic domain, wherein the second TALE DNA binding domain can bind to the second half-site sequence of the target nucleic acid sequence; wherein when the first TALE DNA binding domain binds to the second When the half site sequence and the second TALE DNA binding domain are bound to the second half site sequence, the first TALEN monomer and the second TALEN monomer can form a dimer that cleaves the target nucleic acid sequence, preferably Preferably, the first nuclease catalytic domain is a first FokI nuclease catalytic domain and the second nuclease catalytic domain is a second FokI nuclease catalytic domain.

[0008] In some embodiments, the target nucleic acid sequence is in the sequence encoding HBsAg and HBV polymerase (pol), preferably, (a) the first half-site sequence of the target nucleic acid sequence comprises the same The nucleic acid sequence of SEQ ID NO: 1 is at least about 90% identical, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical A polynucleotide sequence, and the second half-site sequence of the target nucleic acid sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 2, such as at least 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequence; or (b) the first half-site sequence of the target nucleic acid sequence comprises the same sequence as SEQ ID NO: 3 The nucleic acid sequences are at least about 90% identical, such as polynucleotide sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical , and the second half-site sequence of the target nucleic acid sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequence, or (c) the target nucleic acid sequence comprises at least 90% identical to the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 , such as polynucleotide sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0009] In certain embodiments, each of the first mRNA molecule and the second mRNA molecule further comprises a 5' end cap, a 5'-UTR, a sequence encoding a nuclear localization signal, a sequence encoding an N-terminal domain, an encoding C One or more, preferably all, of the sequence of the telodomain, the 3'-UTR and the polyadenosine tail.

[0010] In certain embodiments, respectively, the first TALE DNA binding domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 7 or SEQ ID NO: 9, the second TALE DNA binding domain The domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 8 or SEQ ID NO: 10, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprise the same sequence as SEQ ID NO: 11 At least 90% identical amino acid sequence.

[0011] In some embodiments, the HBV genetic system genotype A, B, C, D, E, F, G, H, I and J in one or more.

[0012] In certain embodiments, the method further comprises administering to the individual a second therapeutic composition, preferably a second therapeutic composition comprising an anti-HBV agent.

[0013] In certain embodiments, the individual has HBV infection, preferably chronic HBV infection.

[0014] In certain embodiments, the individual is co-infected with HBV and HDV.

[0015] In another general aspect, this paper provides a composition comprising a combination of mRNA molecules encapsulated in lipid nanoparticles: (1) a first mRNA molecule comprising A polynucleotide sequence of a transcription activator effector nuclease (TALEN) monomer, the first TALEN monomer comprises a first TALE DNA binding domain and a first nuclease catalytic domain, wherein the first TALE DNA binding domain Can be combined to the first half-site sequence of the target nucleic acid sequence in HBV gene body; And (2) the second mRNA molecule, it comprises the polynucleotide sequence of encoding the second TALEN monomer, and this second TALEN monomer comprises the first two TALE DNA binding domains and a second nuclease catalytic domain, wherein the second TALE DNA binding domain is capable of binding to the second half-site sequence of the target nucleic acid sequence; wherein when the first TALE DNA binding domain binds to the first When the half-site sequence and the second TALE DNA binding domain are bound to the second half-site sequence, the first TALEN monomer and the second TALEN monomer can form a dimer that cleaves the target nucleic acid sequence, preferably Preferably, the first nuclease catalytic domain is a first FokI nuclease catalytic domain and the second nuclease catalytic domain is a second FokI nuclease catalytic domain.

[0016] In some embodiments, the target nucleic acid sequence is in the sequence encoding HBsAg and HBV polymerase (pol), preferably, (a) the first half-site sequence of the target nucleic acid sequence comprises the same The nucleic acid sequence of SEQ ID NO: 1 is at least about 90% identical, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical A polynucleotide sequence, and the second half-site sequence of the target nucleic acid sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 2, such as at least 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequence; or (b) the first half-site sequence of the target nucleic acid sequence comprises the same sequence as SEQ ID NO: 3 The nucleic acid sequences are at least about 90% identical, such as polynucleotide sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical , and the second half-site sequence of the target nucleic acid sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequence, or (c) the target nucleic acid sequence comprises at least 90% identical to the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 , such as polynucleotide sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0017] In certain embodiments, the first mRNA molecule and the second mRNA molecule each further comprise a 5' end cap, a 5'-UTR, a sequence encoding a nuclear localization signal, a sequence encoding an N-terminal domain, an encoding C One or more, preferably all, of the sequence of the telodomain, the 3'-UTR and the polyadenosine tail.

[0018] In certain embodiments, respectively, the first TALE DNA binding domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 7 or SEQ ID NO: 9, and the second TALE DNA binding domain The domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 8 or SEQ ID NO: 10, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprise the same sequence as SEQ ID NO: 11 At least 90% identical amino acid sequence.

[0019] In some embodiments, the HBV genetic system genotype A, B, C, D, E, F, G, H, I and J in one or more.

[0020] In certain embodiments, the lipid nanoparticles encapsulating the combination of mRNA molecules comprise cationic lipids and at least one selected from the group consisting of anionic lipids, zwitterionic lipids, neutral lipids, steroids, Other lipids of the group consisting of polymer-bound lipids, phospholipids, glycolipids, and combinations thereof.

[0021] In another general aspect, a combination is provided herein, the combination has: (1) a first nucleic acid, preferably a first mRNA molecule, comprising a first class of transcriptional activator effector nuclease encoding ( The polynucleotide sequence of the TALEN monomer, the first TALEN monomer comprises the first TALE DNA binding domain and the first FokI nuclease catalytic domain, wherein the first TALE DNA binding domain can be combined to the target nucleic acid in the HBV gene body The first half site sequence of the sequence, and the first half site sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 1, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequences; and (2) a second nucleic acid, preferably a second mRNA molecule, comprising a second class of transcriptional activators encoding A polynucleotide sequence of an effector nuclease (TALEN) monomer comprising a second TALE DNA binding domain and a second FokI nuclease catalytic domain, wherein the second TALE DNA binding domain is capable of binding to the The second half-site sequence of the target nucleic acid sequence, and the second half-site sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 2, such as at least 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequences; wherein when the first TALE DNA binding domain binds to the first half-site sequence and the second TALE When the DNA binding domain binds to the second half-site sequence, the first TALEN monomer and the second TALEN monomer can form a dimer that cleaves the target nucleic acid sequence.

[0022] In some embodiments, the HBV genetic system genotype A, B, C, D, E, F, G, H, I and J in one or more.

[0023] In another general aspect, provided herein is a composition comprising a combination of a first nucleic acid as described herein and a second nucleic acid, wherein the first nucleic acid and the second nucleic acid are encapsulated separately or together Enclosed in lipid nanoparticles.

[0024] In another general aspect, provided herein is a nucleic acid molecule encoding at least one of a first mRNA molecule and a second mRNA molecule as described herein.

[0025] In another general aspect, provided herein is an isolated host cell comprising a nucleic acid molecule as described herein.

[0026] In certain embodiments, the host cell is a hepatocyte.

[0027] In another general aspect, provided herein is a pharmaceutical composition comprising a composition as described herein, a combination as described herein, a nucleic acid molecule as described herein, or a compound as described herein. Isolating host cells and pharmaceutically acceptable carriers.

[0028] In another general aspect, this paper provides a method for cracking the target nucleic acid sequence in the HBV gene body, which comprises making the HBV gene body with a composition as described herein, a combination as described herein, such as A nucleic acid molecule as described herein, an isolated host cell as described herein, or a pharmaceutical composition as described herein is contacted.

[0029] In another general aspect, there is provided herein a method for inducing gene editing of a target nucleic acid sequence in an HBV genome, comprising making an HBV genome with a composition as described herein, as described herein A combination, a nucleic acid molecule as described herein, an isolated host cell as described herein, or a pharmaceutical composition as described herein is contacted.

[0030] In certain embodiments, the HBV genotypes are one or more of genotypes A, B, C, D, E, F, G, H, I and J.

[0031] In another general aspect, provided herein is a method of treating a hepatitis infection in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition as described herein.

[0032] In another general aspect, provided herein is a method of reducing HBV infection in an individual and / or reducing HBV replication in an individual, the method comprising administering to the individual a pharmaceutical composition as described herein.

[0033] In certain embodiments, the method further comprises administering to the individual a second therapeutic composition, preferably a second therapeutic composition comprising an anti-HBV agent.

[0034] In certain embodiments, the individual has HBV infection, preferably chronic HBV infection.

[0035] In certain embodiments, the individual is co-infected with HBV and HDV.

[0036] In certain embodiments, the individual has reduced expression of one or more of HBsAg, HBeAg, HBV DNA, HBV cccDNA, or integrated HBV DNA. In particular embodiments, the expressive quantity is hepatocyte content, nuclear or cellular content, liver content, serum content or plasma content.

[0037] In another general aspect, provided herein is a method of producing a TALEN comprising in vitro or in vivo transcription of a nucleic acid molecule as described herein.

[0038] In another general aspect, provided herein is a pharmaceutical composition as described herein for use in the treatment of a hepatitis B virus (HBV)-induced disease in an individual in need thereof, preferably wherein the individual Suffer from chronic HBV infection.

[0039] In certain embodiments, the HBV-induced disease is selected from the group consisting of advanced fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), and the pharmaceutical composition is optionally combined with another therapeutic agent, preferably Another combination of anti-HBV agents.

[0040] Other aspects, features and advantages of the present invention will be apparent from the following disclosures, including specific implementations, preferred embodiments thereof, and appended patent claims.

Implementation

[0053] Cross reference to related applications

[0054] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 131,145, filed December 28, 2020, which is incorporated herein by reference in its entirety.

[0055] Prior Art and throughout this specification various publications, articles and patents are cited or described; each of these references is incorporated herein by reference in its entirety. Discussions of documents, acts, materials, devices, articles of manufacture, or the like contained in this specification are for the purpose of providing a context for the present invention. This discussion is not an admission that any or all of these matter form part of the prior art with respect to any invention disclosed or claimed.

[0056] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. In addition, certain terms used herein have the meanings as set forth in the specification. All patents, published patent applications, and publications cited herein are hereby incorporated by reference as if fully set forth herein.

[0057] It must also be noted that, as used herein and in the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise things.

[0058] Unless otherwise stated, any value such as % sequence identity or % sequence identity range described herein is to be understood as being modified in all instances by the term "about". Accordingly, a value generally includes ±10% of the stated value. For example, a 10 mg dose includes 9 mg to 11 mg. Unless the context clearly dictates otherwise, the use of numerical ranges as used herein expressly includes all possible subranges, all individual values ​​within that range, including integers within such ranges and fractions of such values.

[0059] As used herein, the connective term "and / or" between multiple stated elements is understood to encompass both individual options and combinations of options. For example, when two elements are linked by "and / or", the first option refers to the applicability of the first element, excluding the second element. The second option refers to the applicability of the second element, excluding the first element. The third option refers to the applicability of the combination of the first element and the second element. Any of these options are understood to fall within that meaning, and thus satisfy the requirements of the term "and / or" as used herein. Simultaneous applicability of more than one of these options is also understood to fall within this meaning, and thus fulfills the requirements of the term "and / or".

[0060] Unless otherwise indicated, the term "at least" preceding a list of elements is understood to refer to each element in the list. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. This invention is intended to cover such equivalents.

[0061] Throughout this specification and subsequent claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises / comprising" should be understood to imply inclusion of a stated integer or step or set of integers or steps, but does not exclude any other integer or step or any other set of integers or steps. When used herein, the term "comprising" may be replaced by the term "containing" or "including", or sometimes when used herein, by the term "having" .

[0062] When used herein, "consisting of" excludes any element, step or ingredient not specified among the claimed elements. When used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the basic and novel features of the technical solution. Whenever used herein in the context of aspects or embodiments of the invention, the aforementioned terms "comprising", "containing", "including" and "having" Any of them may be replaced by the term "consisting of" or "consisting essentially of" to change the scope of the present invention.

[0063] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to both RNA molecules and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, synthetic DNA, and those containing A DNA or RNA molecule that is a nucleic acid analog. The term "complementary nucleotide bases" means a pair of nucleotide bases that form hydrogen bonds with each other. Adenine (A) pairs with thymine (T) or uracil (U) in RNA, and guanine (G) pairs with cytosine (C). Complementary segments or strands of nucleic acids hybridize (ie, join by hydrogen bonding) to each other. "Complementary"means that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence either by conventional Watson-Crick or by other non-traditional binding modes. Nucleic acid molecules can have any three-dimensional structure. Nucleic acid molecules can be double-stranded or single-stranded (eg, sense or antisense). Non-limiting examples of nucleic acid molecules include genes, gene segments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, microRNA, tracrRNA, crRNA, guide RNA, ribonuclease , cDNA, recombinant polynucleotides, branched polynucleotides, nucleic acid probes and nucleic acid primers. Nucleic acid molecules may contain non-conventional nucleotides or modified nucleotides. The terms "polynucleotide sequence" and "nucleic acid sequence" as used interchangeably herein refer to the sequence of a polynucleotide molecule.

[0064] The phrase "percent (%) sequence identity" or "% identity" or "identical to %" when used with reference to amino acid sequences describes two or more aligned amino acids The number of matches ("hits") for consensus amino acids in a sequence compared to the number of amino acid residues making up the total length of the amino acid sequences. In other words, when two or more sequences are compared and aligned for maximum correspondence as measured using sequence comparison algorithms known in the art or when aligned manually and inspected visually, the use of an alignment can Determine the percentage of identical amino acid residues in the sequences (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% over the entire length of the amino acid sequence % Consistency). The same can be determined for nucleotide sequences. Thus, sequences compared to determine sequence identity may differ by substitution(s), addition(s) or deletion(s) of amino acids. Suitable programs for aligning protein sequences are known to those skilled in the art. The percent sequence identity of protein sequences can be determined, for example, with programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, for example using the NCBI BLAST algorithm (Altschul SF et al. (1997), Nucleic Acids Res. 25:3389-3402).

[0065] The term "binding" or "specific binding" as used herein refers to a sequence-specific non-covalent interaction between macromolecules, such as between a protein and a nucleic acid. Not all components of a binding interaction need be sequence-specific (eg, contacts with phosphate residues in the DNA backbone), so long as the overall interaction is sequence-specific.

[0066] As used herein, the terms and phrases "combination", "in combination", "with ... "In combination with", "co-delivery" and "administered together with" refer to the simultaneous administration of two or more therapies or components, such as two, for example, mRNA Molecular nucleic acid molecules, or therapeutic compositions and lipids. "Simultaneous administration" can be at least the administration of the two components on the same day. When the two components are administered "with" or "in combination with", they may be combined independently within a short period of time, such as 24, 20, 16, 12, 8, or 4 hours, or within 1 hour administered sequentially in one composition, or they may be administered simultaneously in a single composition. Use of the term "in combination with" does not limit the order in which the therapies or components are administered to an individual. For example, a first therapy or component (e.g., a first nucleic acid molecule) may be administered prior to (e.g., 5 minutes to one hour prior to), accompanied by, or associated with the administration of a second therapy or component (e.g., a second nucleic acid molecule). Simultaneously or thereafter (eg, 5 minutes to one hour thereafter). In some embodiments, a first therapy or component (eg, a first nucleic acid molecule) and a second therapy or component (eg, a second nucleic acid molecule) are administered in the same composition. In other embodiments, a first therapy or component (eg, a first nucleic acid molecule) and a second therapy or component (eg, a second nucleic acid molecule) are administered in separate compositions.

[0067] The term "cytotoxic" means killing or causing detrimental effects to cells (e.g., mammalian cells (e.g., human cells)), bacteria, viruses, fungi, protozoa, parasites, prions, or combinations thereof , toxic or fatal effects.

[0068] The term "delivery" refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload.

[0069] The term "delivery agent" refers to any substance that facilitates, at least in part, the in vivo delivery of a polynucleotide to a targeted cell.

[0070] The term "engineered" means that a molecule has been designed to have characteristics or properties, whether structural or chemical, that are different from those of the starting, wild-type or native molecule.

[0071] The term "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' capping, and / or processing of the 3' end); (3) translation of the RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

[0072] The term "lipid" means an organic compound comprising esters of fatty acids and characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally divided into at least three categories: (1) "simple lipids", which include fats and oils as well as waxes; (2) "compound lipids", which include phospholipids and glycolipids; and (3) "derived lipids" , such as steroids.

[0073] The term "lipid delivery vehicle" means a lipid formulation that can be used to deliver a therapeutic nucleic acid (eg, mRNA) to a target site of interest (eg, a cell, tissue, organ, and the like). Lipid delivery vehicles can be nucleic acid-lipid particles that can be formed from cationic lipids, non-cationic lipids (such as phospholipids), binding lipids that prevent particle aggregation (such as PEG-lipids), and optionally cholesterol. Typically, the therapeutic nucleic acid (eg, mRNA) can be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.

[0074] The term "encapsulated lipid" means a lipid particle that provides a therapeutic nucleic acid, such as mRNA, fully encapsulated, partially encapsulated, or both. In a preferred embodiment, the nucleic acid (eg, mRNA) is fully encapsulated in the lipid particle.

[0075] A "non-naturally occurring" nucleic acid or polypeptide as used herein refers to a nucleic acid or polypeptide that does not occur in nature. A "non-naturally occurring" nucleic acid or polypeptide can be synthesized, manipulated, manufactured and / or otherwise manipulated in a laboratory and / or manufacturing setting. In some cases, a non-naturally occurring nucleic acid or polypeptide may comprise a naturally occurring nucleic acid or polypeptide that has been treated, processed, or manipulated to exhibit properties that were not present in the naturally occurring nucleic acid or polypeptide prior to the treatment. A "non-naturally occurring" nucleic acid or polypeptide as used herein is one that is isolated or isolated from the natural source in which it is found and which lacks a covalent bond with a sequence with which it is related in the natural source. A "non-naturally occurring" nucleic acid or polypeptide can be produced recombinantly or by other methods such as chemical synthesis.

[0076] As used herein, the term "operably connected" means either keyed or paralleled, wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence operably linked to a nucleic acid sequence of interest can direct the transcription of a nucleic acid sequence of interest, or a signal sequence operably linked to an amino acid sequence of interest can direct the nucleic acid sequence of interest to The amino acid sequence is secreted or translocated to the membrane.

[0077] "Individual" as used herein means any animal, preferably a mammal, most preferably a human, to be treated or has been treated by a method according to an embodiment of the present application . The term "mammal" as used herein encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., and more good place human beings. Human subjects can include patients. The compounds, compositions and methods described herein are applicable both in human therapy and in veterinary applications to species that may be chronically infected with HBV. In some embodiments, the individual has HBV infection, more particularly chronic HBV (CHB) infection. Individuals may develop CHB with or without viral superinfection. "Viral superinfection" as used herein refers to infection with at least two types of viruses. "Virus superinfection" means simultaneous infection with at least two types of viruses. A "viral superinfection" can also be a superinfection in which one or more types of viral infection is added to a pre-existing one or more other types of viral infection. In some embodiments, the individual has HBV-HDV co-infection. Preferably, the individual is co-infected with CHB-HDV. More preferably, the individual has a viral co-infection of CHB and chronic HDV.

[0078] A "target site" or "target sequence" is a nucleic acid sequence that defines a portion of nucleic acid that will bind to a binding molecule or binding domain, providing sufficient conditions for binding to occur. A "half-site sequence" of a target sequence as used herein refers to a portion of the target sequence that will bind to a binding molecule or domain providing sufficient conditions for binding to occur. Typically there are two half-site sequences within the target nucleic acid sequence which may be separated by a spacer sequence. For example, a first TALE DNA binding domain can bind to a first half-site sequence of a target nucleic acid and a second TALE DNA binding domain can bind to a second half-site sequence of the same target nucleic acid. Preferably, the first half-site sequence and the second half-site sequence are different and separated by a spacer sequence.

[0079] The description herein relates to various embodiments of the application. Discussion of any examples is intended to be illustrative only, and is not intended to limit the scope of the invention, including claims, to these examples. For example, although embodiments of the nucleic acid sequences of the present application described herein may contain specific elements, including but not limited to, specific TALE DNA binding domains, nuclease catalytic domains, etc., arranged in a specific order, generally skilled in the art The reader should appreciate that the concepts disclosed herein are equally applicable to other elements arranged in other orders that may be used in the nucleic acid sequences of the present application. This application contemplates the use of any of the applicable components in any combination with any of the nucleic acid sequences useful in this application, whether or not a particular combination is explicitly described. hepatitis

[0080] Hepatitis is inflammation of the liver most commonly caused by viral infection. There are five major hepatitis viruses known as types A, B, C, D and E. Hepatitis A and E are usually caused by ingestion of contaminated food or water. Hepatitis B, C, and D typically occur as a result of parenteral exposure to infected body fluids (eg, from blood transfusions or invasive medical procedures using contaminated equipment). Hepatitis B is also spread through sexual contact.

[0081] Hepatitis A virus (HAV) is an intestinally transmitted viral disease causing fever, malaise, anorexia, nausea, abdominal discomfort and jaundice. HAV is usually acquired by the fecal-oral route, by person-to-person contact, ingestion of contaminated food or water, or transmission of mixed plasma products. The absence of a lipid envelope renders HAV extremely resistant to physicochemical inactivation, and the virus tolerates conventional heat treatments of blood products. The development of sensitive and specific diagnostic assays to identify HAV antigens and / or antibodies in infected individuals and nucleic acid-based assays to detect viremic samples to exclude them from transfusion represents an important public health challenge .

[0082] Hepatitis A virus (HAV) is a non-enveloped small spheroid virus classified in the Hepavirus genus of the family Picornaviridae. The HAV genome consists of a single-stranded linear 7.5 kb RNA molecule encoding a polyprotein precursor, which is processed to produce the structural proteins and enzymatic activities required for viral replication. HAV encodes four capsid proteins (A, B, C and D) that contain the major antigenic domains recognized by antibodies of infected individuals. In addition to capsid proteins, antigenic domains have also been reported in nonstructural proteins such as 2A and in virus-encoded proteases.

[0083] Hepatitis B virus (HBV) infects humans with two possible clinical outcomes. In most adult clinical infections (90%-95%), virus is cleared after weeks or months and patients develop lifelong immunity to reinfection. In the remaining cases, however, the virus is not self-eliminated and the patient remains chronically infected. The sequelae of chronic infection are severe: such individuals are at high risk of scarring (cirrhosis) of the liver tissue and may eventually develop hepatocellular carcinoma. HBV is transmitted during delivery through infected blood or other bodily fluids, especially saliva and semen, sexual activity, or sharing needles contaminated with infected blood.

[0084] Chronic hepatitis B (CHB) infection is the most common cause of cirrhosis and hepatocellular carcinoma (HCC), with an estimated 500,000-900,000 deaths per year. Persistent HBV replication increases the risk of progression to cirrhosis and HCC.

[0085] Hepatitis C virus (HCV) is the causative agent of the major chronic liver infection initially identified as non-A, non-B hepatitis. HCV has infected about four million people in the United States and 170 million people worldwide, about four times as many as HIV and responsible for 90% to 95% of hepatitis attributable to blood transfusions. It is presumed that the primary route of infection is through contact with contaminated body fluids, especially blood, from infected individuals. HCV infection is one of the leading causes of liver transplantation in the United States and other countries. Approximately 40%-50% of liver transplants in the United States are based on HCV infection. The disease usually progresses to chronic liver damage. Although the lesions of HCV infection primarily affect the liver, the virus is found in other cell types in the body including peripheral blood lymphocytes.

[0086] HCV is an RNA virus of the genus Hepacivirus in the Flaviviridae family and is most closely related to plague virus, BVDV and GBV-B. The HCV genome consists of a single strand of RNA approximately 9.6 kb in length. The HCV gene body has a continuously translating open reading frame (ORF) encoding a polymeric protein of approximately 3,000 amino acids. Structural protein(s) appear to be encoded in approximately the first quarter of the N-terminal region of the ORF, with the remainder encoding nonstructural proteins. The polyprotein acts as a precursor for at least 10 individual viral proteins that are critical for the replication and assembly of progeny virions. The organization of structural and nonstructural proteins in HCV polyproteins is as follows: C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b.

[0087] Hepatitis D virus (HDV) is a satellite RNA virus that, depending on the hepatitis B surface antigen, assembles its outer membrane and forms new virions that transmit infection. HDV has a small genome of 1.7 Kb, making it the smallest known human virus. The hepatitis D circular gene body is unique to animal viruses due to its high GC nucleotide content. However, HDV is the most severe form of viral hepatitis. More often than other agents of viral hepatitis, acute HDV infection is associated with fulminant hepatitis, a rapidly progressive, often fatal form of the disease in which large portions of the liver are destroyed. Chronic hepatitis D is often characterized by necrotizing inflammatory lesions, similar to chronic HBV infection but more severe and often rapidly progressive to cirrhosis and liver failure, explaining the asymmetric association of chronic HDV infection with end-stage liver disease. Although HDV infection affects fewer people than HBV alone, the resulting acute or chronic liver failure is a common indication for liver transplantation in Europe and North America. Chronic HDV disease affects 15 million people worldwide, approximately 70,000 of whom are in the United States. The Centers for Disease Control estimates that there are 1,000 deaths in the United States each year from HDV infection.

[0088] The HDV virion consists of a ribonucleoprotein core and an outer membrane. The core contains HDV-RNA and hepatitis D antigen (HDAg), the only protein encoded by this virus. The outer membrane is formed by the surface antigen protein of the hepatitis B helper virus (hepatitis B surface antigen or HBsAg). The mantle is the only accessory functional group provided by HBV. HDV is capable of replicating its RNA intracellularly in the absence of HBV, but requires HBsAg for the encapsulation and release of HDV virions and for the acquisition of infectivity. Since HDV is dependent on HBV, HDV only co-infects individuals with HBV.

[0089] Hepatitis E virus (HEV) is the cause of hepatitis E, an acute form of viral hepatitis prevalent in many resource-limited regions of the world. It is estimated that about two billion people, about one-third of the world's population, live in HEV-endemic regions and are at risk of infection. In these regions, hepatitis E is the predominant form of acute hepatitis; in India, for example, about 50% of acute hepatitis is attributable to HEV.

[0090] HEV is a small non-enveloped virus with a size of 27-34 nm and is classified as a hepatitis virus of the family Hepadnaviridae. The HEV genome is a ~7.2 kb positive-sense single-stranded RNA with a 5'-methylguanine end cap and a 3' poly(adenosine) stretch, and contains three partially overlapping open reading frames (ORFs)—termed orf1 , orf2 and orf3. HEV orf1, a polymeric protein with 1693 amino acids, encodes a viral nonstructural functional group. Domains identified in HEV nonstructural polymeric proteins include (starting at the N-terminus) -methyltransferase (MeT), papain-like cysteine ​​protease (PCP), RNA helicase (Hel), and RNA-dependent Sexual RNA polymerase (RdRp). HEV orf2 encodes a viral capsid protein of 660 amino acids that is believed to coat the viral RNA genome. HEV orf3 is believed to express a 114 amino acid protein that is not essential for replication in vitro and is believed to act as a viral accessory protein, which may influence the host response to infection.

[0091] Such as GBV-C of HCV or hepatitis G virus (HGV) belongs to Flaviviridae. GBV-C has a global distribution, with a high prevalence among donor populations in the United States, and, like HCV and HBV, it can be spread by contaminated blood transfusions and sexual contact. Currently, GBV-C can only be diagnosed by detecting its RNA in serum using polymerase chain reaction. The clinical significance of GBV-C infection relative to acute or chronic hepatitis is not well understood, but other evidence predominates that GBV-C does not cause hepatitis in humans. The viral genome is represented by single-stranded RNA with positive polarity. The GBV-C gene body is similar to HCV RNA in its organization, ie the structural genes are located at the 5' region of the gene body and the nonstructural genes are located at the 3' end. The untranslated region at the 5' end can serve as an internal ribosomal embarkation site, ensuring translation of the RNA coding region. Hepatitis B virus (HBV)

[0092] "Hepatitis B virus" or "HBV" as used herein refers to a specific virus of the hepadnaviridae family. HBV is a (eg 3.2 kb) hepatotropic small DNA virus encoding four open reading frames and seven proteins. Seven proteins encoded by HBV include small (S), medium (M) and large (L) surface antigen (HBsAg) or outer membrane (Env) protein, precore protein, core protein, viral polymerase (Pol) and HBx protein. HBV expresses three surface antigens or outer membrane proteins, namely L, M and S, wherein S is the smallest, L is the largest, and M is the middle. The L protein includes the Pre-S1-Pre-S2-S domain, the M protein includes the Pre-S2-S domain, and the S protein includes the S domain only. The core protein is a subunit of the viral nucleocapsid. Pol is required for viral DNA (reverse transcriptase, RNase H, and primer) synthesis in the nucleocapsid located in the cytoplasm of infected hepatocytes. The precore is the core protein with an N-terminal signal peptide and is proteolytically processed at its N- and C-termini to the so-called hepatitis B e-antigen (HBeAg) before secretion from infected cells. The HBx protein is required for efficient transcription of covalently closed circular DNA (cccDNA). HBx is not a viral structural protein. All HBV viral proteins have their own mRNA except for the core and polymerase which share mRNA. Except for the precore protein, none of these HBV viral proteins undergo post-translational proteolytic processing.

[0093] The HBV virion contains a single copy of the viral envelope, nucleocapsid and part of the double-stranded DNA genome. The nucleocapsid consists of 120 core protein dimers and is covered by a lipid membrane embedded with S, M and L virus outer membrane or surface antigen proteins. After entry into the cell, the viral coat is removed and capsid-containing circular DNA (rcDNA) with covalently bound viral polymerase migrates into the nucleus. During this process, phosphorylation of the core protein induces structural changes that expose nuclear localization signals, allowing the capsid to interact with so-called importins. These importins mediate the association of the core protein with the nuclear pore complex, following which the capsid dissociates and the polymerase / rcDNA complex is released into the nucleus. In the nucleus, rcDNA becomes deproteinized (removal of polymerase) and converted by host DNA repair machinery into covalently closed circular DNA (cccDNA) gene bodies (further converted into minichromosomes via addition of histones and other host factors ), from which overlapping transcripts encode HBeAg, HBsAg, core protein, viral polymerase and HBx protein. Core protein, viral polymerase, and pregenomic RNA (pgRNA) associate in the cytoplasm and self-assemble into immature pgRNA-containing capsid particles, which are further converted into mature rcDNA-capsid and serve as a common intermediate, the The common intermediate is enveloped and secreted as infectious virions or transported back into the nucleus to replenish and maintain a stable cccDNA pool.

[0094] So far, HBV has been divided into four serotypes (adr, adw, ayr, ayw) based on the antigenic epitopes present on the outer membrane protein, and eight genotypes (A , B, C, D, E, F, G and H). Two new genotypes I and J have recently been identified. HBV genotypes were distributed in different geographical regions. For example, the most prevalent genotypes in Asia are genotypes B and C. Genotype D is mainly found in Africa, the Middle East, and India, while genotype A is widely distributed in Northern Europe, sub-Saharan Africa, and West Africa.

[0095] As used herein, a patient or individual suffering from "chronic HBV infection", "chronic hepatitis B virus (CHB) infection", "CHB", "CHB infection" or "CHB virus infection" refers to this The general meaning in the art, more particularly refers to a patient or individual who is chronically infected with HBV and has detectable HBsAg (with or without HBeAg) in the blood for six months or more after HBV detection. CHB infection can be classified into four stages, which usually, but not always, progress from one stage to the next: (I) HBeAg-positive chronic infection (formerly called immune tolerance), (II) HBeAg-positive chronic hepatitis (previously called immunocompetent), (III) HBeAg-negative chronic infection (previously called inactive carrier), and (IV) HBeAg-negative chronic hepatitis (previously called reactivated). The different stages of chronic HBV infection can also be characterized by differences in viral load, liver enzyme levels (necrotizing inflammatory activity), HBeAg or HBsAg load, or the presence of antibodies to these antigens. Even though viremia may vary significantly, cccDNA content in untreated individuals remains relatively constant, roughly 10 to 50 copies per cell, but when suppressed by nucleoside (nucleotide) analog therapy, Can be as low as 1 to 2 replicas per cell. Persistence of cccDNA species leads to chronicity. In some embodiments, chronic HBV infection can be characterized by laboratory guidelines published by the Centers for Disease Control and Prevention (CDC), such as: (i) detection of IgM against hepatitis B core antigen Antibody (IgM anti-HBc) negative and positive for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), or hepatitis B virus DNA nucleic acid test, or (ii) for HBsAg, or HBV DNA Nucleic acid test was positive, or HBeAg was positive twice at least 6 months apart.

[0096] In some embodiments of the present application, the target nucleic acid sequence is within the HBV gene body. In some embodiments, the TALE DNA binding domain is capable of binding to a target nucleic acid sequence within the HBV gene. In some embodiments of the present application, the nucleic acid sequence of interest is within a nucleic acid sequence encoding an HBV antigen. In some embodiments, the TALE DNA binding domain is capable of binding to a target nucleic acid sequence within a nucleic acid sequence encoding an HBV antigen. In some embodiments, the first half-site sequence and the second half-site sequence of the target nucleic acid sequence are within a nucleic acid sequence encoding an HBV antigen. In some embodiments, the TALE DNA binding domain can bind to the half-site sequence of the target nucleic acid sequence in the HBV gene. Throughout this application, a TALEN monomer comprising a TALE DNA binding domain capable of binding to a target nucleic acid sequence within the HBV gene is also referred to as "HBV TALEN". The nucleic acid sequence of the HBV genome of any genotype can be the target nucleic acid sequence for TALE DNA binding. In some embodiments, the nucleic acid sequence of interest is within the HBV gene of one or more of genotypes A, B, C, D, E, F, G, H, I, and J. In some embodiments, one or more of genotypes A, B, C, D, E, F, G and H of the HBV genetic system.

[0097] In some embodiments of the present application, the target nucleic acid sequence is within the nucleic acid sequence encoding the HBV core protein. In certain embodiments, the TALE DNA binding domain is capable of binding to a target nucleic acid sequence within the nucleic acid sequence encoding the HBV core protein. In some embodiments, the first half-site sequence and the second half-site sequence of the target nucleic acid sequence are within a nucleic acid sequence encoding HBV core protein.

[0098] In some embodiments of the present application, the nucleic acid sequence of interest is within a nucleic acid sequence encoding HBV polymerase (pol). In certain embodiments, the TALE DNA binding domain is capable of binding to a target nucleic acid sequence within a nucleic acid sequence encoding HBV pol. In some embodiments, the first half-site sequence and the second half-site sequence of the target nucleic acid sequence are within a nucleic acid sequence encoding HBV pol.

[0099] In some embodiments of the present application, the target nucleic acid sequence is within a nucleic acid sequence encoding HBV surface antigen (HBsAg). In certain embodiments, the TALE DNA binding domain is capable of binding to a target nucleic acid sequence within a nucleic acid sequence encoding HBsAg. In some embodiments, the first half-site sequence and the second half-site sequence of the target nucleic acid sequence are within a nucleic acid sequence encoding HBsAg. TALEN

[0100] Transcription activator-like effector nucleases (TALENs) are artificial restriction enzymes generated by fusing a TAL effector DNA binding domain to a DNA cleavage domain. These reagents enable efficient, programmable and specific DNA cleavage and represent powerful tools for in situ genome editing. Transcription activator effector-like (TALEs) can be rapidly engineered to bind virtually any DNA sequence. The term TALEN as used herein is broad and includes monomeric TALENs that can cleave double-stranded DNA without the assistance of another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs engineered to work together to cleave DNA at the same site. TALENs that work together can be referred to as left TALENs and right TALENs, with reference to the handedness of the DNA. See U.S. Serial No. 12 / 965,590; U.S. Serial No. 13 / 426,991 (U.S. Patent No. 8,450,471); U.S. Serial No. 13 / 427,040 (U.S. Patent No. 8,440,431); U.S. Serial No. 13 / 427,137 (U.S. Patent No. 8,440,432); and US Serial No. 13 / 738,381, all of which are hereby incorporated by reference in their entirety.

[0101] TAL effectors are proteins secreted by phytopathogenic Xanthomonas bacteria. The DNA binding domain contains a highly conserved repeat segment with a sequence of 33-34 amino acids, except for amino acids 12 and 13. These two positions are highly variable (termed repeat variable diresidues (RVDs)) and show a strong correlation with specific nucleotide recognition (see Figure 1C). This simple relationship between amino acid sequence and DNA recognition allows the engineering of specific DNA-binding domains by selecting combinations of repeat segments containing appropriate RVDs. Slight changes in the RVD and incorporation of "non-conventional" RVD sequences can improve targeting specificity.

[0102] The non-specific DNA cleavage domain from the terminus of the FokI endonuclease can be used to construct hybrid nucleases active in yeast assays. These agents are also active in plant and animal cells. Initial TALEN studies used the wild-type Fokl cleavage domain, but some subsequent TALEN studies also used Fokl cleavage domain variants with mutations designed to improve cleavage specificity and cleavage activity. The FokI domain acts as a dimer, requiring two TALEN constructs with unique DNA-binding domains to acquire sites in the target gene body in proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain (C-terminal domain) and the number of bases (spacers) between two individual TALEN binding sites are used to achieve high level activity. The parameter. The number of amino acid residues in the C-terminal domain between the TALE DNA binding domain and the FokI cleavage domain can be modified by introducing or deleting amino acids according to the number of bases in the spacer sequence. The spacer in the DNA target sequence can be about 12 to about 30 nucleotides.

[0103] The relationship between the amino acid sequence and the DNA recognition of the TALE binding domain allows for designable proteins. Once the TALEN gene has been assembled, it can be inserted, for example, into a plastid or viral construct, which is then used to transfect target cells in which the gene product is expressed and into the nucleus for access to the gene body. TALENs can be used to edit gene bodies by inducing double-strand breaks (DSBs) in DNA. Any method and any combination of methods can be used, for example, to express TALENs and perform genome editing in target cells. In some embodiments, DNA, such as plastid DNA, comprising one or more TALEN genes is transfected into the target cell. Cells can be transfected with one or more plastids including one or more TALEN genes. In some embodiments, target cells are transduced with a viral vector delivering the TALEN gene. Cells can be transduced with one or more viral vectors, where each viral vector delivers one or more TALEN genes. In some embodiments, TALEN mRNA is prepared by in vitro transcription (IVT). In some embodiments, in vitro transcribed TALEN mRNA is transfected into target cells. Cells can be transfected with one or more mRNAs encoding one or more TALEN genes.

[0104] In some embodiments, the TAL nuclease monomer binds to one of the two DNA half-site sequences of the target nucleic acid sequence. Preferably, the half-site sequences are separated by spacer sequences. This spacing allows dimerization of the nuclease catalytic domain and creates a DSB in the spacer sequence between the half-site sequences. Preferably, the first half-site sequence and the second half-site sequence are different sequences separated by a spacer sequence.

[0105] In some embodiments, a DSB will induce an insertion / deletion (indel). "Indel" as used herein refers to a mutation resulting from insertion, deletion or a combination thereof. Those skilled in the art will appreciate that insertions / deletions in the coding region of the genome sequence will result in frame-shift mutations unless the length of the insertion / deletion is a multiple of three. In some embodiments, DSBs will induce point mutations. A "point mutation" as used herein refers to one that substitutes one of the nucleotides. Preferably, the insertion / deletion or point mutation is within the spacer sequence between the nucleic acid sequences of interest.

[0106] In some embodiments, cleavage of the target nucleic acid sequence results in decreased expression of the target gene. In some embodiments, the reduction in expression of the target gene is a reduction in mRNA levels. In some embodiments, the reduced expression of the target gene is reduced protein content or reduced protein function. The term "decreased" is generally used herein to mean a statistically significant decrease. However, for the avoidance of doubt, "decrease / weaken" means at least 10% decrease / weaken compared to a reference level, such as at least about 20%, or at least about 30%, or at least about 40%, compared to a reference level, Or at least about 50%, or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 90% reduction / weakening, or up to and including 100% reduction / weakening (also That is, a non-existent level compared to the reference sample), or any reduction / weakening between 10% and 100%. The reference level can be, for example, the performance level of an untreated sample or individual or a control treated sample or individual. The term "statistically significant" or "significantly" refers to statistical significance and generally means two standard deviations (2 SD) or more below the normal concentration of a marker. The term refers to the statistical indication that a difference exists. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. Decisions are often made using p-values.

[0107] In some embodiments, the composition or combination of the present invention comprises: a first TALEN monomer comprising a first TALE DNA binding domain and a first nuclease catalytic domain, wherein the first TALE DNA binding domain is capable of binding to The first half-site sequence of the target nucleic acid sequence in the HBV gene body; and the second TALEN monomer, which comprises a second TALE DNA binding domain and a second nuclease catalytic domain, wherein the second TALE DNA binding domain can bind to the target nucleic acid A second half-site sequence of sequence, wherein when the first TALE DNA binding domain binds to the first half-site sequence and the second TALE DNA-binding domain binds to the second half-site sequence, the first TALEN The monomer and the second TALEN monomer are capable of forming a dimer that cleaves the target nucleic acid sequence. The first TALEN monomer and the second TALEN monomer can form dimers via their catalytic domains. Preferably, the first nuclease catalytic domain is a first FokI nuclease catalytic domain and the second nuclease catalytic domain is a second FokI nuclease catalytic domain. Preferably, the first half-site sequence and the second half-site sequence are different sequences separated by a spacer sequence.

[0108] In some embodiments, the target nucleic acid sequence is within the sequence encoding HBsAg and HBV polymerase (pol). Preferably, the first half-site sequence of the target nucleic acid sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 1, such as at least 90%, 91%, 92%, 93%, 94%, 95% %, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequence, and the second half-site sequence of the target nucleic acid sequence comprises at least about 90% of the nucleic acid sequence of SEQ ID NO: 2 % identical, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical polynucleotide sequences; or the target nucleic acid sequence The first half site sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 3, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequence, and the second half site sequence of the target nucleic acid sequence comprises at least about 90% identical to the nucleic acid sequence of SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequence, or the target nucleic acid sequence comprises the same sequence as SEQ ID NO: 5 or The nucleic acid sequence of SEQ ID NO: 6 is at least 90% identical, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical Nucleotide sequence.

[0109] In some embodiments, the first TALE DNA binding domain and the second TALE DNA binding domain each comprise a repeat unit, and each repeat unit comprises two amino acids that determine the recognition of a base pair in the target nucleic acid sequence The hypervariable region is called repeat variable diresidue (RVD). In some embodiments, the first TALE DNA binding domain and the second TALE DNA binding domain each comprise 15 to 20 RVDs, such as 15, 16, 17, 18, 19 or 20 RVDs.

[0110] In some embodiments, the first TALE DNA binding domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 7, such as at least 90%, 91% identical to the amino acid sequence of SEQ ID NO: 7 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence, and the second TALE DNA binding domain comprises at least 90% of SEQ ID NO: 8 % identity, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 8 Amino acid sequence; or the first TALE DNA binding domain comprises at least 90% identity with the amino acid sequence of SEQ ID NO: 9, such as at least 90%, 91%, 92% with the amino acid sequence of SEQ ID NO: 9 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence, and the second TALE DNA binding domain comprises at least 90% identical to SEQ ID NO: 10, Amino acids such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10 sequence; and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprise at least 90% identity with SEQ ID NO: 11, such as at least 90%, 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity. In some embodiments, the first TALE DNA binding domain comprises the amino acid sequence of SEQ ID NO: 7, the second TALE DNA binding domain comprises the amino acid sequence of SEQ ID NO: 8, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the first TALE DNA binding domain comprises the amino acid sequence of SEQ ID NO: 9, the second TALE DNA binding domain comprises the amino acid sequence of SEQ ID NO: 10, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the first TALE DNA binding domain consists of the amino acid sequence of SEQ ID NO: 7, the second TALE DNA binding domain consists of the amino acid sequence of SEQ ID NO: 8, and the first FokI nuclease The catalytic domain and the second FokI nuclease catalytic domain each consist of the amino acid sequence of SEQ ID NO: 11. In some embodiments, the first TALE DNA binding domain consists of the amino acid sequence of SEQ ID NO: 9, the second TALE DNA binding domain consists of the amino acid sequence of SEQ ID NO: 10, and the first FokI nuclease The catalytic domain and the second FokI nuclease catalytic domain each consist of the amino acid sequence of SEQ ID NO: 11.

[0111] In some embodiments, respectively, the first type of transcription activator effector nuclease (TALEN) monomer comprises a first TALE DNA binding domain and a first FokI nuclease catalytic domain, wherein the first TALE DNA binds The domain can be combined to the first half site sequence of the target nucleic acid sequence in the HBV gene body, and the first half site sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical polynucleotide sequences, and the second type of transcription activator effector nucleic acid The enzyme (TALEN) monomer comprises a second TALE DNA binding domain and a second FokI nuclease catalytic domain, wherein the second TALE DNA binding domain can bind to the second half-site sequence of the target nucleic acid sequence, and the second half-site sequence Comprising at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% identical polynucleotide sequences, wherein when the first TALE DNA binding domain binds to the first half-site sequence and the second TALE DNA binding domain binds to the second half-site sequence, the first TALEN The monomer and the second TALEN monomer are capable of forming a dimer that cleaves the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is within the HBV gene of one or more of genotypes A, B, C, D, E, F, G, H, I and / or J. In some embodiments, one or more of genotypes A, B, C, D, E, F, G and / or H of the HBV genetic system.

[0112] In one aspect, the application provides a method for cleaving a target nucleic acid sequence in an HBV genome, the method comprising combining the HBV genome with any composition, combination, nucleic acid molecule, isolated The host cell or the pharmaceutical composition is contacted. In another aspect, the present application provides a method for inducing gene editing of a target nucleic acid sequence in an HBV genome, the method comprising combining the HBV genome with any composition, combination, nucleic acid molecule, The isolated host cells or the pharmaceutical composition are contacted. In some embodiments, one or more of genotypes A, B, C, D, E, F, G, H, I and J of the HBV genetic system. In some embodiments, one or more of genotypes A, B, C, D, E, F, G and H of the HBV genetic system. polynucleotides and vectors

[0113] In a general aspect, the application provides a combination comprising: a first nucleic acid, preferably a first mRNA molecule, comprising a first class of transcriptional activator effector nuclease (TALEN) encoding The polynucleotide sequence of the monomer, the first TALEN monomer comprises the first TALE DNA binding domain and the first FokI nuclease catalytic domain, wherein the first TALE DNA binding domain can bind to the target nucleic acid sequence in the HBV gene The first half site sequence, and the first half site sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 1, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequences; and a second nucleic acid, preferably a second mRNA molecule, comprising a second class of transcription activator effector nuclease (TALEN ) monomer polynucleotide sequence, the second TALEN monomer comprises a second TALE DNA binding domain and a second FokI nuclease catalytic domain, wherein the second TALE DNA binding domain is capable of binding to the second target nucleic acid sequence A half-site sequence, and the second half-site sequence comprises at least about 90% identity with the nucleotide sequence of SEQ ID NO: 2, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96% %, 97%, 98%, 99% or 100% identical polynucleotide sequences; wherein when the first TALE DNA binding domain binds to the first half-site sequence and the second TALE DNA binding domain binds to the For the second half-site sequence, the first TALEN monomer and the second TALEN monomer can form a dimer that cleaves the target nucleic acid sequence.

[0114] The nucleic acid molecule may comprise any non-naturally occurring polynucleotide sequence encoding the HBV TALEN of the present application, which HBV TALEN may be produced using methods known in the art in light of the present invention. The polynucleotide may be in the form of RNA, preferably mRNA, or in the form of DNA obtained by recombinant techniques (eg, cloning) or produced synthetically (eg, chemical synthesis). DNA can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. DNA may, for example, comprise genomic DNA, cDNA, or combinations thereof. A polynucleotide can also be a DNA / RNA hybrid. The polynucleotides and vectors of the present application can be used to produce recombinant proteins, express proteins in host cells, or produce virions.

[0115] In another general aspect, the application provides a combination of mRNA molecules, the combination comprising: a first mRNA molecule comprising a polynucleotide encoding a first type of transcription activator effector nuclease (TALEN) monomer Nucleotide sequence, the first TALEN monomer comprises a first TALE DNA binding domain and a first nuclease catalytic domain, wherein the first TALE DNA binding domain can bind to the first half site of the target nucleic acid sequence in the HBV gene sequence; and a second mRNA molecule comprising a polynucleotide sequence encoding a second TALEN monomer comprising a second TALE DNA binding domain and a second nuclease catalytic domain, wherein the second TALE DNA The binding domain is capable of binding to the second half-site sequence of the target nucleic acid sequence; wherein when the first TALE DNA-binding domain binds to the first half-site sequence and the second TALE DNA-binding domain binds to the second half-site sequence When targeting a target sequence, the first TALEN monomer and the second TALEN monomer can form a dimer that cleaves the target nucleic acid sequence.

[0116] Preferably, the first nuclease catalytic domain is a first FokI nuclease catalytic domain and the second nuclease catalytic domain is a second FokI nuclease catalytic domain. The first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain can be the same or different. In some embodiments, the FokI nuclease catalytic domain comprises at least about 90% identity to the amino acid sequence of SEQ ID NO: 11, such as 90%, 91%, 92%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity. In some embodiments, each FokI nuclease catalytic domain independently comprises at least about 90% identity to the amino acid sequence of SEQ ID NO: 11, such as 90%, 91%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity. In some embodiments, each FokI nuclease catalytic domain consists of the amino acid sequence of SEQ ID NO: 11.

[0117] In some embodiments, each of the first mRNA molecule and the second mRNA molecule further comprises one or more of the following, preferably all: 5' end cap, 5'-UTR, sequence encoding a nuclear localization signal , the sequence encoding the N-terminal domain, the sequence encoding the C-terminal domain, the 3'-UTR and the polyadenosine tail.

[0118] Preferably, the mRNA as described herein comprises a 5' cap. Capped 5'-ends and the like with various groups are known in the art. An endcap structure present on the 5'-end of mRNA in all eukaryotic organisms (and some viruses) is crucial for stabilizing mRNA in vivo. The naturally occurring endcap structure comprises a ribo-guanosine residue methylated at position N7 of the guanine base. This 7-methylguanosine (m7G) is linked via the 5'-strand to the 5'-triphosphate strand at the 5'-end of the mRNA molecule. The presence of the m7Gppp fragment at the 5'-end is required for mRNA maturation because it protects the mRNA from exonuclease degradation, facilitates its transport from the nucleus to the cytoplasm, and plays a key role in the assembly of the translation initiation complex (Cell 9:645-653, (1976); Nature 266:235, (1977); Federation of Experimental Biologists Society Letter 96:1-11, (1978); Cell 40:223-24, (1985); Prog. Nuc. Acid Res. 35:173-207, (1988); Ann. Rev. Biochem. 68:913-963, (1999); and J Biol. Chem. 274:30337-3040, (1999)).

[0119] Those mRNAs carrying only end caps are active in end cap-dependent translation; "decapitation" of mRNAs results in an almost complete loss of their template activity for protein synthesis (Nature, 255:33- 37, (1975); J. Biol. Chem., vol. 253:5228-5231, (1978); and Proc. Natl. Acad. Sci. USA, 72:1189-1193, (1975)).

[0120] Another element of eukaryotic mRNA is the presence of a 2'-O-methyl nucleoside residue at transcriptional position 1 (end cap 1 ) and in some cases at transcriptional positions 1 and 2 (end cap 2) . 2'-O-methylation of mRNA provides higher mRNA translation efficiency in vivo (Proc. Natl. Acad. Sci. USA, 77:3952-3956 (1980)) and further improves 5'-capped mRNA nuclease stability. mRNAs with cap 1 (and cap 2) are unique markers that allow cells to recognize bona fide mRNA 5' ends and, in some cases, reject transcripts derived from infectious genetic elements (Nucleic Acid Research 43: 482-492 (2015)).

[0121] Some examples of 5' cap structures and methods of preparing mRNAs comprising them are given in WO2015 / 051169A2, WO / 2015 / 061491, US 2018 / 0273576 and US Patent Nos. 8,093,367, 8,304,529 and U.S. 10,487,105 . The 5' end cap can be selected from m7GpppA, m7GpppC; unmethylated end cap analogs (e.g., GpppG); dimethylated end cap analogs (e.g., m2,7GpppG), trimethylated end cap analogs ( For example, m2,2,7GpppG), dimethylated symmetric end cap analogs (e.g., m7Gpppm7G) or anti-reverse end cap analogs (e.g., ARCA; m7, 2'OmeGpppG, m72'dGpppG, m7,3' OmeGpppG, m7, 3'dGpppG and tetraphosphate derivatives thereof) (see e.g. Jemielity, J. et al., RNA 9: 1108-1122 (2003). The 5' end cap may be an ARCA end cap (3'-OMe- m7G(5')pppG). The 5' end cap can be mCAP (m7G(5')ppp(5')G, N7-methyl-guanosine-5'-triphosphate-5'-guanosine). The 5' end cap is resistant to hydrolysis. In some embodiments, the 5' end cap is m7GpppAmpG known in the art. In some embodiments, the 5' end cap is m7GpppG or m7GpppGm, known in the art. Provided below Structural formula of an embodiment of the 5' end cap structure.

[0122] In some embodiments, the mRNA described herein comprises a 5' cap having the following structure (Cap 1): (Cap 1) wherein B1 is a natural or modified nucleobase; R1 and R2 each independently selected from halogen, OH, and OCH3; each L independently selected from the group consisting of phosphoric acid ester, phosphorothioate, and boranophosphate, wherein each L is connected by a diester bond; n is 0 or 1 ; and mRNA means the mRNA of the present invention linked at its 5' end. In some embodiments, B1 is G, m7G or A. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, B1 is A or m6A and R1 is OCH3; wherein G is guanine, m7G is 7-methylguanine, A is adenine and m6A is N6-methyladenine.

[0123] In some embodiments, the mRNA described herein comprises a 5' end cap having the structure (end cap II): (end cap II) wherein B1 and B2 are each independently a natural or modified nucleobase R1, R2, and R3 are each independently selected from halogen, OH, and OCH3; each L is independently selected from the group consisting of phosphoric acid ester, phosphorothioate, and boranophosphate, wherein each L is connected by a diester bond ligated; mRNA means the mRNA of the invention ligated at its 5' end; and n is 0 or 1. In some embodiments, at least one of R1, R2, and R3 is OH. In some embodiments, B1 is G, m7G or A. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, B1 is A or m6A and R1 is OCH3; wherein G is guanine, m7G is 7-methylguanine, A is adenine and m6A is N6-methyladenine.

[0124] In some embodiments, the mRNA described herein comprises a 5' cap having the following structure (Cap III): (Cap III) wherein B1, B2, and B3 are each independently native or modified Nucleobase; R1, R2, R3 and R4 are each independently selected from halogen, OH and OCH3; each L is independently selected from the group consisting of: phosphate, phosphorothioate and boranophosphate, wherein each L is borrowed Linked by a diester bond; mRNA means the mRNA of the present invention linked at its 5' end; n is 0 or 1. In some embodiments, at least one of R1, R2, R3, and R4 is OH. In some embodiments, B1 is G, m7G or A. In some embodiments, B1 is A or m6A and R1 is OCH3; wherein G is guanine, m7G is 7-methylguanine, A is adenine and m6A is N6-methyladenine. In some embodiments, n is 1.

[0125] In some embodiments, the mRNA described herein comprises a m7GpppG 5' cap analog having the structure (cap IV) of the formula: (cap IV) wherein each of R1, R2, and R3 is independently selected from halogen , OH, and OCH3; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate, wherein each L is linked by a diester bond; mRNA represents the base linked at its 5' end Invented mRNA; and n is 0 or 1. In some embodiments, at least one of R1, R2, and R3 is OH. In some embodiments, the 5' endcap is m7GpppG, wherein R1, R2, and R3 are each OH, n is 1, and each L is a phosphate. In some embodiments, n is 1. In some embodiments, the 5' end cap is m7GpppGm, wherein R1 and R2 are each OH, R3 is OCH3, each L is a phosphate, the mRNA is a CFTR mRNA of the invention linked at its 5' end, and n is 1.

[0126] In some embodiments, the mRNA described herein comprises a m7GpppAmpG 5' cap analog having the structure (Cap V) of the following formula: (Cap V) wherein each of R1, R2 and R4 is independently selected from halogen , OH, and OCH3; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate, wherein each L is linked by a diester bond; mRNA represents the base linked at its 5' end Invented mRNA; and n is 0 or 1. In some embodiments, at least one of R1, R2, and R4 is OH. In some embodiments, the endcap V compound is m7GpppAmpG, wherein each of R1, R2, and R4 is OH, n is 1, and each L is a phosphate. In some embodiments, n is 1.

[0127] Preferably, the mRNA described herein further comprises a 5' untranslated region (UTR) sequence. As understood in the art, the 5'-UTR and / or 3'-UTR can affect the translational stability or efficiency of the mRNA. The 5'-UTR can be derived from relatively stable mRNA molecules known in the art (such as histone, tubulin, globulin, glyceraldehyde 1-phosphate dehydrogenase (GAPDH), actin, or citrate cycle enzyme ) to increase the stability of translatable oligomers. In other embodiments, the 5'-UTR sequence may include a partial sequence of the cytomegalovirus (CMV) early-onset 1 (IE1) gene.

[0128] Preferably, the 5'-UTR comprises a sequence of a 5'-UTR selected from the group consisting of human IL-6, alanine aminotransferase 1, human lipoprotein E, human fibrinogen alpha chain, human Transthyretin, human binding globulin, human alpha-1-antichymotrypsin, human antithrombin, human alpha-1-antitrypsin, human albumin, human beta-globulin, human complement C3, human Complement C5, SynK (derived from the thylakoid potassium channel protein of Synechocystis sp. cyanobacteria), mouse beta globulin, mouse albumin, and tobacco etch virus, or any of the foregoing fragments of the Preferably, the 5'-UTR is derived from Tobacco Etch Virus (TEV). Preferably, the mRNA described herein comprises a 5'-UTR sequence derived from a gene expressed by Arabidopsis thaliana. Preferably, the 5'-UTR sequence of the gene expressed by Arabidopsis thaliana is AT1G58420. Preferred 5'-UTR sequences comprise SEQ ID NO: 12-17. Preferably, the 5'-UTR sequence comprises SEQ ID NO: 13 (AT1G58420).

[0129] Preferably, the mRNA as described herein comprises a 3'-UTR. Preferably, the 3'-UTR comprises the sequence of the 3'-UTR selected from the group consisting of alanine aminotransferase 1, human lipoprotein E, human fibrinogen alpha chain, human binding globulin, human anticoagulant Enzyme, human alpha globulin, human beta globulin, human complement C3, human growth factor, human hepcidin, MALAT-1, mouse beta globulin, mouse albumin, and Xenopus Beta globulin, or a fragment of any of the foregoing. Preferably, the 3'-UTR is derived from Xenopus beta globin. Preferred 3'-UTR sequences include SEQ ID NO: 18-24.

[0130] Preferably, the mRNA as described herein comprises a 3' tail region which can be used to protect the mRNA from degradation by exonucleases. The tail region can be a 3' poly(adenosine) region and / or a 3' poly(cytidine) region. Preferably, the tail region is a 3' poly(adenosine) tail. A "3' poly(A) tail" or "poly adenosine tail" as used herein is a contiguous adenosine nucleoside whose size may range, for example, from Polymer of acid: 10 to 250 consecutive adenosine nucleotides; 60-125 consecutive adenosine nucleotides; 90-125 consecutive adenosine nucleotides; 95-125 consecutive adenosine nucleotides; 95 -121 consecutive adenosine nucleotides; 100-121 consecutive adenosine nucleotides; 110-121 consecutive adenosine nucleotides; 112-121 consecutive adenosine nucleotides; 114-121 consecutive adenosine nucleotides nucleotides; and 115-121 consecutive adenosine nucleotides. Preferably, the 3' polyadenosine tail as described herein comprises 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 or 125 consecutive adenosine nucleotides. The 3' poly(adenosine) tail can be added using various methods known in the art, such as using a poly(adenosine) polymerase to add a tail to synthetic or in vitro transcribed RNA. Other methods include the use of transcription vectors to encode polyadenosine tails or the use of ligases (e.g., via splint ligation using T4 RNA ligase and / or T4 DNA ligase), wherein poly(adenosine) can be ligated to the 3' end of the sense RNA. Preferably, a combination of any of the above methods is utilized.

[0131] In some embodiments, the first mRNA molecule and the second mRNA molecule each comprise a 5' end cap, a 5'-UTR, a sequence encoding a nuclear localization signal, a sequence encoding an N-terminal domain, a sequence encoding a DNA binding domain , the sequence encoding the nuclease catalytic domain, the sequence encoding the C-terminal domain, the 3'-UTR and the polyadenosine tail. In some embodiments, the first mRNA molecule and the second mRNA molecule each comprise a 5' cap, a 5'-UTR, a sequence encoding an N-terminal domain, a sequence encoding a DNA binding domain, a sequence encoding a nuclease catalytic domain, a sequence encoding Sequence of C-terminal domain, 3'-UTR and polyadenosine tail. In some embodiments, the first mRNA molecule and the second mRNA molecule each comprise a 5' cap, a 5'-UTR, a sequence encoding a nuclear localization signal, a sequence encoding an N-terminal domain, a sequence encoding a DNA binding domain, a sequence encoding a C Sequence of telodomain, 3'-UTR and polyadenosine tail. In some embodiments, the first mRNA molecule and the second mRNA molecule each comprise a 5' cap, a 5'-UTR, a sequence encoding an N-terminal domain, a sequence encoding a DNA binding domain, a sequence encoding a C-terminal domain, a 3' -UTR and polyadenosine tail.

[0132] The present application also relates to a vector comprising a combination of nucleic acids encoding HBV TALEN. A "vector" as used herein is a nucleic acid molecule used to carry genetic material into another cell where it can be replicated and / or expressed. Any vector known to those skilled in the art may be used in light of the present invention. Examples of vectors include, but are not limited to, plastids, viral vectors (bacteriophage, animal viruses, and plant viruses), myxoplasts, and artificial chromosomes (eg, YAC). Preferably, the vector is a DNA plasmid. A vector can be a DNA vector or an RNA vector. Those of ordinary skill in the art can construct vectors for the present application via standard recombinant techniques in view of the present invention.

[0133] The carrier of the present application may be an expression carrier. The term "expression vector" as used herein refers to any type of genetic construct comprising nucleic acid encoding RNA capable of being transcribed. Expression vectors include, but are not limited to, vectors for expression of recombinant proteins, such as DNA plastids or viral vectors, and vectors, such as DNA plastids or viral vectors, for delivery of nucleic acids into an individual for expression in individual tissues. Those skilled in the art should understand that the design of expression vectors may depend on factors such as the choice of host cells to be transformed, the expression level of the desired protein, and the like.

[0134] The vectors of the present application may contain various regulatory sequences. The term "regulatory sequence" as used herein refers to any sequence that allows, facilitates or regulates the functional regulation of a nucleic acid molecule, including the transfer of one of the nucleic acid or its derivatives (i.e. mRNA) to a host cell or Replication / duplication, transcription, splicing, translation, stability and / or transport in an organism. In the context of the present invention, the term encompasses promoters, enhancers and other expression control elements such as polyadenylation signals and elements affecting mRNA stability.

[0135] In some embodiments of the present application, the vector is a non-viral vector. Examples of non-viral vectors include, but are not limited to, DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophage, and the like. Preferably, the non-viral vector is a DNA plasmid. "DNA plastid" is used interchangeably with "DNA plastid vector", "plastid DNA" or "plastid DNA vector" and refers to a double-stranded, substantially circular shape capable of autonomous replication in a suitable host cell. DNA sequence. DNA plasmids used to express encoded polynucleotides typically include an origin of replication, a multiplex colony site, and a selectable marker, such as an antibiotic resistance gene. Examples of suitable DNA plasmids that may be used include, but are not limited to, commercially available expression vectors used in well known expression systems, including both prokaryotic and eukaryotic systems, such as pSE420 (Invitrogen, San Diego, Calif.), which can be used for protein production and / or expression in E. coli; pYES2 (Invitrogen, Thermo Fisher Scientific), which can be used for production and / or expression in Saccharomyces cerevisiae yeast strains; MAXBAC® complete baculovirus expression system ( Thermo Fisher Scientific), which can be used for production and / or expression in insect cells; pcDNATM or pcDNA3TM (Life Technologies, Thermo Fisher Scientific), which can be used for high-level constitutive protein expression in mammalian cells; and pVAX or pVAX- 1 (Life Technologies, Thermo Fisher Scientific), which can be used for high-level transient expression of proteins of interest in most mammalian cells. The backbone of any commercially available DNA plasmid can be modified to optimize protein expression in the host cell by using conventional techniques and readily available starting materials in order to invert certain elements such as origins of replication and / or antibiotics. resistance cassette), replace the endogenous promoter of the plastid (such as the promoter in the antibiotic resistance cassette), and / or replace the polynucleotide sequence encoding the transcribed protein (such as the antibiotic resistance gene encoding sequence). (See, eg, Sambrook et al., Molecular Cloning a Laboratory Manual, 2nd Ed. Cold Spring Harbor Press (1989)).

[0136] Preferably, the DNA plasmid is an expression vector suitable for protein expression in mammalian host cells. Expression vectors suitable for protein expression in mammalian host cells include, but are not limited to, pcDNA™, pcDNA3™, pVAX, pVAX-1, ADVAX, NTC8454, and the like. Preferably, the expression vector is based on pVAX-1, which can be further modified to optimize protein expression in mammalian cells. pVAX-1 is a commonly used plasmid in DNA vaccines and contains a strong human early-onset cytomegalovirus (CMV-IE) promoter followed by a bovine growth hormone (bGH)-derived polyadenylation sequence (pA) . pVAX-1 further contains the pUC origin of replication and a kamycin resistance gene driven by a small prokaryotic promoter that allows bacterial plastid propagation.

[0137] The vector of the present application may also be a viral vector. Generally speaking, a viral vector is a genetically engineered virus carrying a modified viral DNA or RNA that has been rendered non-infectious but still contains a viral promoter and a transgenic gene, thereby allowing The viral promoter translates the transgene. Because viral vectors typically lack infectious sequences, they require helper viruses or encapsulated strains for large-scale transfection. In certain embodiments, a vector as described herein is, for example, a recombinant adenovirus, a recombinant retrovirus, a recombinant poxvirus such as a pox virus (e.g., Modified Vaccinia Ankara (MVA)), such as Victor A recombinant alphavirus of Semliki forest virus, a recombinant paramyxovirus such as a recombinant measles virus, or another recombinant virus. Examples of viral vectors that can be used include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, enterovirus vectors, Venezuelan Equine Encephalitis virus vectors, Victory Forest virus vectors, tobacco mosaic vectors Virus (Tobacco Mosaic Virus) vector, lentiviral vector, etc. In certain embodiments, vectors as described herein are MVA vectors. The vector can also be a non-viral vector.

[0138] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. The term "AAV" includes such species as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10 and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV of any serotype and AAV of any species of AAV, primate AAV, non-primate AAV, and ovine AAV. In some embodiments, the viral vector is an adenoviral vector such as a recombinant adenoviral vector. Recombinant adenoviral vectors may eg be derived from human adenoviruses (HAdV or AdHu) or monkey adenoviruses such as chimpanzee or gorilla adenoviruses (ChAd, AdCh or SAdV) or rhesus adenoviruses (rhAd). Preferably, the adenovirus vector is a recombinant human adenovirus vector such as recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotypes 5, 4, 35, 7, 48, etc. In other embodiments, the adenoviral vector is a rhAd vector such as rhAd51 , rhAd52 or rhAd53. Recombinant viral vectors useful in this application can be prepared using methods known in the art in view of the present invention. For example, given the degeneracy of the genetic code, several nucleic acid sequences can be designed that encode the same polypeptide. The polynucleotides encoding the HBV TALENs of the present application can optionally be codon-optimized to ensure proper expression in host cells such as bacterial or mammalian cells. Codon optimization is a widely used technique in the art, and methods for obtaining codon-optimized polynucleotides will be known to those skilled in the art in light of the present invention.

[0139] Vectors of the present application, such as DNA plasmids or viral vectors (especially AAV, such as AAV7, AAV8, AAV9, or any other AAV with liver tropism, including hybrid AAV, or adenoviral vectors) may comprise vectors for establishing Any regulatory element of the conventional function(s) of the vector including, but not limited to, the replication and expression of the HBV TALEN(s) encoded by the polynucleotide sequence of the vector. Regulatory elements include, but are not limited to, promoters, enhancers, polyadenylation signals, translation stop codons, ribosome binding elements, transcription terminators, selectable markers, origins of replication, and the like. A carrier may contain one or more presentation cassettes. The "expression cassette" is part of the vector that directs the cellular machinery to make RNA and protein. An expression cassette typically comprises three components: a promoter sequence, an open reading frame, and a 3'-untranslated region (UTR) optionally including a polyadenylation signal. An open reading frame (ORF) is the reading frame comprising the coding sequence of a protein of interest (eg, HBV TALEN) from a start codon to a stop codon. The regulatory element of the expression cassette is operably linked to a polynucleotide sequence encoding the HBV TALEN of interest. As used herein, the term "operably linked" is employed in its broadest reasonable context and refers to a linkage of polynucleotide elements in a functional relationship. A polynucleotide is "operably linked" when it is in a functional relationship with another polynucleotide. For example, a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence. Any of the components suitable for use in the presentation cassettes described herein may be used in any combination and in any order to prepare the carrier of the present application.

[0140] The vector may comprise a promoter sequence, preferably within an expression cassette, to control the expression of the HBV TALEN of interest. The term "promoter" is used in its conventional sense and refers to a nucleotide sequence that initiates the transcription of an operably linked nucleotide sequence. A promoter is located on the same strand near the nucleotide sequence it transcribes. Promoters can be constitutive, inducible or repressible. Promoters can be naturally occurring or synthetic. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects and animals. The promoter can be a homologous promoter (ie, derived from the same gene source as the vector) or a heterologous promoter (ie, derived from a different vector or gene source). For example, if the vector to be employed is a DNA plastid, the promoter may be endogenous to the plastid (homologous) or derived from another source (heterologous). Preferably, the promoter is located upstream of the polynucleotide encoding HBV TALEN in the expression cassette.

[0141] Examples of promoters that can be used include, but are not limited to, the promoter from Simian Virus 40 (SV40), the Mouse Mammary Tumor Virus (MMTV) promoter, such as Human Immunodeficiency Virus (HIV) or Bovine Immunodeficiency Virus ( Lentiviral promoter of BIV) long terminal repeat (LTR) promoter, Moloney virus (Moloney virus) promoter, avian leukemia virus (ALV) promoter, such as CMV early-onset (CMV-IE) promoter Cytomegalovirus (CMV) promoter, Epstein Barr virus (Epstein Barr virus, EBV) promoter or Rous sarcoma virus (Rous sarcoma virus, RSV) promoter. The promoter can also be a promoter from a human gene such as human actin, human myosin, human heme, human muscle creatine, or human metallothionein. The promoter may also be a natural or synthetic tissue specific promoter such as a muscle or skin specific promoter.

[0142] The vector may contain additional polynucleotide sequences that stabilize expressed transcripts, enhance nuclear export of RNA transcripts, and / or improve transcription-translation coupling. Examples of such sequences include polyadenylation signal and enhancer sequences. The polyadenylation signal is usually located downstream of the coding sequence for the protein of interest (eg, HBV TALEN) within the expression cassette of the vector. Enhancer sequences are regulatory DNA sequences that, when bound to a transcription factor, enhance the transcription of an associated gene. The enhancer sequence is preferably located upstream of the polynucleotide sequence encoding the HBV TALEN within the expression cassette of the vector, but downstream of the promoter sequence.

[0143] Any polyadenylation signal known to those skilled in the art may be used in light of the present invention. For example, the polyadenylation signal can be the SV40 polyadenylation signal, the LTR polyadenylation signal, the bovine growth hormone (bGH) polyadenylation signal, the human growth hormone (hGH) polyadenylation signal, or the human β-globin polyadenylation signal.

[0144] Any enhancer sequence known to those skilled in the art may be used in light of the present invention. For example, the enhancer sequence can be human actin, human myosin, human heme, human muscle creatine, or a viral enhancer such as the enhancer from CMV, HA, RSV or EBV. Examples of specific enhancers include, but are not limited to, woodchuck HBV post-transcriptional regulatory elements (WPRE), intron / exon sequences derived from human lipoprotein A1 precursor (ApoAI), human T-cell leukemia virus type 1 (HTLV-1) non-translated R-U5 domain of long terminal repeat (LTR), splice enhancer, synthetic rabbit β-globin intron, or any combination thereof.

[0145] The vector may comprise a polynucleotide sequence encoding a signal peptide sequence for protein localization. Preferably, the polynucleotide sequence encoding the signal peptide sequence is located upstream of the polynucleotide sequence encoding the HBV TALEN, eg in the N-terminal region. Preferably, the signal sequence is a nuclear localization signal (NLS) sequence that can guide the TALEN protein to localize to the nucleus for DNA binding and editing. Any signal peptide known in the art may be used in light of the present invention. For example, the signal peptide can be SV40 large T antigen NLS (PKKKRKV) (SEQ ID NO: 31), nucleoplasmic protein NLS (KRPAATKKAGQAKKKK) (SEQ ID NO: 32), EGL-13 NLS (MSRRRKANPTKLSENAKKLAKEVEN) (SEQ ID NO : 33), c-Myc protooncoprotein NLS (PAAKRVKLD) (SEQ ID NO: 34) or TUS protein NLS (KLKIKRPVK) (SEQ ID NO: 35).

[0146] The vector may comprise a polynucleotide sequence encoding a polypeptide or a peptide affinity tag such as glutathione S-transferase (GST), green fluorescent protein (GFP) and such as Yellow fluorescent protein (YFP) and other fluorescent proteins of mCherry, such as maltose binding protein, protein A, FLAG tag (such as DYKDDDDK (SEQ ID NO: 36) or EYKEEEEK (SEQ ID NO: 37), hexahistidine (eg HHHHHH) (SEQ ID NO: 38), myc tag (eg EQKLISEEDL) (SEQ ID NO: 39), influenza HA tag (eg YPYDVPDYA) (SEQ ID NO: 40), AcV5 tag (SWKDASGWS) (SEQ ID NO: 41), ALFA-tag (eg SRLEEELRRLTE) (SEQ ID NO: 42), Avi-tag (eg GLNDIFEAQKIEWHE) (SEQ ID NO: 43), E-tag (eg GAPVPYPDPLEPR) (SEQ ID NO: 44), S-tag (eg KETAAAKFERQHMDS) (SEQ ID NO: 45), Strep-tag (eg Strep-tag II:WSHPQFEK) (SEQ ID NO: 46), T7-tag (eg MASMTGGQQMG) (SEQ ID NO: 47), Ty1 Tag (for example EVHTNQDPLD) (SEQ ID NO: 48), V5 tag (for example GKPIPNPLLGLDST) (SEQ ID NO: 49), VSV-G tag (for example YTDIEMNRLGK) (SEQ ID NO: 50) or Xpress tag (for example DLYDDDDK) ( SEQ ID NO: 51), in order to carry out purification and / or detection. The reading frame of the polypeptide (such as HBV TALEN) of interest is connected to the reading frame of the gene encoding the affinity tag by the affinity tag or reporter fusion so that Translational fusions are generated. Expression of the fusion gene results in the translation of a single polypeptide that includes both the polypeptide of interest (e.g., HBV TALEN) and an affinity tag. In some cases using an affinity tag, the DNA sequence encoding the protease recognition site The fusion will be between the affinity tag and the reading frame of the polypeptide of interest (eg, HBV TALEN).

[0147] Vectors such as DNA plastids may also include a bacterial origin of replication and an antibiotic resistance expression cassette for plastid selection and maintenance in bacterial cells such as E. coli. The bacterial origin of replication and antibiotic resistance cassette can be located in the vector in the same orientation as the expression cassette encoding the HBV TALEN or in the opposite (reverse) orientation. An origin of replication (ORI) is a sequence that serves as the origin of replication, enabling plastids to replicate and survive within a cell. Examples of ORIs suitable for use in this application include, but are not limited to, ColE1, pMB1, pUC, pSC101, R6K and 15A, preferably pUC.

[0148] Expression cassettes for selection and maintenance in bacterial cells typically include a promoter sequence operably linked to an antibiotic resistance gene. Preferably, the promoter sequence operably linked to the antibiotic resistance gene is different from the promoter sequence operably linked to the polynucleotide sequence encoding the protein of interest, eg, HBV TALEN. Antibiotic resistance genes can be codon-optimized, and the sequence composition of antibiotic resistance genes is often adjusted to the codon usage of bacteria such as E. coli. Any antibiotic resistance gene known to those skilled in the art may be used in view of the present invention, including but not limited to kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr) and tetracycline resistance gene (Tetr ) and genes that confer resistance to chloramphenicol, bleomycin, spectinomycin, carbenicillin, etc.

[0149] The polynucleotides and expression vectors encoding the HBV TALENs of the present application can be produced by any method known in the art in view of the present invention. For example, polynucleotides encoding HBV TALENs can be introduced or "cloned" into expression vectors using standard molecular biology techniques such as polymerase chain reaction (PCR), well known to those skilled in the art.

[0150] As used herein, "percent identity" or "homology" or "consensus sequence identity" or "percent sequence identity (%)" with respect to nucleic acid or polypeptide sequences is defined as The percent identity of a nucleotide or amino acid residue in a candidate sequence to a known polynucleotide or polypeptide after obtaining the maximum percent identity and introducing gaps, if necessary, to achieve the maximum percent identity. N-terminal or C-terminal insertions or deletions should not be interpreted as affecting homology, and have fewer than about 30, fewer than about 20, or fewer than about 10, or fewer than 5 amino acid residues in nucleotides Or internal deletions and / or insertions in the polypeptide sequence should not be interpreted as affecting homology. Can be analyzed by BLAST (Base Local Alignment Search Tool), using the blastp, blastn, blastx, tblastn, and tblastx programs adapted for sequence similarity searches (Altschul (1997), Nucleic Acids Res. 25, 3389-3402 , and the algorithm used by Karlin (1990), Proc. Natl. Acad. Sci. USA 87, 2264-2268) to determine homology or identity at the nucleotide or amino acid sequence position. The method used by the BLAST program is to first consider similar segments with and without gaps between the query sequence and the database sequences, then evaluate the statistical significance of all matches identified, and finally summarize only those satisfying a preselected significance Matching of thresholds. For a discussion of fundamental issues in similarity searching of sequence databases, see Altschul (1994), Nature Genetics 6, 119-129. Search parameters for histograms, descriptions, alignments, expectations (i.e., statistical significance thresholds for reporting matches to database sequences), cutoffs, matrices, and filters (low complexity) can be at default settings down. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix recommended for query sequences longer than 85 (nucleotide bases or amino acids) (Henikoff (1992), Proc. Natl. Acad . Sci. USA 89, 10915-10919).

[0151] For blastn designed to compare nucleotide sequences, the scoring matrix is ​​set by the ratio of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), The default values ​​of M and N can be +5 and -4 respectively. The four blastn parameters can be tuned as follows: Q = 10 (gap creation penalty); R = 10 (gap expansion penalty); wink = 1 (generate word hits at every winkth position along the query); and gapw = 16 (sets the width of the window within which the gapped alignment is generated). An equivalent Blastp parameter set for comparing amino acid sequences would be: Q=9; R=2; wink=1; and gapw=32. BESTFIT® comparisons between sequences available in GCG Suite version 10.0 can use the DNA parameters GAP = 50 (gap creation penalty) and LEN = 3 (gap extension penalty), and the equivalent settings in protein comparisons can be GAP = 8 and LEN = 2.

[0152] When disclosing nucleic acid or polypeptide sequences herein, such as HBV TALEN monomers, TALE DNA binding domains, nuclease catalytic domains, target sequences, spacer sequences, regulatory elements, untranslated regions, enhancer sequences, promoters Sequences are also disclosed as sequences considered based on or derived from the original sequence. The disclosed sequences thus include at least 40%, at least 45%, at least 45%, respectively, of the full-length polynucleotide or polypeptide sequence and fragments thereof of any of the polynucleotide or polypeptide sequences described herein, such as SEQ ID NO: 1-55. At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% sequence identity, for example at least 86%, at least 87%, at least 88%, At least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, or 85- A polynucleotide or polypeptide sequence with 99%, or 85-95%, or 90-99%, or 95-99%, or 97-99%, or 98-99% sequence identity. Fragments or portions of any of the sequences disclosed herein are also disclosed. A fragment or portion of a sequence may comprise at least 5, or at least 7, or at least 10, or at least 20, or at least 30, at least 50, at least 75, at least 100, at least 125, 150 or more, or 5-10, or 10-12, or 10-15, or 15-20, or 20-40, or 20-50, or 30-50, or 30- 75, or 30-100 amino acids or nucleic acid residues of the complete sequence, or at least 100, or at least 200, or at least 300, or at least 400, or at least 500, or at least 600, or at least 700, or at least 800, or at least 900, or at least 1000, or 100-200, or 100-500, or 100-1000, or 500-1000 amino acid or nucleic acid residues, or any of these amounts, but less than 500, or less than 700, or less than 1000, or less than 2000 of any of the SEQ ID NOs disclosed herein: 1-55 or The sequence of contiguous amino acids or nucleic acids of any segment. Variants of these sequences and nucleic acid sequences encoding such variants are also disclosed, for example in which at least one or two or three or four or five amino acid residues have been inserted at the N-terminus and / or C-terminus. Where and / or within the disclosed sequence(s) containing insertions and substitutions. Contemplated variants may additionally or alternatively include variants containing predetermined mutations induced by, for example, homologous recombination or site-directed or PCR mutagenesis, as well as corresponding polypeptides or nucleic acids from other species, including but not limited to: alleles or other naturally occurring variants of polypeptides or nucleic acids, polypeptide or nucleic acid families containing insertions and substitutions; Derivatives that have been covalently modified with moieties other than naturally occurring amino acids (eg, detectable moieties such as enzymes). The nucleic acid sequences described herein may be mRNA sequences.

[0153] As used herein, the term "non-naturally occurring", "recombinant" or "engineered" nucleic acid molecule or polynucleotide sequence refers to a nucleic acid molecule or non-naturally occurring polynucleotide sequence that has been altered by human intervention. Nucleotide sequence. As non-limiting examples, recombinant nucleic acid molecules: 1) have been synthesized using chemical or enzymatic techniques, such as by using chemical nucleic acid synthesis or by using methods for replication, polymerization, exonuclease digestion, endonuclease Enzyme digestion, conjugation, reverse transcription, transcription, base modification (including, for example, methylation) or recombination (including homologous and site-specific recombination) enzymes) synthesis or modification in vitro; 2) including The combined binding nucleotide sequence; 3) has been engineered using molecular breeding techniques so that it does not have one or more nucleotides relative to a naturally occurring nucleic acid molecule sequence; and / or 4) has been using molecular breeding Technology is manipulated to have one or more sequence changes or rearrangements relative to naturally occurring nucleic acid sequences. As a non-limiting example, a cDNA is a recombinant DNA molecule, such as any nucleic acid molecule that has been generated by in vitro polymerase reaction(s) or ligated to a linker or integrated into a vector such as a cloning vector or an expression vector . Lipid-based formulations

[0154] Therapies based on the intracellular delivery of nucleic acids to target cells address both extracellular and intracellular disorders. Indeed, naked nucleic acid substances cannot be easily administered systemically due to their toxicity, low stability in serum, rapid renal clearance, reduced target cell uptake, phagocyte uptake, and their ability to activate immune responses, all of which All hinder its clinical development. When exogenous nucleic acid material, such as mRNA, enters a human biological system, it is recognized by the reticuloendothelial system (RES) as a foreign pathogen and cleared from the blood circulation before it may encounter target cells within or outside the vascular system. The half-life of naked nucleic acid in the bloodstream has been reported to be on the order of minutes (Kawabata K, Takakura Y, Hashida M, Pharm Res. 1995 Jun; 12(6):825-30). Chemical modification and appropriate delivery methods can reduce RES uptake and protect nucleic acids from ubiquitous nuclease degradation, thereby increasing the stability and efficacy of nucleic acid-based therapies. In addition, RNA or DNA are anionic hydrophilic polymers that are unfavorable for uptake by cells, which are also anionic at the surface. Thus, the success of nucleic acid-based therapies depends largely on the development of vehicles or carriers that can efficiently and effectively deliver genetic material to target cells with minimal toxicity for adequate in vivo expression.

[0155] Furthermore, upon internalization into target cells, nucleic acid delivery vectors are subject to challenges including endosomal retention, lysosomal degradation, nucleic acid decapsulation from the vector, translocation across the nuclear membrane (for DNA), and cytoplasmic Release (for RNA) of intracellular barrier attack. Thus, successful nucleic acid-based therapy depends on the ability of the vector to deliver the nucleic acid to a target site inside the cell to achieve a sufficient level of desired activity, such as gene expression.

[0156] Although several gene therapies have been able to successfully utilize viral delivery vectors such as AAV, lipid-based formulations have become increasingly recognized as systemic agents due to their biocompatibility and their ease of large-scale production. One of the most promising delivery systems for RNA and other nucleic acid compounds. When Patisiran (ALN-TTR02) was the first siRNA therapeutic approved by both the Food and Drug Administration (FDA) and the European Commission (EC), lipid-based One of the most notable advances in qualitative nucleic acid therapy came in August 2018. ALN-TTR02 is a siRNA formulation based on the so-called Stable Nucleic Acid Lipid Particle (SNALP) transfection technology. Despite the success of patisran, the delivery of nucleic acid therapeutics, including mRNA, via lipid formulations is still under development.

[0157] According to various embodiments, some art-recognized lipid-formulated delivery vehicles for nucleic acid therapeutics include polymer-based carriers such as polyethylenediimine (PEI), containing Lipidoid formulations, lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, multivesicular liposomes, proteoliposomes, natural extracellular bodies and Both synthetically derived extracellular bodies, natural lamellae, synthetic lamellae and semi-synthetic lamellae, nanoparticles, micelles, and emulsions.

[0158] The structure and composition of these lipid formulations vary, and as might be expected in a rapidly evolving field, several different terms have been used in the art to describe a single type of delivery vehicle. At the same time, the terms used for lipid formulations are often conflated throughout the scientific literature, and this inconsistent use creates confusion with the precise meaning of several terms used for lipid formulations. Among several potential lipid formulations, for the purposes of the present invention, liposomes, cationic liposomes and lipid nanoparticles are specifically described and defined herein in particular. lipid-mRNA formulation

[0159] The combinations of mRNAs, pharmaceutically acceptable salts thereof, and / or nucleic acid encoding HBV TALENs as disclosed herein can be incorporated into lipid formulations (ie, lipid-based delivery vehicles).

[0160] In the context of the present invention, lipid-based delivery vehicles are typically used to transport a desired mRNA molecule or combination of mRNA molecules to a target cell or tissue. The lipid-based delivery vehicle can be any suitable lipid-based delivery vehicle known in the art. In some embodiments, the lipid-based delivery vehicle is a liposome, cationic liposome, or lipid nanoparticle containing an mRNA molecule or combination of mRNA molecules of the invention. In some embodiments, a lipid-based delivery vehicle comprises a nanoparticle or a bilayer of lipid molecules and an mRNA molecule or combination of mRNA molecules of the invention. In some embodiments, the lipid bilayer preferably further comprises neutral lipids or polymers. The term "neutral lipid" means a lipid species that exists in uncharged or neutral zwitterionic form at a chosen pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol. In some embodiments, lipid formulations preferably comprise a liquid medium. In some embodiments, the formulation preferably further encapsulates the nucleic acid. In some embodiments, the lipid formulation preferably further comprises a nucleic acid and a neutral lipid or polymer. In some embodiments, lipid formulations preferably encapsulate nucleic acids.

[0161] The specification provides lipid formulations comprising one or more therapeutic mRNA molecules encapsulated within the lipid formulation. In some embodiments, the lipid formulation comprises liposomes. In some embodiments, the lipid formulation comprises cationic liposomes. In some embodiments, lipid formulations comprise lipid nanoparticles.

[0162] In some embodiments, the mRNA molecule or combination of mRNA molecules is fully encapsulated within the lipid portion of the lipid formulation such that the mRNA molecule or combination of mRNA molecules in the lipid formulation is resistant to nuclease degradation in aqueous solution . The term "fully encapsulated" means that the nucleic acid (eg, mRNA) in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or nuclease assays that significantly degrade free RNA. When fully encapsulated, preferably less than 25% of the nucleic acids in the particles are degraded upon treatment which typically degrades 100% of the free nucleic acids in the particles, more preferably less than 10% and most preferably less than 5% nucleic acid degradation. "Completely encapsulated" as used herein also means that the nucleic acid-lipid particle does not rapidly break down into its constituent parts after in vivo administration. In other embodiments, the lipid formulations described herein are substantially nontoxic to mammals, such as humans. In some embodiments, combinations of mRNA molecules are encapsulated within the same lipid nanoparticle. In some embodiments, each mRNA molecule in the combination of mRNA molecules is independently encapsulated in individual lipid nanoparticles.

[0163] The lipid formulations of the present invention also typically have from about 1:1 to about 100:1, from about 1:1 to about 50:1, from about 2:1 to about 45:1, from about 3:1 to about 40 :1, about 5:1 to about 38:1, or about 6:1 to about 40:1, or about 7:1 to about 35:1, or about 8:1 to about 30:1, or about 10:1 1 to about 25:1, or about 8:1 to about 12:1, or about 13:1 to about 17:1, or about 18:1 to about 24:1, or about 20:1 to about 30:1 Total lipid:RNA ratio (mass / mass ratio). In some preferred embodiments, the total lipid:RNA ratio (mass / mass ratio) is from about 10:1 to about 25:1. The ratio can be any value or subvalue in the stated range, including endpoints.

[0164] The lipid formulations of the present invention generally have a pH of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or About 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm average diameter and is virtually non-toxic. The diameter can be any value or subvalue within the recited range, including endpoints. In addition, when the nucleic acid is present in the lipid nanoparticles of the present invention, the nucleic acid is resistant to nuclease degradation in aqueous solution.

[0165] In preferred embodiments, the lipid formulation comprises an mRNA molecule or combination of mRNA molecules, a cationic lipid (eg, one or more of the cationic lipids described herein or a salt thereof), a phospholipid, and a particle aggregation-inhibiting Lipids are conjugated (eg, one or more PEG-lipid conjugates). Lipid formulations can also include cholesterol. The term "lipid conjugate" means a bound lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates such as: PEG coupled to dialkoxypropyl (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerol (e.g., PEG -DAG conjugates), PEG conjugated to cholesterol, PEG conjugated to phosphatidylethanolamine and PEG conjugated to ceramide, cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligo polymers and their mixtures. PEG or POZ can be bound directly to the lipid or can be attached to the lipid via a linker moiety. Any linker moiety suitable for coupling PEG or POZ to a lipid can be used including, for example, non-ester-containing linker moieties and ester-containing linker moieties. In certain preferred embodiments, non-ester-containing linker moieties such as amides or carbamates are used.

[0166] The term "cationic lipid" as used herein refers to amphiphilic lipids and salts thereof having a positively hydrophilic head group; one, two, three or more hydrophobic (i.e., having a non-polar group) a fatty acid or fatty alkyl chain; and a linker between these two domains. Ionizable or protonatable cationic lipids are typically protonated (ie, positively charged) at pH below their pKa and substantially neutral at pH above their pKa. Preferred ionizable cationic lipids are ionizable cationic lipids with a pKa less than physiological pH, which is typically about 7.4. The cationic lipids of the present invention may also be referred to as titratable cationic lipids. Cationic lipids may be "amine-based lipids" having protonatable tertiary amine (eg, pH titratable) headgroups. Some amine groups Exemplary amino lipids can include C18 alkyl chains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and a head group and an alkyl chain ether, ester or ketal linkages between them. Such cationic lipids include, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA, γ-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin - K-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), DLin-M-C3 - DMA (also known as MC3) and (DLin-MP-DMA) (also known as 1-Bl 1).

[0167] The term "anionic lipid" as used herein refers to a lipid that is negatively charged at physiological pH. Such lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecylphosphatidylethanolamine, N-butacylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleylphosphatidylglycerol (POPG) and other anionic modifying groups conjugated to neutral lipids.

[0168] In nucleic acid-lipid formulations, the mRNA molecule or combination of mRNA molecules can be fully encapsulated within the lipid portion of the formulation, thereby protecting the nucleic acid from nuclease degradation. In preferred embodiments, lipid formulations comprising mRNA molecules or combinations of mRNA molecules are fully encapsulated within the lipid portion of the lipid formulation, thereby protecting nucleic acids from nuclease degradation. In certain instances, the mRNA molecule or combination of mRNA molecules in the lipid formulation is not substantially degraded after exposing the particle to a nuclease for at least 20, 30, 45, or 60 minutes at 37°C. In certain other instances, the mRNA molecule or combination of mRNA molecules in the lipid formulation is incubated in serum at 37°C for at least 30, 45, or 60 minutes or at least 2, 3, 4, 5, 6, 7, Not substantially degraded after 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 hours. In other embodiments, the mRNA molecule or combination of mRNA molecules is complexed with the lipid portion of the formulation.

[0169] In the case of nucleic acids, complete encapsulation can be determined by performing a membrane-impermeable fluorescent dye exclusion assay using dyes that have enhanced fluorescence when associated with nucleic acids. Encapsulation was determined by adding the dye to the lipid formulation, measuring the resulting fluorescence, and comparing the resulting fluorescence to that observed after addition of a small amount of non-ionic detergent. Detergent-mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation can be calculated as E=(I0-I) / I0, where I and I0 refer to the fluorescence intensity before and after addition of detergent.

[0170] In other embodiments, the present invention provides a nucleic acid-lipid composition comprising a plurality of nucleic acid-liposomes, nucleic acid-cationic liposomes or nucleic acid-lipid nanoparticles. In some embodiments, the nucleic acid-lipid composition comprises a plurality of mRNA-liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of mRNA-cationic liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of mRNA-lipid nanoparticles.

[0171] In some embodiments, the lipid formulation comprises an mRNA molecule or combination of mRNA molecules fully encapsulated within the lipid portion of the formulation such that about 30% to about 100%, about 40% to about 100% , about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 30% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, or at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or any fraction thereof or Particles in the range of ) have mRNA molecules or combinations of mRNA molecules encapsulated therein. The amount can be any value or subvalue within the recited range, including endpoints.

[0172] Depending on the intended use of the lipid formulation, the proportions of the components may vary, and the delivery efficiency of a particular formulation can be measured using assays known in the art.

[0173] According to some embodiments, the polynucleotide and mRNA architectures described herein can be expressed lipid-formulated. The lipid formulation is preferably selected from, but not limited to, liposomes, cationic liposomes and lipid nanoparticles. In a preferred embodiment, the lipid formulation is a cationic liposome or lipid nanoparticle (LNP) comprising: (a) the mRNA molecule or combination of mRNA molecules of the present invention, (b) a cationic lipid, (c) aggregation reducing agents (such as polyethylene glycol (PEG) lipids or PEG-modified lipids), (d) optionally non-cationic lipids (such as neutral lipids), and (e) optionally The sterol of choice.

[0174] Preferably, the lipid nanoparticles encapsulating mRNA molecules or combinations of mRNA molecules comprise cationic lipids and at least one selected from the group consisting of anionic lipids, zwitterionic lipids, neutral lipids, steroids, polymer binding Other lipids of the group consisting of type lipids, phospholipids, glycolipids and combinations thereof.

[0175] In some embodiments, the cationic lipid is an ionizable cationic lipid. In one embodiment, the lipid nanoparticle formulation consists of: (i) at least one cationic lipid; (ii) a helper lipid; (iii) a sterol (such as cholesterol); and (iv) PEG- Lipids in a molar ratio of about 20% to about 40% ionizable cationic lipid: about 25% to about 45% helper lipid: about 25% to about 45% sterol; about 0.5-5% PEG- Lipid. Exemplary cationic lipids (including ionizable cationic lipids), helper lipids (eg, neutral lipids), sterols, and ligand-containing lipids (eg, PEG-lipids) are described herein below.

[0176] The choice of specific lipids and their relative composition % will depend on several factors including the desired therapeutic effect, the intended in vivo delivery target, and the planned dosing regimen and frequency. In general, lipids that correspond to both high potency (ie, therapeutic effect, such as gene knockdown activity or translational efficiency) and biodegradability that result in rapid tissue clearance are optimal. However, biodegradability may be less important for formulations intended for only one or two administrations in a subject. In addition, the lipid composition may require careful engineering so that the lipid formulation maintains its morphology and its course towards the intended target during in vivo administration, but will then be able to release the active agent upon uptake into the target cells. Therefore, it is often necessary to evaluate several formulations in order to find the best possible lipid combination in the best possible lipid molar ratio as well as in the total lipid:active ingredient ratio.

[0177] Suitable lipid components and methods of making lipid nanoparticles are well known in the art and described, for example, in PCT / US2020 / 023442, U.S. 8,058,069, U.S. 8,822,668, U.S. 9,738,593, U.S. / 066242, PCT / US2015 / 030218, PCT / 2017 / 015886, and PCT / US2017 / 067756, the contents of which are incorporated by reference. cationic lipid

[0178] The lipid formulation preferably includes cationic lipids suitable for the formation of cationic liposomes or lipid nanoparticles. Cationic lipids have been extensively studied for nucleic acid delivery because of their ability to bind to negatively charged membranes and induce uptake. In general, cationic lipids are amphiphiles containing a positively hydrophilic headgroup, two (or more) lipophilic tails or steroid moieties, and a linker between these two domains. Preferably, cationic lipids carry a net positive charge at about physiological pH. Cationic liposomes have traditionally been the most commonly used non-viral delivery system for oligonucleotides including plastid DNA, antisense oligonucleotides and siRNA / small hairpin RNA-shRNA. Such as DOTAP (1,2-dioleyl-3-trimethylammonium-propane) and DOTMA (N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl -ammonium methylsulfate) cationic lipids can form complexes or lipoplexes with negatively charged nucleic acids through electrostatic interactions, thereby providing high in vitro transfection efficiency.

[0179] In the lipid formulation disclosed in the present invention, the cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl - N,N-dimethylammonium bromide (DDAB), 1,2-dioleyltrimethylpropaneammonium chloride (DOTAP) (also known as N-(2,3-dioleyloxy )propyl)-N,N,N-trimethylammonium chloride and l,2-dioleoxy-3-trimethylaminopropane chloride), N-(l-(2,3-dioleoxy (yl)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), l,2-diethylene Oleoxy-N,N-Dimethylaminopropane (DLinDMA), 1,2-Di-Linenoxy-N,N-Dimethylaminopropane (DLenDMA), 1,2-Di-y-Linen Oxy-N,N-dimethylaminopropane (γ-DLenDMA), 1,2-Dilinoleylamineformyloxy-3-dimethylaminopropane (DLin-C-DAP), l,2 -Dilinoleyloxy-3-(dimethylamino)acetyloxypropane (DLin-DAC), l,2-dilinoleyloxy-3-(N-alphalinyl)propane (DLin-MA ), l,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l -Linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA .Cl ), l,2-Dilinoleyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), l,2-Dilinoleyloxy-3-(N-methylpiperyl ) propane (DLin-MPZ) or 3-(N,N-dioleylamino)-l,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-l,2- Propylene Glycol (DOAP), l,2-Dilinoleyloxy-3-(2-N,N-Dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl -4-Dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA) or its analogs, (3aR,5s,6aS)-N,N-dimethyl -2 ,2-bis((9Z,12Z)-octadec-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5- Amine, (6Z,9Z,28Z,31Z)-Heptadecta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), l,l'- (2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperone-l-yl) Ethylazanediyl)docos-2-ol (C12-200), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxo Heterocyclopentane (DLin-K-C2-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA ), (6Z,9Z,28Z,31Z)-Heptadec-6,9,28 31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-Thirtyheptac-6,9,28,3 l-tetraen-19-yloxy)-N,N-dimethylpropan-l-amine (MC3 ether), 4-((6Z,9Z,28Z,31 Z)-heptaka-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-l- Amines (MC4 ethers) or any combination thereof. Other cationic lipids include, but are not limited to, N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P-(N-(N',N'-dimethylaminoethane) -carbamoyl)cholesterol (DC-Choi), N-(1-(2,3-dioleyloxy)propyl)-N-2-(spermidylcarbamoyl)ethyl)-N, N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylaminoglycylcarboxyspermine (DOGS), 1,2-dioleyl-sn-3-phosphoethanolamine ( DOPE ), 1,2-dioleyl-3-dimethylammoniumpropane (DODAP), N-(1,2-dimyristyloxypropan-3-yl)-N,N-dimethyl - N-Hydroxyethylammonium bromide (DMRIE) and 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC). Additionally, commercial cationic lipid formulations such as LIPOFECTIN (including DOTMA and DOPE available from GIBCO / BRL) and Lipofectamine (including DOSPA and DOPE available from GIBCO / BRL) can be used .

[0180] Other suitable cationic lipids are disclosed in International Publication Nos. WO 09 / 086558, WO 09 / 127060, WO 10 / 048536, WO 10 / 054406, WO 10 / 088537, WO 10 / 129709 and WO 2011 / 153493; U.S. Patent Publication Nos. 2011 / 0256175, 2012 / 0128760 and 2012 / 0027803; U.S. Patent No. 8,158,601; and Love et al., PNAS, 107( 5 ), 1864-69, 2010, these cases and the content of this document are incorporated herein by reference.

[0181] Other suitable cationic lipids include cationic lipids with alternative fatty acid groups and other dialkylamino groups, including those in which the alkyl substituents differ (e.g., N-ethyl-N-methylamino- and N -Propyl-N-ethylamino-) cationic lipid. These lipids are part of a subclass of cationic lipids known as amino lipids. In some embodiments of the lipid formulations described herein, the cationic lipid is an amino lipid. In general, amino lipids with less saturated acyl chains are easier to size, especially when complexes must be sized less than about 0.3 microns for filter sterilization purposes. Amino lipids containing unsaturated fatty acids with carbon chain lengths ranging from C14 to C22 can be used. Other scaffolds can also be used to separate the amine group and the fatty acid or fatty alkyl moiety of the aminolipid.

[0182] In some embodiments, the lipid formulation comprises a cationic lipid of Formula I according to patent application PCT / EP2017 / 064066. In this context, the disclosure of PCT / EP2017 / 064066 is also incorporated herein by reference.

[0183] In some embodiments, the amine-based or cationic lipids of the invention are ionizable and have at least one protonatable or deprotonatable group such that the lipid is at or below physiological pH ( It is positively charged at a pH such as pH 7.4) and neutral at a second pH which is preferably at or above physiological pH. Of course, it will be understood that the addition or removal of protons as a function of pH is an equilibrium process and that reference to charged or neutral lipids refers to the nature of the dominant species and does not require all lipids to be present in charged or neutral form . Lipids having more than one protonatable or deprotonatable group or being zwitterionic are not excluded from use in the present invention. In certain embodiments, the pKa of the protonatable group of the protonatable lipid is in the range of about 4 to about 11. In some embodiments, the pKa of the ionizable cationic lipid is from about 5 to about 7. In some embodiments, the pKa of the ionizable cationic lipid is from about 6 to about 7.

[0184] In some embodiments, the lipid formulation comprises an ionizable cationic lipid of formula I: or a pharmaceutically acceptable salt or solvate thereof, wherein R5 and R6 are each independently selected from linear or branched A group consisting of chain C1-C31 alkyl, C2-C31 alkenyl or C2-C31 alkynyl and cholesteryl; L5 and L6 are each independently selected from the group consisting of straight chain C1-C20 alkyl and C2-C20 alkenyl; X5 is -C(O)O-, thereby forming -C(O)O-R6, or X5 is -OC(O)-, thereby forming -OC(O)-R6; X6 is -C(O) O-, thus forming -C(O)O-R5, or X6 is -OC(O)-, thereby forming -OC(O)-R5; X7 is S or O; L7 does not exist or is a low carbon number Alkyl; R4 is straight chain or branched C1-C6 alkyl; and R7 and R8 are each independently selected from the group consisting of hydrogen and straight chain or branched C1-C6 alkyl.

[0185] In some embodiments, X7 is S.

[0186] In some embodiments, X5 is -C(O)O-, thereby forming -C(O)O-R6, and X6 is -C(O)O-, thereby forming -C(O) O-R5.

[0187] In some embodiments, R7 and R8 are each independently selected from the group consisting of methyl, ethyl and isopropyl.

[0188] In some embodiments, L5 and L6 are each independently C1-C10 alkyl. In some embodiments, L5 is C1-C3 alkyl, and L6 is C1-C5 alkyl. In some embodiments, L6 is C1-C2 alkyl. In some embodiments, L5 and L6 are each linear C7 alkyl. In some embodiments, L5 and L6 are each linear C9 alkyl.

[0189] In some embodiments, R5 and R6 are each independently alkenyl. In some embodiments, R6 is alkenyl. In some embodiments, R6 is C2-C9 alkenyl. In some embodiments, an alkenyl group contains a single double bond. In some embodiments, R5 and R6 are each alkyl. In some embodiments, R5 is branched chain alkyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of C9 alkyl, C9 alkenyl and C9 alkynyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of C11 alkyl, C11 alkenyl and C11 alkynyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of C7 alkyl, C7 alkenyl and C7 alkynyl. In some embodiments, R5 is -CH((CH2)pCH3)2 or -CH((CH2)pCH3)((CH2)p-1CH3), wherein p is 4-8. In some embodiments, p is 5 and L5 is C1-C3 alkyl. In some embodiments, p is 6 and L5 is C3 alkyl. In some embodiments, p is 7. In some embodiments, p is 8 and L5 is C1-C3 alkyl. In some embodiments, R5 consists of -CH((CH2)pCH3)((CH2)p-1CH3), where p is 7 or 8.

[0190] In some embodiments, R4 is ethylidene or propylidene. In some embodiments, R4 is n-propyl or iso-butyl.

[0191] In some embodiments, L7 is absent, R4 is ethylidene, X7 is S and R7 and R8 are each methyl. In some embodiments, L7 is absent, R4 is n-propyl, X7 is S and R7 and R8 are each methyl. In some embodiments, L7 is absent, R4 is ethylidene, X7 is S and R7 and R8 are each ethyl.

[0192] In some embodiments, X7 is S, X5 is -C(O)O-, thereby forming -C(O)O-R6, and X6 is -C(O)O-, thereby forming -C (O)O-R5, L5 and L6 are each independently a linear C3-C7 alkyl group, L7 is absent, R5 is -CH((CH2)pCH3)2, and R6 is a C7-C12 alkenyl group. In some further embodiments, p is 6 and R6 is C9 alkenyl.

[0193] In some embodiments, the lipid formulation comprises an ionizable cationic lipid selected from the group consisting of.

[0194] In some embodiments, any one or more of the lipids described herein can be specifically excluded. Helper lipids and sterols

[0195] The mRNA-lipid formulations of the present invention may comprise helper lipids, which may be referred to as neutral lipids, neutral helper lipids, non-cationic lipids, non-cationic helper lipids, anionic lipids , an anionic helper lipid or a zwitterionic lipid. Lipid formulations, especially cationic liposomes and lipid nanoparticles, have been found to have increased cellular uptake if helper lipids are present in the formulation. (Curr. Drug Metab. 2014; 15(9):882-92). For example, some studies indicate that cationic lipids such as 1,2-dioleyl-sn-glycero-3-phosphatidylcholine (DOPC), di-oleoylcholine, are more fusogenic (i.e., facilitate fusion) than cationic lipids. Acyl-phosphatidyl-ethanolamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) neutral and zwitterionic lipids may affect lipid- The pleomorphic nature of nucleic acid complexes facilitates the transition from lamellar to hexagonal phases and thus induces fusion and membrane rupture. (Nanomedicine (Lond). 2014 Jan;9(1):105-20). In addition, the use of helper lipids can help reduce any potential deleterious effects of using many common cationic lipids, such as toxicity and immunogenicity.

[0196] Non-limiting examples of non-cationic lipids suitable for use in the lipid formulations of the invention include phospholipids such as: lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, Phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine ( DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG) , dioleyl phosphatidylethanolamine (DOPE), palmityl oleyl-phosphatidylcholine (POPC), palmityl oleyl-phosphatidylethanolamine (POPE), palmityl oleyl-phosphatidylglycerol (POPG ), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine ( DPPE), dimyristyl-phosphatidylethanolamine (DMPE), distearyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, di-eirayl - Phosphatidylethanolamine (DEPE), stearyloleyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleylphosphatidylcholine and mixtures thereof. Other diacylphosphatidylcholines and diacylphosphatidylethanolamine phospholipids may also be used. The acyl group in these lipids is preferably an acyl group derived from a fatty acid having a C10-C24 carbon chain such as lauryl, myristyl, palmityl, stearyl or oleyl.

[0197] Additional examples of non-cationic lipids include sterols such as cholesterol and its derivatives. One study concluded that cholesterol as a helper lipid increases the charge spacing of the lipid layer interfacing with nucleic acids such that the charge distribution more closely matches that of nucleic acids. (J. R. Soc. Interface. 2012 March 7; 9(68): 548-561). Non-limiting examples of cholesterol derivatives include polar analogs such as: 5α-cholestanol, 5α-calosterol, cholesteryl-(2′-hydroxy)-ether, cholesteryl-(4′-hydroxy) -Butyl ether and 6-ketocholestanol; nonpolar analogues such as: 5α-cholestane, cholestenone, 5α-cholestanone, 5α-cholestanone, and cholesteryl decanoate ; and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.

[0198] In some embodiments, the helper lipid present in the lipid formulation comprises or consists of a mixture of one or more phospholipids and cholesterol or derivatives thereof. In other embodiments, the helper lipid present in the lipid formulation comprises or consists of one or more phospholipids, eg, cholesterol-free lipid formulations. In yet other embodiments, the helper lipid present in the lipid formulation comprises or consists of cholesterol or a derivative thereof, such as a phospholipid-free lipid formulation.

[0199] Other examples of helper lipids include non-phospholipids such as stearylamine, laurylamine, cetylamine, acetyl palmitate, glyceryl ricinoleate, cetyl stearate, carnitine Isopropyl Myristate, Amphoteric Acrylic Polymer, Triethanolamine-Lauryl Sulfate, Alkyl-Aryl Sulfate Polyethoxylated Fatty Acid Amide, Dioctadecyldimethylammonium Bromide, Ceramide Amines and sphingomyelin.

[0200] In some embodiments, the helper lipid comprises about 20 mol% to about 50 mol%, about 22 mol% to about 48 mol%, about 24 mol% to about 24 mol% of the total lipids present in the lipid formulation 46 mol%, about 25 mol% to about 44 mol%, about 26 mol% to about 42 mol%, about 27 mol% to about 41 mol%, about 28 mol% to about 40 mol%, or about 29 mol%, About 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol% or about 39 mol% ( or any fraction thereof or range therein).

[0201] In some embodiments, the total helper lipids in the formulation comprise two or more helper lipids, and the total amount of helper lipids is about 20 mol% of the total lipids present in the lipid formulation to about 50 mol%, about 22 mol% to about 48 mol%, about 24 mol% to about 46 mol%, about 25 mol% to about 44 mol%, about 26 mol% to about 42 mol%, about 27 mol% to about 41 mol%, about 28 mol% to about 40 mol%, or about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol %, about 36 mol%, about 37 mol%, about 38 mol% or about 39 mol% (or any fraction or range thereof). In some embodiments, the helper lipid is a combination of DSPC and DOTAP. In some embodiments, the helper lipid is a combination of DSPC and DOTMA.

[0202] The cholesterol or cholesterol derivatives in the lipid formulation can account for up to about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the total lipids present in the lipid formulation. mol%. In some embodiments, the cholesterol or cholesterol derivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 30 mol% to about 40 mol% of the total lipids present in the lipid formulation. mol%, or about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, or about 40 mol%.

[0203] The percentage of helper lipid present in the lipid formulation is a target amount, and the actual amount of helper lipid present in the formulation may vary, eg, ± 5 mol%.

[0204] A lipid formulation containing a cationic lipid compound or an ionizable cationic lipid compound can be about 20-40% cationic lipid compound, about 25-40% cholesterol, about 25-50% accessory Lipid and about 0.5-5% polyethylene glycol (PEG) lipid, where the percentage is the percentage of total lipid present in the formulation. In some embodiments, the composition is about 22-30% cationic lipid compound, about 30-40% cholesterol, about 30-40% helper lipid, and about 0.5-3% PEG-lipid, wherein the percentages are formulated Percentage of total lipids present in the substance. lipid conjugate

[0205] The lipid formulations described herein may further comprise lipid conjugates. Conjugated lipids are suitable to prevent particle aggregation. Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, cationic polymer-lipid conjugates, and mixtures thereof. In addition, lipid delivery vehicles can be used for specific targeting by attaching ligands such as antibodies, peptides, and carbohydrates to their surface or to the termini of attached PEG chains (Front. Pharmacol. Dec. 2015 1st; 6:286).

[0206] In a preferred embodiment, the lipid conjugate is PEG-lipid. Inclusion of polyethylene glycol (PEG) in lipid formulations as a coating or surface coordination system A technique called pegylation that helps protect nanoparticles from the immune system and helps them escape RES absorption (Nanomedicine (Lond). 2011 Jun;6(4):715-28). Pegylation has been widely used to stabilize lipid formulations and their payloads through physical, chemical and biological mechanisms. Detergent-like PEG lipids such as PEG-DSPE can enter lipid formulations to form hydration layers and steric barriers on surfaces. Based on the degree of PEGylation, surface layers can generally be divided into two types: brush-like layers and mushroom-like layers. For formulations stabilized by PEG-DSPE, PEG will assume a mushroom conformation with a low degree of PEGylation (typically less than 5 mol%) and will convert to a brush conformation as the content of PEG-DSPE increases beyond a certain threshold ( J. Nanomaterials. 2011;2011:12). It has been shown that increased PEGylation causes a significant increase in the circulating half-life of lipid formulations (Annu. Rev. Biomed. Eng. 2011 Aug 15; 13:507-30; J. Control Release. 2010 Aug 3;145(3):178-81).

[0207] Suitable examples of PEG-lipids include, but are not limited to, PEG coupled to dialkoxypropyl (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), and lipids such as phosphatidylethanolamine Phospholipid-conjugated PEG (PEG-PE), ceramide-conjugated PEG, cholesterol-conjugated PEG or derivatives thereof, and mixtures thereof.

[0208] PEG is a linear water-soluble polymer having repeating units of ethylene PEG with two terminal hydroxyl groups. PEGs are classified according to their molecular weight and include the following: Monomethoxypolyethylene glycol (MePEG-OH), Monomethoxypolyethylene glycol-succinate (MePEG-S), Monomethoxypolyethylene glycol Diol-butanediimide succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH2), monomethoxypolyethylene glycol-trifluoroethanesulfonic acid Esters (MePEG-TRES), monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), and compounds containing terminal hydroxyl groups instead of terminal methoxy groups (such as HO-PEG-S, HO-PEG- S-NHS, HO-PEG-NH2).

[0209] The PEG moiety of the PEG-lipid conjugates described herein can comprise an average molecular weight in the range of about 550 Daltons to about 10,000 Daltons. In some cases, the PEG moiety has a range of about 750 Daltons to about 5,000 Daltons (e.g., about 1,000 Daltons to about 5,000 Daltons, about 1,500 Daltons to about 3,000 Daltons, about 750 Daltons Daltons to about 3,000 Daltons, about 750 Daltons to about 2,000 Daltons). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 Daltons or about 750 Daltons. The average molecular weight can be any value or subvalue within the recited range, inclusive of endpoints.

[0210] In some cases, the PEG monomers are optionally substituted with alkyl, alkoxy, acyl, or aryl groups. PEG can be directly bound to the lipid or can be attached to the lipid via a linker moiety. Any linker moiety suitable for coupling PEG to a lipid can be used including, for example, non-ester-containing linker moieties and ester-containing linker moieties. In a preferred embodiment, the linker moiety is a non-ester-containing linker moiety. Suitable non-ester-containing linker moieties include, but are not limited to, amido (-C(O)NH-), amine (-NR-), carbonyl (-C(O)-), carbamate (- NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-) , succimidyl (-NHC(O)CH2CH2C(O)NH-), ethers, and combinations thereof (such as linkers containing both a carbamate linker moiety and an amide-based linker moiety). In a preferred embodiment, a carbamate linker is used to couple the PEG to the lipid.

[0211] In other embodiments, the PEG is coupled to the lipid using an ester-containing linker moiety. Suitable ester-containing linker moieties include, for example, carbonate (-OC(O)O-), succinyl, phosphate (-O-(O)POH-O-), sulfonate, and combinations thereof.

[0212] Phosphatidylethanolamines with various acyl chain groups of different chain lengths and degrees of saturation can be conjugated with PEG to form lipid conjugates. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. Phosphatidylethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths ranging from C10 to C20 are preferred. Phosphatidylethanolamines with monounsaturated or diunsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyrisyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearyl -phosphatidylethanolamine (DSPE).

[0213] In some embodiments, the PEG-DAA conjugate is PEG-didecyloxypropyl (C10) conjugate, PEG-dilauryloxypropyl (C12) conjugate, PEG-dimyristyloxy Propyl (C14) conjugate, PEG-dipalmityloxypropyl (C16) conjugate or PEG-distearoyloxypropyl (C18) conjugate. In such embodiments, the PEG preferably has an average molecular weight of about 750 to about 2,000 Daltons. In certain embodiments, the terminal hydroxyl groups of PEG are substituted with methyl groups.

[0214] In addition to the foregoing, other hydrophilic polymers may also be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl, methacrylamide, polymethacrylamide Amines and polydimethylacrylamides, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0215] In some embodiments, the lipid conjugate (eg, PEG-lipid) comprises about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol% of the total lipids present in the lipid formulation %, about 1 mol% to about 2 mol%, about 0.6 mol% to about 1.9 mol%, about 0.7 mol% to about 1.8 mol%, about 0.8 mol% to about 1.7 mol%, about 0.9 mol% to about 1.6 mol% %, about 0.9 mol% to about 1.8 mol%, about 1 mol% to about 1.8 mol%, about 1 mol% to about 1.7 mol%, about 1.2 mol% to about 1.8 mol%, about 1.2 mol% to about 1.7 mol %, about 1.3 mol% to about 1.6 mol%, or about 1.4 mol% to about 1.6 mol% (or any fraction or range thereof). In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprises about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2% of the total lipid present in the lipid formulation , 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5% or 5% (or any fraction thereof or range). The amount can be any value or subvalue within the recited range, including endpoints.

[0216] In some preferred embodiments, the PEG-lipid is PEG550-PE. In some preferred embodiments, the PEG-lipid is PEG750-PE. In some preferred embodiments, the PEG-lipid is PEG2000-DMG.

[0217] The percentage of lipid conjugate (eg, PEG-lipid) present in the lipid formulations of the invention is a target amount, and the actual amount of lipid conjugate present in the formulation may vary, eg, ± 0.5 mol%. Those of ordinary skill in the art will appreciate that the concentration of the lipid conjugate may vary depending on the lipid conjugate employed and the rate at which the lipid formulation becomes fusogenic. Mechanism of Action for Cellular Uptake of Lipid Formulations

[0218] Lipid formulations for intracellular delivery of nucleic acids, especially liposomes, cationic liposomes, and lipid nanoparticles, are designed to carry out cellular Absorption, wherein the contents of the lipid delivery vehicle are delivered to the cytosol of the target cell. (Nucleic Acid Therapeutics, 28(3):146-157, 2018). In particular, in the case of hepatocyte-targeted mRNA-lipid formulations described herein, the mRNA-lipid formulation enters hepatocytes via receptor-mediated endocytosis. Functionalized ligands such as PEG-lipids at the surface of the lipid delivery vehicle are shed from the surface prior to endocytosis, triggering internalization into the target cell. During endocytosis, some portion of the cell's plasma membrane surrounds the carrier and engulfs it into a vesicle, which is then pinched from the cell membrane, enters the cytosol, and ultimately undergoes the endolysosomal pathway. For ionizable cationic lipid-containing delivery vehicles, the increased acidity of endosomes with aging produces vehicles with strong positive charges on the surface. Interaction between the delivery vehicle and the endosomal membrane then leads to a membrane fusion event, resulting in cytosolic delivery of the payload. For mRNA payloads, the cell's own internal translation process then translates the mRNA molecule or combination of mRNA molecules into the encoded protein (eg HBV TALEN). The encoded protein may further undergo post-translational processing including transport to targeted organelles or locations within the cell. In the case of the HBV TALENs described herein, the HBV TALEN proteins translocate into the nucleus where the HBV gene body can be edited.

[0219] By controlling the composition and concentration of the lipid conjugate, we can control the rate at which the lipid conjugate is exchanged from the lipid formulation and in turn control the rate at which the lipid formulation becomes fusogenic. Additionally, other variables including, for example, pH, temperature or ionic strength can be used to alter and / or control the rate at which the lipid formulation becomes fusogenic. Other methods that can be used to control the rate at which lipid formulations become fusogenic will become apparent to those of skill in the art upon reading this disclosure. Furthermore, by controlling the composition and concentration of lipid conjugates, one can control liposome or lipid particle size. Lipid formulation manufacturing

[0220] There are many different methods of preparing lipid formulations comprising nucleic acids, eg, mRNA molecules or combinations of mRNA molecules. (Curr. Drug Metabol. 2014, 15, 882-892; Chem. Phys. Lipids 2014, 177, 8-18; Int. J. Pharm. Stud. Res. 2012, 3, 14-20). A brief description of thin film hydration, double emulsification, reverse phase evaporation, microfluidic preparation, double asymmetric centrifugation, ethanol injection, detergent dialysis, spontaneous vesicle formation by ethanol dilution and encapsulation in preformed liposomes is briefly described. technology. film hydration

[0221] In thin film hydration (TFH) or the Bangham method, lipids are dissolved in an organic solvent followed by evaporation via the use of a rotary evaporator, causing a thin lipid layer to form. After layer hydration by aqueous buffer containing the compound to be loaded, multilamellar vesicles (MLVs) are formed which can be reduced in size by extrusion through the membrane or by sonication of the starting MLV to produce small or large vesicles. Unilamellar vesicles (LUV and SUV). double emulsification

[0222] Lipid formulations can also be prepared via double emulsification techniques, which involve dissolving lipids in water / organic solvent mixtures. Mixing an organic solution containing water droplets with an excess of aqueous medium causes the formation of a water-in-oil-in-water (W / O / W) double emulsion. After vigorous mechanical shaking, one of the water droplets partially ruptures, resulting in large unilamellar vesicles (LUVs). reversed phase evaporation

[0223] The reverse evaporation (REV) method also allowed us to achieve LUVs loaded with nucleic acids. In this technique, biphasic systems are formed by dissolving phospholipids in organic solvents and aqueous buffers. Subsequently, the resulting suspension was sonicated briefly until the mixture became a single-phase clear dispersion. Lipid formulations were achieved after evaporation of the organic solvents under reduced pressure. This technology has been used to encapsulate different hydrophilic macromolecules and small molecules including nucleic acids. Microfluidic preparation

[0224] Microfluidic methods differ from other bulk technologies in that they give the possibility to control lipid hydration methods. The method can be classified into continuous flow microfluidics and droplet-based microfluidics according to the way the flow is manipulated. In the microfluidic hydrodynamic focusing (MHF) method operated in continuous flow mode, lipids are dissolved in isopropanol, which hydrodynamically focuses at the microchannel cross-junction between two aqueous buffer streams. in the face. Vesicle size can be controlled by adjusting the flow rate, thereby controlling the lipid solution / buffer dilution process. This method can be used to generate oligonucleotide (ON) lipid formulations by using a microfluidic device consisting of three inlets and one outlet port. double asymmetric centrifugal

[0225] Double asymmetric centrifugation (DAC) differs from the more commonly used centrifugation in that it uses additional rotation about its own vertical axis. Effective homogenization is achieved due to the creation of two overlapping movements: pushing the sample outwards as in a normal centrifuge, and then pushing the sample towards the center of the vial due to the extra spin. Lipid formulation dispersions were obtained by mixing lipids with NaCl-solution to achieve a viscous vesicular phospholipid gel (VPC), which was subsequently diluted. Lipid formulation size can be adjusted by optimizing DAC speed, lipid concentration and homogenization time. ethanol injection

[0226] The ethanol injection (EI) method can be used to encapsulate nucleic acids. This method involves rapid injection, using a needle, of an ethanol solution in which lipids are dissolved into an aqueous medium containing the nucleic acid to be encapsulated. Vesicles form spontaneously when phospholipids are dispersed throughout the medium. Detergent Dialysis

[0227] Detergent dialysis methods can be used to encapsulate nucleic acids. Briefly, lipids and plastids are dissolved in a detergent solution of appropriate ionic strength to form a stable lipid formulation after removal of the detergent by dialysis. Subsequently, unencapsulated nucleic acids were removed by ion exchange chromatography and empty vesicles were removed by sucrose density gradient centrifugation. The technique is highly sensitive to cationic lipid content and the salt concentration of the dialysis buffer, and the method is also difficult to scale. Spontaneous vesicle formation by ethanol dilution

[0228] Stable lipid formulations can also be generated by the method of spontaneous vesicle formation by ethanol dilution by controlled addition of lipids dissolved in ethanol to rapid nucleic acid-containing Mix in aqueous buffer to transiently form nucleic acid-loaded vesicles. Encapsulated in preformed liposomes

[0229] Nucleic acid retention can also be obtained by two different methods starting from pre-formed liposomes: (1) simply mixing cationic liposomes with nucleic acids to obtain electrostatic complexes called "liposomes". complexes, wherein these electrostatic complexes can be successfully used to transfect cell cultures but are characterized by low encapsulation efficiency and poor performance in vivo; and (2) destabilization of liposomes towards cationic vesicles Loaded vesicles were achieved by slow addition of pure ethanol to a suspension of vesicles to a concentration of 40% v / v, followed by dropwise addition of nucleic acid; however, the two main steps characterizing the encapsulation method were also sensitive, and Particle size must be reduced.

[0230] In certain embodiments, lipids and lipid nanoparticles, pharmaceutical compositions comprising lipids, methods of making lipids or formulating pharmaceutical compositions comprising lipids and nucleic acid molecules, and using pharmaceutical compositions to treat or methods of preventing disease are described in US or international patent application publications such as: US2017 / 0190661, US2006 / 0008910, US2015 / 0064242, US2005 / 0064595, WO / 2019 / 036030, US2019 / 0022247, WO / 2019 / 036028, WO / 2019 / 036008, WO / 2019 / 036000, US2016 / 0376224, US2017 / 0119904, WO / 2018 / 200943, WO / 2018 / 191657, WO / 2018 / 118102, US20180169268, WO2 018118102, WO2018119163, US2014 / 0255472 and US2013 / 0195968, the relevant contents of each of these cases are hereby incorporated by reference in their entirety. Cells and Peptides

[0231] The application also provides cells, preferably isolated cells, comprising any of the polynucleotides and vectors described herein. Such cells can be used, for example, for the production of recombinant proteins or for the production of virions. In some embodiments, the cells can be used to produce HBV TALENs. The term "isolated" as used herein refers to a cell or a descendant of such a cell that has been removed from the organism in which the cell was originally found. In some embodiments, a cell comprising a polynucleotide or vector described herein may be in or part of an individual rather than isolated. In these embodiments, individual cells can be used to produce recombinant proteins or produce virions in vivo. In some embodiments, cells are cultured ex vivo, eg, in the presence of other cells. In some embodiments, the cell is then introduced into or reintroduced into the second organism from which the cell was isolated (or the cell from which the cell was propagated).

[0232] Host cells comprising the HBV TALENs of the present application or nucleic acids encoding HBV TALENs also form part of the present invention. HBV TALENs can be produced via recombinant DNA techniques involving expression of molecules in host cells or transgenic animals or plants, such as Chinese Hamster Ovary (CHO) cells, tumor cell lines, BHK cells, such as HEK293 cells, human cell lines of PER.C6 cells or yeast, bacteria, fungi, insect cells and similar cells. In certain embodiments, the cell lines are from multicellular organisms, and in certain embodiments, the cells are of vertebrate or invertebrate origin. In certain embodiments, the cells are mammalian cells such as human cells or insect cells. In certain embodiments, the cells are primary cells, such as hepatocytes, more specifically infected hepatocytes, HBV-infected hepatocytes or hepatocytes with HBV cccDNA. In general, production of recombinant proteins such as the HBV TALENs of the present invention in a host cell comprises introducing a heterologous nucleic acid molecule encoding the protein in an expressible format into the host cell, culturing the cell under conditions favorable for expression of the nucleic acid molecule, and The protein is allowed to express in the cell. A nucleic acid molecule encoding a protein in an expressible format may be in the form of an expression cassette, and generally requires sequences such as enhancer(s), promoter(s), polyadenylation signals, and the like that enable expression of the nucleic acid. Those skilled in the art understand that a variety of promoters can be used to achieve gene expression in host cells. Promoters can be constitutive or regulated, and can be obtained from a variety of sources including viral, prokaryotic or eukaryotic sources, or artificially designed. Additional regulatory sequences may be added. Many promoters are available for expression of the transgene(s) and are known to the skilled artisan, for example such promoters may include viral, mammalian, synthetic promoters and the like. A non-limiting example of a suitable promoter for obtaining expression in eukaryotic cells is the CMV promoter (US 5,385,839), such as the CMV early onset promoter, e.g. comprising nt from the CMV early onset gene enhancer / promoter . CMV early-onset promoter from -735 to +95. A polyadenylation signal such as the bovine growth hormone polyadenosine signal (US 5,122,458) may be present behind the transgene(s). Alternatively, several widely used expression vectors are available in the art and from commercial sources, such as Invitrogen's pcDNA and pEF vector series, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc. , which can be used to recombinantly express a protein of interest or to obtain suitable promoter and / or transcription terminator sequences, polyadenylation sequences and the like.

[0233] The cell culture can be any type of cell culture, including adherent cell cultures, such as cells attached to the surface of a culture vessel or to microcarriers, and suspension cultures. Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the easiest to operate and they can be scaled up. Today, continuous methods based on perfusion principles are becoming more common and suitable. Suitable media are also well known to the skilled artisan and are generally available in large quantities from commercial sources or customized according to standard protocols. Cultivation can be performed, for example, in vessels, roller bottles, or bioreactors using batch, fed-batch, continuous systems, and the like. Conditions suitable for culturing cells are known (see for example Tissue Culture, Academic Press, Kruse and Paterson eds. (1973), and R.I. Freshney, Culture of animal cells: A manual of basic technique, Fourth Edition (Wiley-Liss Co. , 2000, ISBN 0-471-34889-9)). Cell culture media are available from a variety of suppliers, and suitable media can be routinely selected for host cell expression of the protein of interest, here HBV TALEN. Suitable media may or may not contain serum.

[0234] Accordingly, the embodiments of the present application also relate to methods of making the HBV TALENs of the present application. The method comprises transfecting a host cell with an expression vector comprising a polynucleotide encoding the HBV TALEN of the present application operably linked to a promoter, allowing the transfected cell to grow under conditions suitable for expressing the HBV TALEN , and optionally purify or isolate the HBV TALEN expressed in the cells. HBV TALENs can be isolated or collected from cells by any method known in the art, including affinity chromatography, size exclusion chromatography, and the like. Techniques for expressing recombinant proteins will be well known to those of ordinary skill in the art in light of the present invention. Expressed proteins can also be studied without purification or isolation of expressed proteins, e.g., by analyzing supernatants of cells transfected with expression vectors encoding HBV TALENs and grown under conditions suitable for expressing HBV TALENs. HBV TALEN.

[0235] Accordingly, non-naturally occurring or recombinant polypeptides comprising an amino acid sequence at least 90% identical to the amino acid sequence of one or more of SEQ ID NO: 25, 26, 27 or 28 are also provided. In some embodiments, the non-naturally occurring or recombinant polypeptide having a TALE DNA binding domain comprises the amino acid sequence of one or more of SEQ ID NO: 25, 26, 27 or 28. In some embodiments, the combination of non-naturally occurring polypeptides or recombinant polypeptides having a TALE DNA binding domain comprises the amino acid sequences of SEQ ID NO: 25 and SEQ ID NO: 26. In some embodiments, the combination of non-naturally occurring polypeptides or recombinant polypeptides having a TALE DNA binding domain comprises the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO: 28. As described above and below, the present application also encompasses isolated nucleic acid molecules encoding these sequences, vectors comprising these sequences operably linked to a promoter, and compositions comprising polypeptides, polynucleotides or vectors.

[0236] In one embodiment of the present application, the recombinant polypeptide comprises at least 90% identity with the amino acid sequence of one or more of SEQ ID NO: 25, 26, 27 or 28, such as with SEQ ID NO : One or more of 25, 26, 27 or 28 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical amino acid sequences. In some embodiments, the combination of recombinant polypeptides comprises at least 90% identity to the amino acid sequences of SEQ ID NO: 25 and 26, respectively, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6% , 99.7%, 99.8%, 99.9% or 100% identical amino acid sequences. In some embodiments, the combination of recombinant polypeptides comprises at least 90% identity to the amino acid sequences of SEQ ID NO: 27 and 28, respectively, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6% , 99.7%, 99.8%, 99.9% or 100% identical amino acid sequences. Preferably, the non-naturally occurring or recombinant polypeptide consists of one or more of SEQ ID NO: 25, 26, 27 or 28. Preferably, the non-naturally occurring polypeptide or combination of recombinant polypeptides consists of SEQ ID NO: 25 and 26 or SEQ ID NO: 27 and 28. combination

[0237] The present application is also concerned with polynucleotides (including RNA and DNA, preferably mRNA), vectors, LNPs and and / or compositions of host cells, more particularly pharmaceutical compositions. Any of the HBV TALENs, combinations, nucleic acids, vectors, LNPs and / or host cells of the present application described herein may be used in the compositions or pharmaceutical compositions of the present application.

[0238] The present application provides, for example, pharmaceutical compositions comprising any of the nucleic acid molecules, vectors, combinations, LNPs or host cells described herein and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are non-toxic and should not interfere with the efficacy of the active ingredients. Pharmaceutically acceptable carriers may include one or more excipients such as: binders, disintegrating agents, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavoring agents, sweetening agents Agents, preservatives, dyes, solubilizers and coating agents. The exact nature of the carrier or other material may depend on the route of administration eg intramuscular, intradermal, subcutaneous, oral, intravenous, transdermal, intramucosal (eg enteral), intranasal or intraperitoneal routes. For liquid injectable preparations such as suspensions and solutions, suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like. For solid oral preparations such as powders, capsules, sachets, caplets and lozenges, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrants and the like . For nasal spray / inhalant mixtures, aqueous solutions / suspensions may contain water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavorings and the like as suitable carriers and additives.

[0239] The pharmaceutical compositions of the present application may be formulated for ease of administration and enhanced efficacy in any material suitable for administration to a subject, including but not limited to oral (enteral) administration and parenteral injection. Parenteral injection includes intravenous injection or infusion, subcutaneous injection, intradermal injection and intramuscular injection. The pharmaceutical compositions of the present application can also be formulated for administration including transmucosal, ophthalmic, rectal, long-acting implants, sublingual administration, under the tongue, from the oral mucosa bypassing the portal circulation, inhalation, or intranasal. Other ways of investing.

[0240] In one preferred embodiment of the present application, the pharmaceutical composition of the present application is formulated for parenteral injection, preferably subcutaneous injection, intradermal injection or intramuscular injection, more preferably intramuscular injection .

[0241] According to an embodiment of the present application, the pharmaceutical composition for administration will generally comprise a buffered solution, such as buffered saline and its analogs, such as phosphoric acid, contained in a pharmaceutically acceptable carrier, such as an aqueous carrier. Salt buffered saline (PBS). The compositions may also contain pharmaceutically acceptable substances for approximating physiological conditions such as pH adjusting agents and buffering agents as desired. For example, a pharmaceutical composition of the present application comprising plastid DNA may contain phosphate buffered saline (PBS) as a pharmaceutically acceptable carrier. Plastid DNA can be according to for example 0.5 mg / mL to 5 mg / mL, such as 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL or 5 mg / mL, preferably present at a concentration of 1 mg / mL.

[0242] In some embodiments, the pharmaceutical composition of the present application comprising lipid nanoparticles can be, for example, from about 20 µg / mL to about 200 µg / mL, such as 20 µg / mL, 30 µg / mL, 40 µg / mL, 50 µg / mL, 60 µg / mL, 70 µg / mL, 80 µg / mL, 90 µg / mL, 100 µg / mL, 110 µg / mL, 120 µg / mL, 130 µg / mL, 140 The concentration of µg / mL, 150 µg / mL, 160 µg / mL, 170 µg / mL, 180 µg / mL, 190 µg / mL or 200 µg / mL is administered. In some embodiments, the pharmaceutical compositions of the present application comprising lipid nanoparticles can be administered at a concentration of less than 20 μg / mL. In some embodiments, the pharmaceutical compositions of the present application comprising lipid nanoparticles can be administered at concentrations greater than 200 μg / mL.

[0243] The present application also provides methods of making the pharmaceutical compositions of the present application. The method of manufacturing the pharmaceutical composition comprises mixing the isolated polynucleotide encoding the HBV TALEN, the carrier and / or the LNP of the present application with one or more pharmaceutically acceptable carriers. Those of ordinary skill in the art will be familiar with conventional techniques for preparing such compositions. treatment method

[0244] This application provides methods of treating hepatitis. In some embodiments, the method is treating hepatitis B virus (HBV), particularly HBV infection, in an individual in need thereof, the method comprising administering to the individual an effective amount of a pharmaceutical composition of the present application. Some embodiments relate to methods of treating HBV / HDV co-infection. Some embodiments relate to methods of reducing HBV surface antigen. Some embodiments relate to methods of reducing or eradicating HBV cccDNA. Some embodiments relate to methods for targeted integration of HBV DNA. Any of the pharmaceutical compositions of the present application described herein can be used in the methods of the present application. The present application also provides methods of reducing HBV infection in an individual and / or reducing HBV replication in an individual, the methods comprising administering to the individual a pharmaceutical composition of the present application. The term "reducing / reduced" as used herein with respect to viral infection or replication generally means a statistically significantly reduced infection compared to a reference level such as an untreated individual or an individual administered a control treatment and / or a statistically significant reduction in the number of replicates. However, for the avoidance of doubt, "decrease / weaken" means at least 10% decrease / weaken compared to a reference level, such as at least about 20%, or at least about 30%, or at least about 40%, compared to a reference level, Or at least about 50%, or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 90% reduction / weakening, or up to and including 100% reduction / weakening (also That is, a non-existent level compared to a reference level), or any reduction / weakening between 10% and 100%. The level of infection and / or replication can be determined by one of ordinary skill in the art in light of the present invention.

[0245] The term "treat / treated / treating" as used herein refers to both therapeutic treatment and prophylactic or preventive measures, wherein the aim is to prevent (partially or completely) or slow down (for example to alleviate or delay onset) an undesired physiological condition, disorder or disease, or the achievement of a beneficial or desirable clinical outcome such as partial or complete restoration or inhibition of attenuation of a parameter, value, function, or result that has become or will become abnormal . For purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, relief of symptoms; reduction in the extent or activity or rate of progression of a condition, disorder or disease; stabilization (ie, not worsening) of a condition, disorder or disease state; Delaying the onset or slowing the progression of a condition, disorder, or disease; ameliorating a condition, disorder, or disease state; and alleviating a condition, disorder, or disease, whether partial or complete, whether or not this translates into immediate relief of actual clinical symptoms, or enhancement or improvement Condition, disease or disease. Treatment strives to elicit a clinically significant response without excessive side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0246] The term "infection" as used herein refers to the invasion of a host by a pathogen. A pathogen is considered "infectious" when it is capable of invading a host and replicating or multiplying within the host. Examples of infectious agents include viruses such as HBV, HDV, and certain species of adenoviruses, prions, bacteria, fungi, protozoa, and the like. "HBV infection" refers specifically to HBV invasion of a host organism, such as cells and tissues of the host organism. In some embodiments, the infection is a co-infection of HBV and HDV.

[0247] "Therapeutically effective amount" or "effective amount" as used herein means an amount of a composition, polynucleotide or vector sufficient to induce a desired effect or response in a subject in need thereof. An effective amount may be an amount sufficient to provide a therapeutic effect against a disease such as HBV infection. An effective amount may vary depending on factors such as the physical condition, age, weight, health, etc. of the individual; the particular application, such as therapeutic treatment; and the particular disease requiring immunity, such as viral infection. An effective amount can be readily determined by one of ordinary skill in the art in light of the present invention. A therapeutically effective amount can be determined experimentally, for example by analyzing the blood concentration of the compound, or theoretically by calculating the bioavailability in view of the present invention by one of ordinary skill in the art.

[0248] In certain embodiments of the present application, an effective amount refers to an amount of a composition or combination sufficient to achieve one, two, three, four or more of the following effects: (i) reducing Or improve the severity of HBV infection or its related symptoms; (ii) reduce the duration of HBV infection or its related symptoms; (iii) prevent the deterioration of HBV infection or its related symptoms; (iv) make HBV infection or its related symptoms subside; (v) prevent the development or onset of HBV infection or its associated symptoms; (vi) prevent the recurrence of HBV infection or its associated symptoms; (vii) reduce the hospitalization of individuals with HBV infection; (ix) prolong the survival of individuals with HBV infection; (x) eliminate HBV infection in individuals; (xi) inhibit or reduce HBV replication in individuals; and / or (xii) enhance or improve another therapy The (multiple) preventive or therapeutic effects.

[0249] The therapeutically effective amount may also be that amount of the compound that is sufficient to reduce HBsAg levels consistent with the evolution of clinical seroconversion; and / or achieve sustained HBsAg clearance associated with the reduction of infected hepatocytes by the individual's immune system; induce HBV- Antigen-specific activation of T cell populations; and / or achieving a decrease in detectable HBsAg during or after treatment, which then persists preferably 6 months or more after the end of treatment, optimally lifetime. Examples of target indices include serum HBsAg levels below a threshold such as 100 IU / mL HBsAg and / or HBV-specific CD8 T cell responses in greater number or multipotency than, eg, at the start of treatment. Additional examples of target indices include, but are not limited to, serum HBV RNA levels below the lower limit of quantitation (LLoQ); and / or 3-fold lower than the upper limit of normal or, in the case of a male individual, less than 129 U / L or A serum ALT concentration of less than 108 U / L in the case of a female individual, more particularly less than 120 U / L in the case of a male individual or less than 105 U / L in the case of a female individual A serum ALT concentration, more particularly a serum ALT concentration of less than 90 U / L if the subject is a male subject or less than 57 U / L if the subject is a female subject; and / or HBeAg-negative serum; and / or or 100 IU / mL or less, more particularly considered a normalized serum HBsAg level of 10 IU / mL or less; and / or HB seroconversion; and / or core-associated antigen (crAg) below LLoQ. In some preferred embodiments, the effective amount is an amount sufficient to reduce the expression of one or more of HBsAg, HBeAg, HBV DNA, HBV cccDNA or integrated HBV DNA in an individual. In some embodiments, the expressive quantity is hepatocyte content, nuclear or cellular content, liver content, serum content, or plasma content. In some preferred embodiments, the effective amount is an amount sufficient to reduce serum and / or plasma levels of one or more of HBsAg, HBeAg, and HBV DNA in an individual.

[0250] As a general guide, when used with respect to nucleic acid molecules or carriers, an effective amount may be in the range of about 0.1 mg / kg nucleic acid molecule or carrier to about 5 mg / kg nucleic acid molecule or carrier, such as about 0.1 mg / kg, about 0.25 mg / kg, about 0.5 mg / kg, about 0.75 mg / kg, about 1.0 mg / kg, about 1.5 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, about 3.0 mg / kg, about 3.5 mg / kg, about 4.0 mg / kg, about 4.5 mg / kg or about 5.0 mg / kg nucleic acid molecule or vector. Preferably, the effective amount of the nucleic acid molecule or vector is about 0.25 mg / kg to about 3 mg / kg. When used with respect to a nucleic acid molecule or carrier in a pharmaceutical composition, the effective amount may be within a concentration range of about 0.01 mg / mL to about 2 mg / mL nucleic acid molecule or carrier in total, such as 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, 1.5 mg / mL or 2 mg / mL nucleic acid molecule or carrier. Preferably, the effective amount of the molecule or carrier is less than 1 mg / mL, more preferably less than 0.05 mg / mL. An effective amount can be from one nucleic acid molecule or vector or from multiple nucleic acid molecules or vectors. An effective amount may be in a single composition or in multiple compositions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 compositions (e.g. lozenges, capsules or injectables or suitable for skin Intradermal delivery, such as any composition suitable for intradermal delivery using an intradermal delivery patch), wherein administration of multiple capsules or injections together provides an effective amount to the individual. For example, when two DNA plasmids are used, the effective amount may be 3-4 mg / mL, with 1.5-2 mg / mL of each plasmid. The effective amount can be administered in a single therapeutically effective dose or in a series of therapeutically effective doses, such as 1, 2, 3, 4, 5 or more doses, or wherein the series of doses together provide a therapeutically effective amount to the individual . When multiple doses are administered, each dose may be the same amount and / or concentration or different amounts and / or concentrations. Optimization of dosing strategies will be readily understood and practiced by one of ordinary skill in the art. Combinations comprising two nucleic acid molecules or vectors, for example a first vector encoding a first HBV TALEN and a second vector encoding a second HBV TALEN can be achieved by mixing the two vectors or nucleic acid molecules and delivering the mixture to a single anatomy site to administer to individuals. Alternatively, two separate administrations can be performed, each delivering a single vector or nucleic acid molecule. In these embodiments, whether the two vectors or nucleic acid molecules are administered as a mixture in a single administration or in two separate administrations, the first vector or nucleic acid molecule and the second vector or nucleic acid molecule can be mixed 10:1 to 1:10 weight ratio such as 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2 , 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 by weight ratio. Preferably, the first vector and the second vector or the first nucleic acid molecule and the second nucleic acid molecule are administered in a weight ratio of 1:1.

[0252] Preferably, the subject treated according to the method of the present application is a subject infected with hepatitis, preferably a subject infected with HBV, especially a subject suffering from chronic HBV infection. In some embodiments, the individual is co-infected with HBV and HDV. Acute HBV infection is characterized by efficient activation of the innate immune system coupled with subsequent broad adaptive responses (eg, HBV-specific T cells, neutralizing antibodies), which often result in successful inhibition of replication or removal of infected hepatocytes . In contrast, these responses are attenuated or diminished by high viral and antigenic loads, such as HBV outer membrane protein, which is produced in large quantities and can be released in subvirions in 1,000-fold excess relative to infectious virus.

[0253] Chronic HBV infection is described in stages characterized by viral load, liver enzyme levels (necrotizing inflammatory activity), HBeAg or HBsAg load or the presence of antibodies against these antigens. cccDNA content remains relatively constant, roughly 10 to 50 copies per cell, even though viremia may vary significantly. Persistence of cccDNA species leads to chronicity. More specifically, the stages of chronic HBV infection include: (i) a stage of immune tolerance, characterized by a high viral load and normal or minimally increased liver enzymes; (ii) an immune-activated HBeAg-positive stage, in which lower or decreased levels of viral replication and significantly increased liver enzymes; (iii) the inactive HBsAg carrier phase, which is a low replicative state with traceable HBeAg seroconversion and low viral load and liver enzyme levels in normal serum; and (iv) HBeAg-negative stage, in which viral replication (reactivation) occurs periodically with fluctuations in liver enzyme levels, mutations in the pre-core and / or basal-core promoters are common, rendering infected cells unable to produce HBeAg. Preferably, the effective amount refers to the amount of the composition or combination of the present application that is sufficient to treat chronic HBV infection.

[0254] In some embodiments, the individual with chronic HBV infection is undergoing nucleoside analog (NUC) therapy and is inhibited by NUC. As used herein, "inhibited by NUC" means that an individual has undetectable levels of HBV virus and stable levels of alanine aminotransferase (ALT) for at least six months. Examples of nucleoside / nucleotide analog therapy include HBV polymerase inhibitors such as entecavir and tenofovir. Preferably, individuals with chronic HBV infection do not develop advanced liver fibrosis or cirrhosis. The individual typically has a METAVIR score for fibrosis of less than 3 and a fibroscan result of less than 9 kPa. The METAVIR score is a scoring system commonly used to assess the degree of inflammation and fibrosis by histopathological evaluation of liver biospecimens from hepatitis B patients. The scoring system assigns two standardized values: one reflecting the degree of inflammation and one reflecting the degree of fibrosis.

[0255] It is believed that elimination or attenuation of chronic HBV may allow early disease interception of severe liver disease including virus-induced cirrhosis and hepatocellular carcinoma. Therefore, the method of the present application can also be used as a therapy for the treatment of HBV-induced diseases. Examples of HBV-induced diseases include, but are not limited to, cirrhosis, cancer (eg, hepatocellular carcinoma), and fibrosis, particularly advanced fibrosis characterized by a METAVIR score for fibrosis of 3 or higher. In these embodiments, the effective amount is an amount sufficient to achieve sustained reduction of HBsAg and significant relief of clinical disease (eg, cirrhosis, hepatocellular carcinoma, etc.) within 12 months.

[0256] The phrase "inducing an immune response" when used in connection with the methods described herein encompasses providing therapeutic immunity for the treatment of pathogens such as HBV. In one embodiment, "inducing an immune response" means generating immunity in an individual in need, for example to provide a therapeutic effect against a disease such as HBV infection or HBV and HDV co-infection. In certain embodiments, "inducing an immune response" refers to eliciting or improving cellular immunity, eg, HBV-specific CD4+ and CD8+ T cell responses. In certain embodiments, this T cell response can result in a functional cure of a treated patient with CHB. In some embodiments, "inducing an immune response" refers to causing or improving a humoral immune response against HBV infection or HBV and HDV co-infection. In certain embodiments, "inducing an immune response" refers to eliciting or improving cellular and humoral immune responses against HBV infection or HBV and HDV co-infection.

[0257] The term "functional cure" or "FC" as used herein refers to the state of an individual with CHB in which the individual's serum remains free of detectable HBV DNA and HBsAg. For example, 6 months after cessation of HBV therapy, sera of FC individuals had undetectable HBV DNA and were HBsAg negative. HBsAg reduction in FC individuals may or may not be accompanied by HBsAg seroconversion. In some embodiments, FC lasts for at least 1 year, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 years. Optimally, FC lasts a lifetime.

[0258] The term "restoration of T cell function" as used herein refers to the reactivation of exhausted and otherwise non-functional HBV-specific T cells so that they can carry out their normal effector functions. Examples of restored HBV-specific T cell responses include, but are not limited to: killing or non-cytolytic control of HBV-infected hepatocytes, killing of hepatocytes with integrated HBV DNA expressing HBsAg, and once FC is achieved, subsequent Infected cells that reactivate over time perform surveillance and kill. The net effect is to keep the serum free of viral products such as HBV DNA, HBsAg and other HBV proteins.

[0259] In some embodiments, the disclosed mRNA molecules can be used in combination with one or more additional agents for the treatment and / or inhibition of replicating hepatitis infections such as HBV and / or HDV. Additional agents can be one or more immunomodulators. Additional agents include, but are not limited to, interferons, nucleoside / nucleotide analogs, capsid assembly modulators, sequence-specific oligonucleotides (such as antisense oligonucleotides and / or siRNA), entry inhibitors, and / or or therapeutic HBV vaccine. In some embodiments, the disclosed mRNA molecules are administered in combination with cytokines. In some embodiments, the disclosed mRNA molecules are administered in combination with thymosin alpha-1. In some embodiments, the disclosed mRNA molecules are administered in combination with standard-of-care treatments for hepatitis infection. Standard-of-care treatment for HBV infection may include viral polymerase inhibitors such as: nucleotides / nucleotide analogues (e.g., Lamivudine, Telbivudine, Entecavir, Adefo Adefovir, Tenofovir and Clevudine, Tenofovir alafenamide (TAF), CMX157 and AGX-1009) and interferons (such as Peg-IFN-2a and IFN-a-2b, interferon lambda, recombinant interferon alpha 2b, IFN-a). In some embodiments, the disclosed mRNA molecules are administered in combination with an interferon selected from the group consisting of: interferon alpha-2a; interferon alpha-2b; interferon alpha-N3; interferon beta-1a; interferon Interferon β-1b; Interferon γ-1b; Interferon λ-1; Interferon λ-2; Interferon λ-3; Pegylated Interferon α-2a; Pegylated Interferon α-2b; Diolated interferon lambda-1; and pegylated interferon lambda-2. In some embodiments, the disclosed mRNA molecules are administered in combination with one or more TLR7 agonists. In some embodiments, the disclosed mRNA molecules are administered in combination with one or more TLR8 agonists. In some embodiments, the disclosed mRNA molecules are combined with GS-962O (4-amino-2-butoxy-8-3-(pyrrolidin-1-ylmethyl)phenyl)methyl-5,7 - dihydropterin-6-one) or GS-9688 (Selgantolimod) in combination administration. In some embodiments, the disclosed mRNA molecules are administered in combination with Bulevirtide.

[0260] In some embodiments, a disclosed mRNA molecule is administered in combination with one or more oligonucleotides either simultaneously (co-administration) or sequentially. Oligonucleotides may include siRNA, ASO, Gapmers and / or Nucleic Acid Polymers (NAP). In some embodiments, the disclosed mRNA molecules are administered in combination with one or more antiviral agents, such as inhibitors of viral replication. In some embodiments, the disclosed mRNA molecules are administered in combination with an HBV capsid assembly modulator (CAM). HBV CAM molecules may include JNJ 6379, NVR 3-778, AB-423, GLS-4, Bayer 41-4109, HAP-1 and AT-1. In some embodiments, the disclosed oligonucleotide constructs are administered in combination with one or more immunomodulators, such as TLR agonists. TLR agonists may include GS-9620, GS-9688, ARB-1598, ANA975, RG7795 (ANA773), MEDI9197, PF-3512676, and IMO-2055. In some embodiments, the disclosed mRNA molecules are administered in combination with HBV vaccines. HBV vaccines may include Heplislav, ABX203 and INO-1800.

[0261] In one aspect, the siRNA component comprises one or more siRNA agents. Each siRNA agent disclosed herein includes at least one sense and one antisense strand. Sense shares and antisense shares may be partially, substantially or fully complementary to each other. The length of the sense and antisense strands of the siRNA agents described herein can each be 16 to 30 nucleotides in length. In some embodiments, the sense and antisense strands are independently 17-26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 19-26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21 to 24 nucleotides in length. Sense shares and antisense shares may be of the same length or of different lengths. The HBV siRNA agents disclosed herein have been designed to include antisense strand sequences that are at least partially complementary to sequences in the HBV gene body that are conserved in most known serotypes of HBV. Following delivery to cells expressing HBV, the siRNA agents described herein inhibit the expression of one or more HBV genes in vivo or in vitro. In some embodiments, the siRNA molecule can be JNJ 3989, VIR-2218, or ARB-1467.

[0262] siRNA agents include a sense strand comprising a first sequence (also referred to as a follower strand) and an antisense strand comprising a second sequence (also referred to as a leader strand). The sense strands of the HBV siRNA agents described herein include a core stretch that is at least about 85% identical to a nucleotide sequence of at least 16 contiguous nucleotides in an HBV mRNA. In some embodiments, the sense strand core nucleotide stretch having at least about 85% identity to the sequence in the HBV mRNA is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length . The antisense strand of the HBV siRNA agent comprises a nucleotide sequence that is at least about 85% complementary to the sequence in the HBV mRNA and the corresponding sense strand over a core stretch of at least 16 contiguous nucleotides. In some embodiments, there are 16, 17, 18, 19, 20, 21, 22, or 23 antisense strand core nucleotide sequences that are at least about 85% complementary to a sequence in the HBV mRNA or to the corresponding sense strand nucleotide length.

[0263] The method according to the embodiments of the present application further comprises administering another anti-HBV agent (such as a nucleoside analog or other anti-HBV agent) and the pharmaceutical composition of the present application to the individual in need. For example, another anti-HBV agent can be a small molecule or antibody including, but not limited to: immune checkpoint inhibitors (such as anti-PD1, anti-TIM-3, etc.), toll-like receptor agonists (such as TLR7 promoting agonist and / or TLR8 agonist), RIG-1 agonist, IL-15 superagonist (Altor Bioscience), mutant IRF3 and IRF7 gene adjuvant, STING agonist (Aduro), FLT3L gene adjuvant , IL-12 gene adjuvant, IL-7-hyFc; CAR-T combined with HBV env (S-CAR cells); capsid assembly regulator; cccDNA inhibitors, HBV polymerase inhibitors (such as entecavir and tenofovir Wei). The one or other anti-HBV active agents can be, for example, small molecules, antibodies or antigen-binding fragments thereof, polypeptides, proteins or nucleic acids. delivery method

[0264] The pharmaceutical compositions and combinations of the present application may be administered to an individual in view of the present invention by any method known in the art, including but not limited to parenteral administration (e.g., intramuscular, subcutaneous, intravenous or dermal administration) Intravenous injection), oral administration, transdermal administration and nasal administration. Preferably, the pharmaceutical compositions and combinations are administered parenterally (eg, by intravenous, intramuscular or intradermal injection) or transdermally.

[0265] In some embodiments of the present application where the pharmaceutical composition or combination comprises one or more DNA plasmids, administration may be by injection through the skin, such as intramuscular or intradermal injection, preferably intramuscular injection. carry on. Intramuscular injection can be combined with electroporation, ie, application of an electric field to facilitate delivery of DNA plasmids into cells. The term "electroporation" as used herein refers to the induction of microscopic pathways (pores) in biological membranes using transmembrane electric field pulses. During in vivo electroporation, an electric field of appropriate magnitude and duration is applied to cells, thereby inducing a transient state of increased cell membrane permeability, thereby enabling cells to take up molecules that cannot cross the cell membrane alone. Creating such pores by electroporation facilitates the passage of biomolecules such as plastids, oligonucleotides, siRNA, drugs, etc., from one side of the cell membrane to the other. In vivo electroporation for DNA delivery has been shown to significantly increase the uptake of plastids by host cells, while also causing mild to moderate inflammation at the injection site. Thus, using intradermal or intramuscular electroporation results in significantly higher transfection efficiencies (eg, up to 1,000-fold and 100-fold, respectively) compared to conventional injection.

[0266] In a typical embodiment, electroporation is combined with intramuscular injection. However, it is also possible to combine electroporation with other forms of parenteral administration such as intradermal injection, subcutaneous injection and the like.

[0267] Administration of the pharmaceutical compositions, combinations or vaccines of the present application via electroporation can be accomplished using electroporation devices that can be configured to deliver a pulse of energy effective to cause the formation of reversible pores in the cell membrane to the Requires mammalian tissue. An electroporation device may include an electroporation assembly and an electrode assembly or a handle assembly. The electroporation component may include one or more of the following electroporation device components: a controller, a current waveform generator, an impedance tester, a waveform recorder, an input element, a status reporting element, a communication port, a memory element, a power supply, and a power supply switch. Electroporation can be achieved using an in vivo electroporation device. Examples of electroporation devices and electroporation methods that may facilitate delivery of the compositions and immunogenic combinations of the present application, particularly those comprising DNA plasmids, include CELLECTRA® (Inovio Pharmaceuticals, Blue Bell, PA) , Elgen Electroporator (Inovio Pharmaceuticals), Tri-GridTM Delivery System (Ichor Medical Systems, San Diego, CA 92121) and U.S. Patent No. 7,664,545, U.S. Patent No. 8,209,006, U.S. Patent No. 9,452,285, U.S. Patent No. 5,273,525, US Patent 6,110,161, US Patent 6,261,281, US Patent 6,958,060, US Patent 6,939,862, US Patent 7,328,064, US Patent 6,041,252, US Patent 5,873,849, US Patent 6,278,895 No. , the electroporation devices described in US Patent No. 6,319,901, US Patent No. 6,912,417, US Patent No. 8,187,249, US Patent No. 9,364,664, US Patent No. 9,802,035, US Patent No. 6,117,660 and International Patent Application Publication WO2017172838 and electroporation methods, all of which are incorporated herein by reference in their entirety. The present application also contemplates the use of pulsed electric fields to deliver the compositions and combinations of the present application, as described, for example, in US Patent No. 6,697,669, which is incorporated herein by reference in its entirety.

[0268] In other embodiments of the present application where the pharmaceutical composition or combination comprises one or more DNA plasmids, the method of administration is transdermal. Transdermal administration can be combined with epidermal abrasion to facilitate delivery of DNA plasmids to cells. For example, epidermal abrasion can be performed using a dermal patch. After the skin patch is removed, the composition or combination can be deposited on the abraded skin.

[0269] The method of delivery is not limited to the examples described above, and any means of intracellular delivery may be used. Other intracellular delivery methods contemplated by the methods of the present application include, but are not limited to, liposomal encapsulation, liposomes, nanoparticles, and the like. For example, mRNA encoding one or more HBV TALENs of the present application can be formulated in a composition comprising one or more lipid molecules, preferably positively charged lipid molecules. In some embodiments, mRNA encoding one or more HBV TALENs of the invention can be formulated using one or more liposomes, liposomes, and / or lipid nanoparticles. In some embodiments, the liposome or lipid nanoparticle formulations described herein can comprise polycationic compositions. In some embodiments, formulations comprising polycationic compositions are useful for in vivo and / or ex vivo delivery of the HBV TALENs described herein. Example

[0270] Embodiment 1 includes a method of treating a hepatitis infection in a subject in need thereof, the method comprising administering to the subject a combination of mRNA molecules comprising: (1) a first mRNA molecule comprising a first mRNA molecule encoding a first type of transcriptional activation A polynucleotide sequence of a sub-effector nuclease (TALEN) monomer, the first TALEN monomer comprising a first TALE DNA binding domain and a first nuclease catalytic domain, wherein the first TALE DNA binding domain is capable of binding to HBV The first half-site sequence of the target nucleic acid sequence in the gene; and (2) a second mRNA molecule comprising a polynucleotide sequence encoding a second TALEN monomer comprising a second TALE DNA binding domain and a second nuclease catalytic domain, wherein the second TALE DNA binding domain is capable of binding to the second half-site sequence of the target nucleic acid sequence; wherein when the first TALE DNA binding domain binds to the first half-site sequence And when the second TALE DNA binding domain binds to the second half-site sequence, the first TALEN monomer and the second TALEN monomer can form a dimer that cleaves the target nucleic acid sequence, preferably, the second A nuclease catalytic domain is a first FokI nuclease catalytic domain and the second nuclease catalytic domain is a second FokI nuclease catalytic domain.

[0271] Embodiment 1a comprises the method of embodiment 1, wherein the first FokI nuclease catalytic domain is identical to the second FokI nuclease catalytic domain.

[0272] Embodiment 1b comprises the method of embodiment 1, wherein the first FokI nuclease catalytic domain is different from the second FokI nuclease catalytic domain.

[0273] Embodiment 2 includes the method of any one of embodiments 1 to 1b, wherein the target nucleic acid sequence is within the sequence encoding HBsAg and HBV polymerase (pol).

[0274] Embodiment 2a comprises the method of embodiment 2, wherein the first half site sequence of the target nucleic acid sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 1, such as at least 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequence, and the second half-site sequence of the target nucleic acid sequence comprises the same The nucleic acid sequence of SEQ ID NO: 2 is at least about 90% identical, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequence.

[0275] Embodiment 2b comprises the method of embodiment 2, wherein the first half-site sequence of the target nucleic acid sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 3, such as at least 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequence, and the second half-site sequence of the target nucleic acid sequence comprises the same The nucleic acid sequence of SEQ ID NO: 4 is at least about 90% identical, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequence.

[0276] Embodiment 2c comprises the method of embodiment 2, wherein the target nucleic acid sequence comprises at least 90% identity with the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, such as at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequences.

[0277] Embodiment 3 comprises the method of any one of embodiments 1 to 2c, wherein each of the first mRNA molecule and the second mRNA molecule further comprises a 5' end cap, a 5'-UTR, a sequence encoding a nuclear localization signal , one or more of the sequence encoding the N-terminal domain, the sequence encoding the C-terminal domain, the 3'-UTR and the polyadenosine tail, preferably all of them.

[0278] Embodiment 3a comprises the method of embodiment 3, wherein the first mRNA molecule and the second mRNA molecule each comprise a 5' end cap, a 5'-UTR, a sequence encoding the N-terminal domain, a sequence encoding the C-terminal domain sequence, 3'-UTR and polyadenosine tail.

[0279] Embodiment 3b comprises the method of embodiment 3, wherein the first mRNA molecule and the second mRNA molecule each comprise a 5' end cap, a 5'-UTR, a sequence encoding a nuclear localization signal, a sequence encoding an N-terminal domain Sequence, sequence encoding C-terminal domain, 3'-UTR and polyadenosine tail.

[0280] Embodiment 4 comprises the method of any one of embodiments 1 to 3b, wherein respectively, the first TALE DNA binding domain comprises an amine that is at least 90% identical to SEQ ID NO: 7 or SEQ ID NO: 9 an amino acid sequence, the second TALE DNA binding domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 8 or SEQ ID NO: 10, and the first FokI nuclease catalytic domain and the second FokI nuclease The catalytic domains each comprise an amino acid sequence at least 90% identical to SEQ ID NO: 11.

[0281] Embodiment 4a comprises the method of embodiment 4, wherein the first TALE DNA binding domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 7, and the second TALE DNA binding domain comprises an amino acid sequence identical to SEQ ID NO: 7 NO: 8 at least 90% identical amino acid sequence, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprise an amino acid sequence at least 90% identical to SEQ ID NO: 11.

[0282] Embodiment 4a1 comprises the method of embodiment 4a, wherein the first TALE DNA binding domain comprises the amino acid sequence of SEQ ID NO: 7, and the second TALE DNA binding domain comprises the amino acid sequence of SEQ ID NO: 8 acid sequence, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprise the amino acid sequence of SEQ ID NO: 11.

[0283] Embodiment 4a2 comprises the method of embodiment 4a, wherein the first TALE DNA binding domain consists of the amino acid sequence of SEQ ID NO: 7, and the second TALE DNA binding domain consists of the amine of SEQ ID NO: 8 amino acid sequence, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain are each composed of the amino acid sequence of SEQ ID NO: 11.

[0284] Embodiment 4b comprises the method of embodiment 4, wherein the first TALE DNA binding domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 9, and the second TALE DNA binding domain comprises an amino acid sequence identical to SEQ ID NO: 9 NO: 10 at least 90% identical amino acid sequence, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprise an amino acid sequence at least 90% identical to SEQ ID NO: 11.

[0285] Embodiment 4b1 comprises the method of embodiment 4b, wherein the first TALE DNA binding domain comprises the amino acid sequence of SEQ ID NO: 9, and the second TALE DNA binding domain comprises the amino acid sequence of SEQ ID NO: 10 acid sequence, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprise the amino acid sequence of SEQ ID NO: 11.

[0286] Embodiment 4b2 comprises the method of embodiment 4b, wherein the first TALE DNA binding domain consists of the amino acid sequence of SEQ ID NO: 9, and the second TALE DNA binding domain consists of the amine of SEQ ID NO: 10 amino acid sequence, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain are each composed of the amino acid sequence of SEQ ID NO: 11.

[0287] Embodiment 5 includes the method of any one of embodiments 1 to 4b2, wherein one or more of the HBV gene system genotypes A, B, C, D, E, F, G, H, I and J By.

[0288] Embodiment 5a includes the method as in Embodiment 5, wherein the HBV genotypes are one or more of genotypes A, B, C, D, E, F, G and H.

[0289] Embodiment 6 comprises the method of any one of embodiments 1-5a, the method further comprising administering to the individual a second therapeutic composition, preferably a second therapeutic composition comprising an anti-HBV agent.

[0290] Embodiment 7 comprises the method of any one of embodiments 1 to 6, wherein the individual has HBV infection, preferably chronic HBV infection.

[0291] Embodiment 8 includes the method of any one of embodiments 1-7, wherein the individual is co-infected with HBV and HDV.

[0292] Embodiment 9 comprises a composition comprising a combination of mRNA molecules encapsulated in lipid nanoparticles comprising: (3) a first mRNA molecule comprising a first mRNA molecule encoding a first type of transcriptional activation A polynucleotide sequence of a sub-effector nuclease (TALEN) monomer, the first TALEN monomer comprising a first TALE DNA binding domain and a first nuclease catalytic domain, wherein the first TALE DNA binding domain is capable of binding to HBV The first half-site sequence of the target nucleic acid sequence in the gene; and (4) a second mRNA molecule comprising a polynucleotide sequence encoding a second TALEN monomer comprising a second TALE DNA binding domain and a second nuclease catalytic domain, wherein the second TALE DNA binding domain is capable of binding to the second half-site sequence of the target nucleic acid sequence; wherein when the first TALE DNA binding domain binds to the first half-site sequence And when the second TALE DNA binding domain binds to the second half-site sequence, the first TALEN monomer and the second TALEN monomer can form a dimer that cleaves the target nucleic acid sequence, preferably, the second A nuclease catalytic domain is a first FokI nuclease catalytic domain and the second nuclease catalytic domain is a second FokI nuclease catalytic domain.

[0293] Embodiment 9a comprises the composition of embodiment 9, wherein the first FokI nuclease catalytic domain is identical to the second FokI nuclease catalytic domain.

[0294] Embodiment 9b comprises the composition of embodiment 9, wherein the first FokI nuclease catalytic domain is different from the second FokI nuclease catalytic domain.

[0295] Embodiment 9c comprises the composition of any one of embodiments 9-9b, wherein each mRNA molecule is individually encapsulated in lipid nanoparticles.

[0296] Embodiment 9d comprises the composition of any one of embodiments 9-9b, wherein a plurality of mRNA molecules are encapsulated in individual lipid nanoparticles.

[0297] Embodiment 10 comprises the composition of any one of embodiments 9-9d, wherein the target nucleic acid sequence is within the sequence encoding HBsAg and HBV polymerase (pol).

[0298] Embodiment 10a comprises the composition of embodiment 10, wherein the first half site sequence of the target nucleic acid sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 1, such as at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequences, and the second half-site sequence of the target nucleic acid sequence comprises At least about 90% identical to the nucleic acid sequence of SEQ ID NO: 2, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical the polynucleotide sequence.

[0299] Embodiment 10b comprises the composition of embodiment 10, wherein the first half-site sequence of the target nucleic acid sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 3, such as at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequences, and the second half-site sequence of the target nucleic acid sequence comprises At least about 90% identical to the nucleic acid sequence of SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical the polynucleotide sequence.

[0300] Embodiment 10c comprises the composition of embodiment 10, wherein the target nucleic acid sequence comprises at least 90% identity with the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, such as at least 90%, 91%, 92% %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical polynucleotide sequences.

[0301] Embodiment 11 comprises the composition of any one of embodiments 9 to 10c, wherein each of the first mRNA molecule and the second mRNA molecule further comprises a 5' end cap, a 5'-UTR, an encoding nuclear localization signal One or more of the sequence, the sequence encoding the N-terminal domain, the sequence encoding the C-terminal domain, the 3'-UTR and the polyadenosine tail.

[0302] Embodiment 12 comprises the composition of any one of embodiments 9 to 11, wherein respectively, the first TALE DNA binding domain comprises at least 90% identical to SEQ ID NO: 7 or SEQ ID NO: 9 an amino acid sequence, the second TALE DNA binding domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 8 or SEQ ID NO: 10, and the first FokI nuclease catalytic domain and the second FokI nucleic acid The enzyme catalytic domains each comprise an amino acid sequence at least 90% identical to SEQ ID NO: 11.

[0303] Embodiment 12a comprises the composition of embodiment 12, wherein the first TALE DNA binding domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 7, and the second TALE DNA binding domain comprises an amino acid sequence identical to SEQ ID NO: 7 ID NO: 8 at least 90% identical amino acid sequence, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprise an amino acid sequence at least 90% identical to SEQ ID NO: 11.

[0304] Embodiment 12a1 comprises the composition of embodiment 12a, wherein the first TALE DNA binding domain comprises the amino acid sequence of SEQ ID NO: 7, and the second TALE DNA binding domain comprises the amine of SEQ ID NO: 8 amino acid sequence, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprise the amino acid sequence of SEQ ID NO: 11.

[0305] Embodiment 12a2 comprises the composition of embodiment 12a, wherein the first TALE DNA binding domain consists of the amino acid sequence of SEQ ID NO: 7, and the second TALE DNA binding domain consists of the amino acid sequence of SEQ ID NO: 8 amino acid sequence, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain are each composed of the amino acid sequence of SEQ ID NO: 11.

[0306] Embodiment 12b comprises the composition of embodiment 12, wherein the first TALE DNA binding domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 9, and the second TALE DNA binding domain comprises an amino acid sequence identical to SEQ ID NO: 9 ID NO: 10 has an amino acid sequence that is at least 90% identical, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 11.

[0307] Embodiment 12b1 comprises the composition of embodiment 12b, wherein the first TALE DNA binding domain comprises the amino acid sequence of SEQ ID NO: 9, and the second TALE DNA binding domain comprises the amine of SEQ ID NO: 10 amino acid sequence, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprise the amino acid sequence of SEQ ID NO: 11.

[0308] Embodiment 12b2 comprises the composition of embodiment 12b, wherein the first TALE DNA binding domain consists of the amino acid sequence of SEQ ID NO: 9, and the second TALE DNA binding domain consists of the amino acid sequence of SEQ ID NO: 10 amino acid sequence, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain are each composed of the amino acid sequence of SEQ ID NO: 11.

[0309] Embodiment 13 comprises the composition of any one of embodiments 9 to 12b2, wherein the HBV genotype genotypes A, B, C, D, E, F, G, H, I and J are one or many.

[0310] Embodiment 13a comprises the composition of Embodiment 13, wherein the HBV genotypes are one or more of genotypes A, B, C, D, E, F, G and H.

[0311] Embodiment 14 comprises the composition of any one of embodiments 9 to 13a, wherein the lipid nanoparticles encapsulating the combination of mRNA molecules comprise cationic lipids and at least one selected from the group consisting of anionic lipids, amphoteric Other lipids of the group consisting of ionic lipids, neutral lipids, steroids, polymer-bound lipids, phospholipids, glycolipids, and combinations thereof.

[0312] Embodiment 15 comprises a combination comprising: (1) a first nucleic acid, preferably a first mRNA molecule, comprising a polynucleotide encoding a first type of transcription activator effector nuclease (TALEN) monomer Nucleotide sequence, the first TALEN monomer comprises a first TALE DNA binding domain and a first FokI nuclease catalytic domain, wherein the first TALE DNA binding domain can bind to the first half of the target nucleic acid sequence in the HBV gene body point sequence, and the first half-site sequence comprises at least about 90% identity with the nucleic acid sequence of SEQ ID NO: 1, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequences; and (2) a second nucleic acid, preferably a second mRNA molecule, comprising a second class of transcription activator effector nuclease (TALEN ) monomer polynucleotide sequence, the second TALEN monomer comprises a second TALE DNA binding domain and a second FokI nuclease catalytic domain, wherein the second TALE DNA binding domain can bind to the second target nucleic acid sequence A half-site sequence, and the second half-site sequence comprises at least about 90% identity with the nucleotide sequence of SEQ ID NO: 2, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96% %, 97%, 98%, 99% or 100% identical polynucleotide sequences; wherein when the first TALE DNA binding domain binds to the first half-site sequence and the second TALE DNA binding domain binds to the For the second half-site sequence, the first TALEN monomer and the second TALEN monomer can form a dimer that cleaves the target nucleic acid sequence.

[0313] Embodiment 15a comprises the combination of embodiment 15, wherein the first half-site sequence comprises the nucleic acid sequence of SEQ ID NO: 1 and the second half-site sequence comprises the nucleic acid sequence of SEQ ID NO: 2.

[0314] Embodiment 15b comprises a combination as in embodiment 15, wherein the first half-site sequence is made up of the nucleotide sequence of SEQ ID NO: 1 and the second half-site sequence is made up of the nucleotide sequence of SEQ ID NO: 2 .

[0315] Embodiment 16 comprises the combination of any one of embodiments 15 to 15b, wherein one or more of the HBV gene system genotypes A, B, C, D, E, F, G, H, I and J By.

[0316] Embodiment 16a comprises the combination as in Embodiment 16, wherein the HBV genotypes are one or more of genotypes A, B, C, D, E, F, G and H.

[0317] Embodiment 17 comprises a composition comprising the combination of any one of embodiments 15 to 16b, wherein the first nucleic acid and the second nucleic acid are separately or jointly encapsulated in lipid nanoparticles middle.

[0318] Embodiment 18 comprises a nucleic acid molecule encoding at least one of the first mRNA molecule and the second mRNA molecule of any one of Embodiments 15-15b.

[0319] Embodiment 19 comprises an isolated host cell comprising the nucleic acid molecule of Example 18.

[0320] Embodiment 20 comprises the isolated host cell of embodiment 19, wherein the host cell is a hepatocyte.

[0321] Embodiment 21 comprises a pharmaceutical composition comprising the composition of any one of embodiments 9 to 14 or 17, the combination of any one of embodiments 15 to 16a, the combination of any one of embodiments 18 Nucleic acid molecule or isolated host cell as in embodiment 19 or 20 and a pharmaceutically acceptable carrier.

[0322] Embodiment 22 includes a method for cracking a target nucleic acid sequence in an HBV gene body, the method comprising making the HBV gene body and a composition as in any one of embodiments 9 to 14 or 17, such as in embodiments 15 to 17 The combination of any one of 16a, the nucleic acid molecule of Example 18, the isolated host cell of Example 19 or 20, or the pharmaceutical composition of Example 21.

[0323] Embodiment 23 includes a method for inducing gene editing of a target nucleic acid sequence in an HBV gene body, the method comprising making the HBV gene body and a composition such as any one of embodiments 9 to 14 or 17, such as implementing The combination of any one of Examples 15-16a, the nucleic acid molecule of Example 18, the isolated host cell of Example 19 or 20, or the pharmaceutical composition of Example 21 are contacted.

[0324] Embodiment 24 includes the method of embodiment 22 or 23, wherein the HBV genotypes are one or more of genotypes A, B, C, D, E, F, G, H, I and J.

[0325] Embodiment 24a includes the method as in Embodiment 24, wherein the HBV genotypes are one or more of genotypes A, B, C, D, E, F, G and H.

[0326] Embodiment 25 includes a method of treating a hepatitis infection in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of Example 21.

[0327] Embodiment 26 includes a method of reducing HBV infection in a subject and / or reducing HBV replication in a subject, the method comprising administering the pharmaceutical composition of Example 21 to the subject.

[0328] Embodiment 27 includes the method of Embodiment 25 or 26, the method further comprising administering to the individual a second therapeutic composition, preferably a second therapeutic composition comprising an anti-HBV agent.

[0329] Embodiment 28 comprises the method of any one of embodiments 25-27, wherein the individual has HBV infection, preferably chronic HBV infection.

[0330] Embodiment 29 includes the method of any one of embodiments 25-28, wherein the individual is co-infected with HBV and HDV.

[0331] Embodiment 30 includes the method of any one of embodiments 25-29, wherein the individual has reduced expression of one or more of HBsAg, HBeAg, HBV DNA, HBV cccDNA, or integrated HBV DNA.

[0332] Embodiment 30a includes the method of embodiment 30, wherein the individual has reduced serum and / or plasma levels of one or more of HBsAg, HBeAg, and HBV DNA.

[0333] Embodiment 31 comprises the method of embodiment 30, wherein the expression is hepatocyte content, nuclear or cell content, liver content, serum content or plasma content.

[0334] Embodiment 32 includes a method of producing a TALEN comprising in vitro or in vivo transcription of the nucleic acid molecule of Embodiment 18.

[0335] Embodiment 33 comprises the pharmaceutical composition of Embodiment 21 for the treatment of a hepatitis B virus (HBV)-induced disease in an individual in need thereof, preferably wherein the individual has chronic HBV Infect.

[0336] Embodiment 33a comprises the pharmaceutical composition of Embodiment 33 and another therapeutic agent, preferably another anti-HBV agent.

[0337] Embodiment 34 comprises the pharmaceutical composition of embodiment 33 or 33a, wherein the HBV-induced disease is selected from the group consisting of advanced fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Materials and Methods for Examples 1-6 Cells and Culture

[0338] Cryopreserved HepG2.2.15 cells (Fox Chase Cancer Center (Fox Chase Cancer Center)) were thawed and incubated in Dulbecco containing 10% FBS supplemented with antibiotics, 2 mM L-glutamine and G418. Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen). Cells were cultured on collagen I coated flasks and plates.

[0339] The cryopreserved primary human hepatocytes (PHH) (Lonza, batch number 8317) were thawed and mixed with 10% FBS containing HEPES, L-proline, insulin, epidermal growth factor, dexamethasone (dexamethasone) , ascorbic acid-2-phosphate and 2% DMSO (Sigma) supplemented with Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen). HBV inoculum

[0340] Genotype (GT) D HBV inoculum was pooled and concentrated from the supernatant of HepG2.2.15 human hepatoblastoma cells (Genbank U95551 ) constitutively expressing GT D HBV. GTA (PS 3.57), B (PS 3.4), C (PS 3.7) and D (PS 3.56) HBV inoculum from patient sera were purchased from American Red Cross. HBV DNA plasmid

[0341] Plastid DNA containing the 1.1x genotype B HBV genotype under the control of the CMV promoter was previously cloned from the serum of an HBV-infected patient (Genbank AY220698, Fudan University, China). HBV sequences from genotypes A (Genbank AF305422), C (Genbank AB033550), D (Genbank V01460), E (Genbank AB274971), F (Genbank AB036912), G (Genbank AF160501) and H (Genbank AB375159) were 1.1 Gene synthesized in the x-genome form and includes a portion of the CMV promoter at the 5' end. The gene synthesis segment was confirmed by sequencing and sub-cloned into pCMV-HBV plastids using the restriction sites Sail and NdeI (Genewiz, USA). HBV TALEN mRNA

[0342] TALEN DNA sequences were cloned into plastids with a T7 promoter for in vitro transcription. Plastids were linearized using BspQI restriction sites and transcribed in vitro. Briefly, nucleotide triphosphates (NTPs) and T7 RNA polymerase are combined with linearized templates in aqueous buffer and incubated. The resulting mRNA transcripts are purified using column chromatography, DNA and double-stranded RNA contaminants are removed from the mRNA using an enzymatic reaction, the mRNA is concentrated, and the buffer is exchanged. Using HepG2.2.15 cells to transfect HBVTALENmRNA

[0343] On the day of transfection, mRNAs encoding right or left TALEN proteins were combined at a 1:1 ratio in OptiMEM medium before mixing with Lipofectamine MessengerMAX Transfection Reagent (Invitrogen). Transfection mixtures were also prepared in the absence of HBV TALEN mRNA as no TALEN controls. Place the transfection mixture on top of the transfection mixture for reverse transfection by placing the transfection mixture on top of the transfection mixture at a density of 30,000 / well and maintain overnight at 37°C before removing Medium containing the transfection mixture and supplemented with fresh cell culture medium. Transfected cells were maintained at 30°C, and another medium was changed on day 3 after transfection. Gene editing and secreted HBsAg were monitored at day 6 post-transfection. HBV infection and HBV TALEN mRNA transfection using PHH

[0344] At least one day prior to HBV infection, PHH were seeded at a density of 240,000 cells / well in collagen-coated 24-well dishes. PHH were infected with an HBV inoculum at 200 genosome equivalents / cell in medium containing 4% PEG-8000 (Sigma). The next day, the HBV inoculum was removed and cccDNA and virus replication were allowed to establish for at least 5 days before transfection of mRNA encoding HBV TALENs. On the day of transfection, mRNAs encoding right or left TALEN proteins were combined at a 1:1 ratio in OptiMEM medium before being mixed with Lipofectamine messenger Max transfection reagent (Invitrogen). Transfection mixtures were also prepared in the absence of HBV TALEN mRNA as no TALEN controls. HBV-infected PHH were transfected with or without HBV TALEN mRNA overnight at 37°C, after which the medium containing the transfection mixture was removed and fed with fresh PHH medium. Transfected cells were maintained at 30°C, and another medium was changed on day 3 after transfection. Gene editing and secreted HBsAg and HBeAg were monitored on day 6 post-transfection. HBVTALEN protein and HBV antigen detection in cell culture

[0345] Expression of HBV TALEN proteins in transfected PHH was detected by immunofluorescence 6 hours after transfection. Cells were fixed with perfusion fixative (Electron Microscopy Sciences, VWR), permeabilized by using 1% Triton-X 100 and blocked with 3% normal goat serum (NGS) / PBS. Immunostaining was performed by using primary peptide tag-specific antibodies with 3% NGS, followed by secondary staining with goat anti-mouse Alexa Fluor 488 (Invitrogen) diluted 1:1000 in PBS with 3% NGS. Nuclear staining was performed in combination with secondary staining in which DAPI (ThermoFisher) diluted 1:1000 was mixed with secondary antibodies. HBsAg and HBeAg contents in supernatants from HBV-infected PHH or HepG2.2.15 cells were monitored by using a chemiluminescent immunoassay kit (Autobio Diagnostics) corresponding to the quantitative detection of HBsAg and HBeAg, respectively. Immunofluorescent staining and imaging were performed on an Opera system (Perkin Elmer). HBVcccDNA gene editing detection

[0346] On day 6 after HBV TALEN mRNA transfection, PHH was treated with a lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP40 for 10 minutes at room temperature. Cells were centrifuged briefly to isolate nuclei from which gene bodies and HBV DNA were extracted by using the NucleoSpin Tissue kit (Macherey-Nagel). To remove the loose circular form of HBV DNA from cccDNA, the extracted DNA was treated with 10 units of T5 exonuclease (New England Biolabs) for 60 minutes at 37°C. Subsequently, the T5 exonuclease was inactivated by heating at 90°C for 5 minutes. By using the forward primer 5'-CCT AGG ACC CCT TCT CGT GT (SEQ ID NO: 29) and the reverse primer 5'-ACT GAG CCA GGA GAA ACG GG (SEQ ID NO: 30) and Platinum high-fidelity PCR ultra Mixture (Invitrogen), using 1 cycle of 95°C for 10 minutes, 45 cycles of 95°C for 30 seconds (denaturation step), 64°C for 30 seconds (bonding step), 72°C for 30 seconds (extension step), followed by 72°C Cycling conditions for a final extension of 10 minutes were used to amplify the targeted HBV sequence within the HBsAg coding region. 10× NEB2 buffer and water were added to 10 μL of the PCR reaction to a final concentration of 1× and denatured at 95° C. for 10 minutes, and then by first lowering the temperature at 3° C. / sec to 85° C. and then adding 0.3°C / sec further lowering to 25°C for rebonding. Mismatched DNA sequences were cleaved by adding 5 units of T7E1 nuclease (New England Biolabs) and incubating at 37°C for 1 hour. Digested and undigested products were analyzed by electrophoresis using Novex 10% TBE gels (Thermo Fisher) and stained with SYBR green (Thermo Fisher) for visualization by UV using a FluorChem M imager (Protein Simple). In addition, amplicons were submitted for next generation sequencing (Genewiz) to determine changes within the TALEN target region. Deployment of mRNA encoding HBVTALEN in lipid nanoparticles Preparation of lipid-encapsulated mRNA by mixing lipids in ethanol (cationic lipid:DSPC:cholesterol:PEG-DMG) with mRNA encoding the desired polypeptide dissolved in citrate buffer particle. Dilute the combined material instantaneously with phosphate buffered saline. Migration by dialysis against phosphate buffered saline using regenerated cellulose membranes (100 kD MWCO) or by tangential flow filtration (TFF) using modified polyethersulfone (mPES) hollow fiber membranes (100 kD MWCO) In addition to ethanol. Once the ethanol was completely removed, the buffer was exchanged with HEPES (4-(2-hydroxyethyl)-1-piperoneethanesulfonic acid) buffer containing 50 mM NaCl and 9% sucrose, pH 7.3. The formulation was concentrated followed by 0.2 µm filtration using a PES filter. Subsequently, the mRNA concentration in the formulation was measured by Ribogreen fluorescence analysis, and thereafter adjusted to the final desired concentration by diluting with HEPES buffer containing 50 mM NaCl, 9% sucrose, pH 7.3, containing glycerol. concentration. Next, the final formulation was filtered through a 0.2 μm filter and filled into glass vials, stoppered, capped and placed at -70±5°C. mRNA content and percent encapsulation for frozen formulations were analyzed by Ribogreen, for mRNA integrity by Fragment Analyzer, for lipid content by high performance liquid chromatography (HPLC), for particle size on a Malvern Zetasizer Nano Dynamic light scattering on ZS, frozen formulations were characterized for pH and osmolality. Mouse model infected with AAV-HBV

[0348] C57B16 male mice (Taconic) were infected with 1 x 10^11 of AAV adenovirus infectious particles-1.3 x HBV (FivePlus) by intravenous tail injection. Four weeks post-infection, serum was collected and analyzed to quantify HBsAg and HBeAg levels. Mice with 4 or more logs of HBsAg were selected for in vivo efficacy studies. Detection and quantification of HBV antigen and HBV DNA in serum

[0349] The levels of HBsAg and HBeAg in mouse serum of AAV-HBV mice were measured by using a chemiluminescence immunoassay kit (Autobio Diagnostics). Sera were diluted 1:100 and 1:250 in PBS for HBeAg and HBsAg CLIA detection, respectively.

[0350] HBV DNA was extracted from 5 µl of serum following the instructions of the QIAamp DNA Mini Kit (Qiagen, Cat. No. 51306), ie, the accessory protocol for viral DNA. Briefly, after incubation in proteinase K buffer for 10 min at 56°C, samples were mixed with 100% ethanol and applied to a QIAmp mini spin column. The spin column was centrifuged and washed, and the isolated DNA was resuspended in 30 μl of water as indicated in the instructions.

[0351] A mixture of 2× TaqMan Rapid Development Mix (Thermo Fisher, Cat. No. 4444557) and 20× sAg probes (Thermo Fisher, Analytical Id Vi03453406_s1 TaqMan Gene Expression Analysis (FAM) Dye Label and Assay Concentration FAM-MGB) was used for amplification. Increase the purified serum HBV DNA, and use QuantStudio 6 Flex qPCR machine and use 50°C 1 cycle for 2 minutes and 95°C for 2 minutes, 95°C for 45 cycles for 10 seconds (denaturation step), 60°C for 20 seconds (gluing step) , 72° C. for 30 seconds (extension step) with a cycle condition of 1.6° C. / second rising speed. For quantification, a standard curve was run in parallel using serial dilutions of known numbers of replicates of synthetic HBV DNA performed by AATC (Quantitative Synthetic Hepatitis B Virus DNA (ATCC® VR-3232SD™)). Data were extracted and analyzed using QuantStudio software, while statistical analysis was performed using GraphPad software. Detection of HBV DNA gene editing in mouse liver tissue

[0352] For gene editing assays, HBV DNA was extracted from snap-frozen mouse livers using reagents and protocols from the Quick-DNA Miniprep Plus Kit (ZymoResearch Cat# D4068). First, snap-frozen mouse liver tissue was placed in proteinase K digestion buffer and disrupted by two mechanisms using 0.5 ml tubes from the Precellys Lysis Kit for Soft Tissue (Precellys, cat. no. P000933-LYSK0-A) Homogenization was performed in a Precellys Evolution Homogenizer (Precellys, Bertin Instruments) and samples were subsequently incubated at 55°C for 20 minutes by digestion. Subsequently, HBV DNA was purified in the Quick-DNA miniprep Plus kit by spin column cleanup as indicated. Genome DNA was eluted in 50 μl DNA elution buffer and quantified.

[0353] By PCR, use 5 ng / μl of purified DNA mixed in 25 μl of PCR reaction and 2× SuperFI Platinum master mix (Invitrogen, Cat# 12358250); forward primer 5'- ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT TAT CGC TGG ATG TGT CTG CG-3' (SEQ ID NO: 29) and reverse primer 5'- GAC TGG AGT TCA GAC GTG TGC TCT TCC GAT CTG TCC GAA GGT TTG GTA CAG C -3 ' (SEQ ID NO: 30) and using 98°C for 1 cycle for 2 minutes, 95°C for 35 cycles for 15 seconds (denaturation step), 60°C for 15 seconds (bonding step), 72°C for 30 seconds (extension step), and then Cycling conditions for a final extension of 5 minutes at 72°C amplified the HBV DNA target sequence. PCR products were cleaned up with NucleoSpin® gel and PCR cleanup kit (Macherey-Nagel) following the kit instructions, eluted in NE buffer, quantified and diluted to 20 ng / μl if necessary for NGS sequencing. Submit samples for amplicon sequencing (Amplicon-EZ) and analysis at Genewiz. Examples Example 1: TALEN target site and TALEN structure

[0354] Publicly available bioinformatics tools were used to predict potential TALEN target sites within the common HBV genome sequence. TALEN target sites were selected from within the gene encoding HBsAg / pol to reduce HBsAg secretion and viral replication (Fig. 1A). TALEN 28 and TALEN 33 target the HBV gene body region with a length of 50 and 53 base pairs, respectively, including the left-arm TALEN sequence, the right-arm TALEN sequence and the spacer sequence between the two arms (Fig. 1B). All TALEN proteins are encoded by mRNA molecules that include an end cap, a 5' untranslated region (UTR), a sequence encoding a tag peptide, a sequence encoding a nuclear localization signal, a sequence encoding a TALEN N-terminal domain, an encoding sequence containing Sequence of TALEN DNA binding domain of repeat variable double residue (RVD) domain, sequence encoding C-terminal domain, sequence encoding Fokl nuclease, 3'UTR and polyadenosine tail. Optionally, constructs can be made that include an end cap, a 5' untranslated region (UTR), a sequence encoding a nuclear localization signal, a sequence encoding a TALEN N-terminal domain, a sequence encoding a variable double residue containing repeats. The sequence of the TALEN DNA binding domain of the repeat domain of the base (RVD), the sequence encoding the C-terminal domain, the sequence encoding the FokI nuclease, the 3' UTR and the polyadenosine tail. TALEN 28 and TALEN 33 RVDs for targeting four DNA nucleotide bases are NN (targeting A and G), NI (targeting T), NG (targeting C) and HD (targeting G) ( Figure 1C). Example 2: TALEN target sites are conserved and TALENs show broad functional genotype coverage

[0355] Conservation analysis of the HBV target sequences of the left and right arms of TALEN 28 and 33 was performed among hundreds of HBV genotype sequences including all different HBV genotypes. Alignment of HBV target sequences across different genotypes showed extremely high conservation, 97-100% for TALEN 28 and TALEN 33 (Table 1 and Table 2 below, respectively). For the sequences targeted by the right arms of TALEN 28 and TALEN 33, two positions showed mismatches for genotypes A and C (Tables 1 and 2 below). Table 1. Similarity percentage analysis of the left and right arms of TALEN28 across HBV genotypes A to H TALEN28 left target sequence (similarity to HBV genotype %) HBV GT Number of isolates T G C T G C T A T G C C T C A T C T T C total 6341 A 855 100.0 99.9 100.0 99.8 100.0 99.9 99.9 99.9 99.9 99.9 99.8 99.9 99.8 100.0 99.9 100.0 100.0 99.9 100.0 100.0 B 1749 99.9 99.8 100.0 99.8 99.9 99.9 99.9 99.8 99.9 99.8 99.8 99.9 99.8 99.9 99.9 99.7 99.9 99.9 99.2 100.0 C 2179 99.9 99.9 99.9 99.8 100.0 100.0 99.8 100.0 100.0 100.0 100.0 100.0 99.7 99.9 99.9 99.0 100.0 100.0 99.8 100.0 D. 954 100.0 100.0 100.0 100.0 97.5 99.9 99.8 99.9 100.0 99.9 99.9 100.0 100.0 99.9 100.0 98.8 100.0 100.0 99.9 99.9 E. 290 100.0 100.0 100.0 100.0 100.0 99.3 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 f 248 100.0 100.0 100.0 100.0 100.0 100.0 99.6 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 99.6 100.0 100.0 100.0 100.0 G 40 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 h 26 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 96.2 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 TALEN28 right target sequence (similarity to HBV genotype %) HBV GT Number of isolates C T G G A C T A T C A A G G T A total 6341 A 855 99.9 99.9 99.9 99.9 99.8 1.3 99.9 93.1 91.2 99.9 100.0 97.5 99.1 99.8 99.9 99.8 B 1749 99.8 99.9 99.9 99.9 99.9 99.5 99.9 98.7 99.3 99.9 99.8 97.6 99.9 99.9 99.9 99.9 C 2179 93.5 99.7 100.0 100.0 99.8 99.2 100.0 98.3 9.9 98.7 98.4 98.2 99.8 100.0 100.0 99.9 D. 954 100.0 100.0 100.0 100.0 100.0 99.8 99.9 99.4 99.0 100.0 98.5 97.1 100.0 100.0 99.9 99.9 E. 290 100.0 99.7 100.0 100.0 99.0 100.0 100.0 99.7 100.0 100.0 99.3 96.9 100.0 100.0 99.7 100.0 f 248 94.8 100.0 100.0 100.0 100.0 100.0 100.0 99.6 92.3 100.0 99.6 97.6 100.0 100.0 100.0 100.0 G 40 100.0 100.0 100.0 100.0 100.0 95.0 100.0 100.0 100.0 100.0 100.0 97.5 100.0 100.0 100.0 100.0 h 26 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 Table 2. Similarity percentage analysis of the left and right arms of TALEN33 across HBV genotypes A to H TALEN33 left target sequence (similarity to HBV genotype %) HBV GT Number of isolates T C C T G C T G C T A T G C C T C A T total 6341 99.9 99.9 100.0 99.9 99.9 100.0 99.8 99.6 99.9 99.9 99.9 99.9 99.9 99.9 100.0 99.8 99.9 99.9 99.4 A 855 99.9 100.0 100.0 100.0 99.9 100.0 99.8 100.0 99.9 99.9 99.9 99.9 99.9 99.8 99.9 99.8 100.0 99.9 100.0 B 1749 99.9 100.0 99.9 99.9 99.8 100.0 99.8 99.9 99.9 99.9 99.8 99.9 99.8 99.8 99.9 99.8 99.9 99.9 99.7 C 2179 99.8 99.8 100.0 99.9 100.0 99.9 99.8 100.0 100.0 99.8 100.0 100.0 100.0 100.0 100.0 99.7 99.9 99.9 99.0 D. 954 99.8 100.0 99.9 100.0 100.0 100.0 100.0 99.9 99.9 99.8 99.9 100.0 99.9 99.9 100.0 100.0 99.9 100.0 98.8 E. 290 100.0 100.0 100.0 100.0 100.0 100.0 100.0 99.3 99.3 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 f 248 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 99.6 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 99.6 G 40 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 h 26 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 96.2 100.0 100.0 100.0 100.0 100.0 100.0 100.0 TALEN33 right target sequence (similarity to HBV genotype %) HBV GT Number of isolates C T T C T G G A C T A T C A A G G T A total 6341 99.9 100.0 99.6 97.5 99.8 100.0 100.0 99.8 86.2 100.0 98.0 67.2 99.5 99.1 97.7 99.8 99.9 99.9 99.9 A 855 99.8 100.0 98.2 99.9 99.9 99.9 99.9 99.8 1.3 99.9 93.1 91.2 99.9 100.0 97.5 99.1 99.8 99.9 99.8 B 1749 99.9 100.0 99.9 99.8 99.9 99.9 99.9 99.9 99.5 99.9 98.7 99.3 99.9 99.8 97.6 99.9 99.9 99.9 99.9 C 2179 100.0 100.0 99.8 93.5 99.7 100.0 100.0 99.8 99.2 100.0 98.3 9.9 98.7 98.4 98.2 99.8 100.0 100.0 99.9 D. 954 99.9 99.8 99.5 100.0 100.0 100.0 100.0 100.0 99.8 99.9 99.4 99.0 100.0 98.5 97.1 100.0 100.0 99.9 99.9 E. 290 100.0 100.0 100.0 100.0 99.7 100.0 100.0 99.0 100.0 100.0 99.7 100.0 100.0 99.3 96.9 100.0 100.0 99.7 100.0 f 248 100.0 100.0 100.0 94.8 100.0 100.0 100.0 100.0 100.0 100.0 99.6 92.3 100.0 99.6 97.6 100.0 100.0 100.0 100.0 G 40 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 95.0 100.0 100.0 100.0 100.0 100.0 97.5 100.0 100.0 100.0 100.0 h 26 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0

[0356] In order to graphically represent the conservation of the sequences targeted by TALEN 28 and TALEN 33, conservation markers were generated based on the abundance of each targeted nucleotide in different HBV gene bodies, where the higher the conservation, the (multiple ) The larger the size of the abundant nucleotide. The left arms of TALEN 28 and TALEN 33 showed very high conservation across all targeted nucleotides (Figure 2A and Figure 3A). For the HBV sequences targeted by the right arms of both TALEN 28 and TALEN 33, two positions were found showing two alternative bases (C or T) (positions 6 and 9 for TALEN 28, Figure 2B; at positions 9 and 12 of TALEN 33, Figure 3B), depending on the HBV genotype.

[0357] To understand how changes in the HBV target sequence may affect TALEN efficacy, biochemical assays were used to assess TALEN 28 and TALEN 33 genotype coverage. Colonization of HBV sequences from 8 different genotypes into plastids and alignment of their TALEN 28 and TALEN 33 target sequences revealed a single mismatch for genotypes A and C (Figure 4A and Figure 4B). Left and right arms from TALEN 28 and TALEN 33 mRNAs were translated in vitro using the reticulocyte in vitro translation system (Promega) and grown with linearized plastids containing the sequences of the different HBV genotypes. TALEN digestion of plastids containing HBV target sequences was analyzed using polyacrylamide gels and detected by the presence of new DNA fragments not present in uncut DNA. TALEN 28 and TALEN 33 showed DNA cleavage for all HBV genotypes (A to H) tested, thus exhibiting broad genotype coverage (Figure 4C). The genotype coverage of TALEN 33 observed in biochemical analysis was verified by infecting primary human hepatocytes with HBV of different genotypes (A, B, C and D) and transfecting these cells with TALEN 33 mRNA . HBV DNA fragmentation was observed in all TALEN 33 mRNA transfected samples (Fig. 8, Table 3). Table 3. TALEN33 induces cccDNA disruption of PHH infected with HBVGTA to D* GTA (PS 3.57) HBsAg target sequence read segment TCCTGCTGCTATGCCTCATCTTCTTATTGGTTTCTTCTGGATTATCAAGGTA 40692 TCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTA 2017 TCCTGCTGCTATGCCTCATCTTCT--TTGGTTCTTCTGGATTATCAAGGTA 1934 TCCTGCTGCTATGCCTCATCTTC-TATTGGTTTCTTCTGGATTATCAAGGTA 1069 TCCTGCTGCTATGCCTCATCTTCTTA-TGGTTCTTCTGGATTATCAAGGTA 1000 TCCTGCTGCTATGCCTCATCTTC---TTGGTTCTTCTGGATTATCAAGGTA 856 GT B (PS 3.4) HBsAg target sequence Read segment TCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTA 66704 TCCTGCTGCTATGCCTCATCTTCT--TTGGTTCTTCTGGACTATCAAGGTA 4073 TCCTGCTGCTATGCCTCATCTTC---TTGGTTCTTCTGGACTATCAAGGTA 2355 TCCTGCTGCTATGCCTCATCTTCTTG-TGGTTCTTCTGGACTATCAAGGTA 2162 TCCTGCTGCTATGCCTCATCTTC----TGGTTCTTCTGGACTATCAAGGTA 1679 TCCTGCTGCTATGCCTCATCTTC-TGTTGGTTCTTCTGGACTATCAAGGTA 1074 GT C (PS 3.7) HBsAg target sequence Read segment TCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCAAGGTA 60493 TCCTGCTGCTATGCCTCATCTTC-TGTTGGTTTCTTCTGGACTACCAAGGTA 4323 TCCTGCTGCTATGCCTCATCTTCT--TTGGTTCTTCTGGACTACCAAGGTA 3384 TCCTGCTGCTATGCCTCATCTTC---TTGGTTCTTCTGGACTACCAAGGTA 2863 TCCTGCTGCTATGCCTCATCTTC----TGGTTCTTCTGGACTACCAAGGTA 1087 GT D (PS 3.56) HBsAg target sequence Read segment TCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTA 24933 TCCTGCTGCTATGCCTCATCTTCT--TTGGTTCTTCTGGACTATCAAGGTA 3649 TCCTGCTGCTATGCCTCATCTTC---TTGGTTCTTCTGGACTATCAAGGTA 3191 TCCTGCTGCTATGCCTCATCTTC----TGGTTCTTCTGGACTATCAAGGTA 1790 TCCTGCTGCTATGCCTCATCTTC-TGTTGGTTCTTCTGGACTATCAAGGTA 1672 TCCTGCTGCTATGCCTCATCTTCTTG-TGGTTCTTCTGGACTATCAAGGTA 1286 TCCTGCTGCTATGCCTCATCTTCTT----GTTCTTCTGGACTATCAAGGTA 497 HBV gene # TCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTA Pol P A A M P H L L V G S S G L S R HBsAg # L L L C L I F L L V L L D Y Q G *TALEN 33-induced breakage of targeted cccDNA sequences was examined by next-generation sequencing of targeted amplicons. Left and right TALEN targeting sequences are bolded and spacer sequences are underlined. Typically detected breaks and corresponding number of sequenced reads are indicated. Dashed lines indicate deletions and base substitutions are shown in larger font. # Open reading frames of HBV Pol and HBsAg within the TALEN 33 target sequence. Example 3: HBVTALENHBsAg reduction and gene editing in HepG2.2.15 cells

[0358] The efficacy of TALEN mRNA was tested in the HepG2.2.15 cell line constitutively expressing HBV. Briefly, TALEN mRNA was transcribed in vitro from linearized TALEN plastids. TALEN mRNA was transfected into HepG2.2.15 cells using lipofectamine reagent. After 24 hours, TALEN expression could be detected in transfected cells by immunofluorescence using TALEN protein-specific antibodies (FIG. 5A). Six days after transfection, TALEN 28 and TALEN 33 mRNA were transfected into HepG2.2.15 cells at different concentrations, and the HBsAg content in the cell culture medium was evaluated. The reduction of HBsAg content in the cell culture medium was dose-dependent (Fig. 5B). Transfection of high concentrations of mRNA had a negative impact on cell viability (Fig. 5C). Six days after mRNA transfection, HBV DNA editing activity in the same samples was assessed by next-generation sequencing. TALEN 28 and TALEN 33 gene editing activities were quantified by measuring the abundance of insertions and deletions in the spacer (Fig. 5D). As shown in Figure 5D, gene editing activity was dose-dependent and correlated with HBsAg reduction. Example 4: HBVTALENHBsAg Reduction and Gene Editing in Primary Human Hepatocytes (PHH)

[0359] Primary human hepatocytes were infected for 5 days before being transfected with TALEN 28 or TALEN 33 mRNA. After changing the medium 3 days after transfection, HBV cccDNA and cell culture medium were collected for gene editing and antiviral analysis (for TALEN 28 and TALEN 33, respectively, Figure 6A and Figure 7A ). TALEN 28 expression was detected by immunofluorescence 24 hours after transfection of different amounts of mRNA ( FIG. 6C ). A robust reduction in HBsAg and HBeAg content in cell culture medium was detected 6 days after TALEN 28 mRNA transfection compared to HBsAg and HBeAg content in non-transfected cells ( FIG. 6C ). HBV DNA cleavage activity was analyzed in HBV cccDNA extracted from the same treated and untreated cells by T7E1 digestion assay (Fig. 6D). As shown in Figure 6D, DNA corresponding to edited HBV DNA could be detected in the transfected cells. In contrast, edited DNA was absent in untransfected cells (Fig. 6D). To confirm the nature of the HBV cccDNA modification introduced by TALEN 28, HBV DNA regions were targeted by NGS sequencing. Most of the modifications detected corresponded to the deletion of the predicted FokI cleavage site (Fig. 6E), confirming the efficacy of TALEN 28 to modify HBV cccDNA.

[0360] The same analysis was performed on TALEN 33 (FIG. 7A). First, 6 hours after mRNA transfection, TALEN protein expression in PHH was confirmed by immunofluorescence staining ( FIG. 7B ). TALEN 33 efficacy in infected cells was assessed 6 days after transfection (11 days after infection with HBV genotype D). TALEN 33 mRNA transfection reduced both HBsAg and HBeAg levels in the culture medium of treated PHH cells (Fig. 7C). Following extraction of HBV cccDNA from the same PHH samples, T7 endonuclease 1 digestion analysis confirmed the presence of modified HBV DNA in cells transfected with TALEN 33 mRNA ( FIG. 7D ). Furthermore, TALEN 33 mRNA transfection was able to disrupt HBV cccDNA from PHH cells infected with HBV of genotype A, B, C or D, as analyzed by T7E1 digestion (Figure 8) and quantified by gene editing by NGS (Table 3) as shown. These results confirm the ability of TALEN mRNA transfection to reduce the secretion of HBV viral markers into the cell culture medium and modify HBV DNA of different genotypes by gene editing. Example 5: LNP-TALENs reduce HBV markers in vivo in an AAVHBV mouse model

[0361] To evaluate the therapeutic efficacy of TALEN mRNA in vivo, AAV-HBV mice were injected with different concentrations of TALEN 28 and TALEN 33 mRNA formulated in lipid nanoparticles (LNP) or PBS as a negative control. After a single dose on day 0 (D0), in mice injected with 0.5 mg / kg and 1 mg / kg, the serum HBsAg content decreased significantly from the 7th day after administration, and after a small injection of 0.25 mg / kg decreased to a lesser extent in mice (Fig. 9A). HBsAg reduction was maintained until the last time point assessed, which was Day 71 after dosing. At the highest dose (1 mg / kg) of LNP-TALEN 28 and 33, the HBsAg reduction reached 2 log HBsAg, close to the lower limit of quantitation of the CLIA system used for quantification. In parallel with the decrease in HBsAg content, HBV DNA in serum at all tested doses, that is, before the 7th day after treatment at the doses of 0.25 mg / kg, 0.5 mg / kg and 1 mg / kg, until the 71st day The content was also significantly reduced (Fig. 9B). Reductions in HBsAg and HBV DNA markers in serum were maintained until day 71 after dosing, consistent with irreversible reductions. To verify the HBV DNA modification activity of TALEN proteins in treated AAV-HBV mice, total DNA was extracted from liver tissue and HBV TALEN target sites were sequenced by NGS. NGS data analysis showed that TALEN 28 and TALEN 33 induced insertion / deletion modification in the HBV DNA target sequence, and the amount of detected modified DNA increased with the dose of LNP-TALEN (Fig. 9C). Without being bound by theory, because TALEN-induced HBV DNA modification is irreversible, the likelihood of a rebound in serum HBsAg or HBV DNA levels is reduced. Example 6: Compared with nucleoside inhibitors, the reduction of HBV serum markers in vivo after single LNP-TALEN treatment is irreversible

[0362] To compare nucleoside inhibitor treatments with LNP-TALEN treatments, an AAV-HBV mouse model was used. Select mice whose serum content is higher than 4 logarithms of HBsAg and HBV DNA, and use daily administration, continuous seven days (QD) nucleoside inhibitor entecavir (ETV, 0.01 mg / kg), single dose at 2 mg / kg Treatment was either with HBV-targeted LNP-TALEN 33 administered as an IV injection or with a non-HBV-targeted TALEN TTR administered as a single 2 mg / kg IV injection. A single injection of LNP-TALEN 33 permanently reduced plasma HBsAg ( FIG. 10A ) and HBV DNA ( FIG. 10B ) levels over 91 days. No effect on HBV serum markers was detected with non-HBV-targeting LNP-TALEN TTR treatment (Fig. 10A, 10B). ETV treatment only reduced plasma HBV DNA levels but not HBsAg levels during the treatment period (Figures 10A and 10B), and at the end of treatment (Day 7), viral rebound was seen (Figure 10B). sequence listing SEQ ID NO: name sequence 1 TALE 28 left DNA binding target sequence TGCTGCTATGCCTCATCTTC 2 TALE 28 right DNA binding target sequence CTGGACTATCAAGGTA 3 TALE 33 left DNA binding target sequence TCCTGCTGCTATGCCTCAT 4 TALE 33 right DNA binding target sequence CTTCTGGACTATCAAGGTA 5 TALE 28 DNA Binding Target Sequence TGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTA 6 TALE 33 DNA binding target sequence TCCTGCTGCTATGCCTCATCTTCTTATTGGTTCTTCTGGACTATCAAGGTA 7 TALE 28 left DNA binding sequence LTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVAIAASHD GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGG 8 TALE 28 right DNA binding sequence LTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGG GKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGG 9 TALE 33 left DNA binding sequence LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIAASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGK QALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGG 10 TALE 33 right DNA binding sequence LTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGG GKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIAASNIGGKQALETV QRLLPVLCQAHGLTPEQVVAIAASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGG 11 FokI nuclease QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLI GGEMIKAGTLTLEEVRRKFNNGEINFRS 12 TEV 5'UTR UCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUUGCAGCAAUUUAAAUCAUUUCUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAG 13 AT1G58420 5'UTR AUUAUUACAUCAAAACAAAAAGCCGCCA 14 ARC5-2 5'UTR CUUAAGGGGGCGCUGCCUACGGAGGUGGCAGCCAUCCCUUCUCGGCAUCAAGCUUACCAUGGUGCCCCAGGCCCUGCUCUUGGUCCCGCUGCUGGUGUUCCCCCCUCUGCUUCGGCAAGUUCCCCAUCUACACCAUCCCCCGACAAGCUGGGGCCGUGGAGCCCCAUCGACAUCCACCACCUGUCCUGCCCCAACAA CCUCGUGGUCGAGGACGAGGGCUGCACCAACCUGAGCGGGUUCUCCUAC 15 HCV 5'UTR UGAGUGUCGUACAGCCUCCAGGCCCCCCCCUCCCGGGAGAGCCAUAGUGGUCUGCGGAACCGGUGAGUACACCGGAAUUGCCGGGAAGACUGGGUCCUUUCUUGGAUAAACCCACUCUAUGCCCGGCCAUUUGGGCGUGCCCCCGCAAGACUGCUAGCCGAGUAGUGUUGGGUUGCG 16 human albumin 5'UTR AAUUAUUGGUUAAAGAAGUAUAUUAGUGCUAAUUUCCCUCCGUUUGUCCUAGCUUUUCUCUUCUGUCAACCCCCACACGCCUUUGGCACA 17 EMCV 5'UTR CUCCCUCCCCCCCCCUAACGUUACUGGCCGAAGCCGCUUGGAAUAAGGCCGGUGUGCGUUUGUCUAUAUGUUAUUUUCCACCAUAUUGCCGUCUUUUGGCAAUGUGAGGGCCCGGAAACCUGGCCCUGUCUUCUUGACGAGCAUUCCUAGGGGUCUUUCCCCUCUCGCCAAAGGAAUGCAAGG UCUGUUGAAUGUCGUGAAGGAAGCAGUUCCUCUGGAAGCUUCUUGAAGACAACAACGUCUGUAGCGACCCUUUGCAGGCAGCGGAACCCCCACCUGGCGACAGGUGCCUCUGCGGCCAAAAGCCACGUGUAUAAGAUACACCUGCAAAGGCGGCACACCCCAGUGCCACGUUGUGAGUUGGAUAGUUGUGGA AAGAGUCAAAUGGCUCUCCUCAAGCGUAUUCAACAAGGGGCUGAAGGAUGCCCAGAAGGUACCCCAUUGUAUGGGAUCUGAUCUGGGGCCUCGGUGCACAUGCUUUACGUGUGUUUAGUCGAGGUUAAAAAACGUCUAGGCCCCCCGAACCACGGGGACGUGGUUUUUCCUUUGAAAAACACGAU GAUAAU 18 XBG 3'UTR CUAGUGACUGACUAGGAUCUGGUUACCACUAAACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAU 19 human binding globulin 3'UTR UGCAAGGCUGGCCGGAAGCCCUUGCCUGAAAGCAAGAUUUCAGCCUGGAAGAGGGCAAAGUGGACGGGAGUGGACAGGAGUGGAUGCGAUAAGAUGUGGUUUGAAGCUGAUGGGUGCCAGCCCUGCAUUGCUGAGUCAAUCAAUAAAGAGCUUUCUUUUGACCCAU 20 human lipoprotein E 3'UTR ACGCCGAAGCCUGCAGCCAUGCGACCCCACGCCACCCCGUGCCUCCUGCCUCCGCGCAGCCUGCAGCGGGAGACCCUGUCCCCGCCCCAGCCGUCCUCCUGGGGUGGACCCUAGUUUAAUAAAGAUUCACCAAGUUUCACGCA twenty one HCV 3'UTR UAGAGCGGCAAAACCCUAGCUACACUCCAUAGCUAGUUUCUUUUUUUUUGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUCCUUUCUUUUCCUUCUUUUUUUCCUCUUUUCUUGGUGGCUCCAUCUUAGCCCUAGUCACGGCUAGCUGUGAAAG GUCCGUGAGCCGCAUGACUGCAGAGAGUGCCGUAACUGGUCUCUCUGCAGAUCAUGU twenty two mouse albumin 3'UTR ACACAUCACAACCACAACCUUCUCAGGCUACCCUGAGAAAAAAAAGACAUGAAGACUCAGGACUCAUCUUUUCUGUUGGUGUAAAAUCAACACCCUAAGGAACACAAAUUUCUUUAAACAUUUGACUUCUUGUCUCUGUGCUGCAAUUAAUAAAAAAAUGGAAAGAAUCUAC twenty three human alpha globulin 3'UTR GCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGAAUAAAGUCUGAGUGGGCAGCA twenty four EMCV 3'UTR UAGUGCAGUCACUGGCACAACGCGUUGCCCGGUAAGCCAAUCGGGUAUACACGGUCGUCAUACUGCAGACAGGGUUCUUCUACUUUGCAAGAUAGUCUAGAGUAGUAAAAUAAAUAGUAUAAG 25 TALEN 28 left protein MDYKDGDGDYKDIDIDDDDDDDDDDDKGSGSGSGSGSGKKRKRKVGIHGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGFTHAHPAALGTVAVMIAIVGVGKQWSGAR AleallTVAGELRGPLQLDTGQLLLLKIAKRGGVTAVEAWRNAWRNAPLNLTPEQVVAIASNNGGKQRLLCQAHGLTPEQVVALETVQAHGLCQAHGLTPEQVVVVVVV AIASNGGGKQALETPVLCQAHGLTPEQVVAIASNNNGGKQRLLCQAHGLTPEQVVAIASHDGGKQALALLLCQAHGLCQAHGLCQAHGKQALETVQRLLCQA HgltpeqvaiasSiggkqaletvqrllpvLCQAHGLTPEQVVVAIASNGGGKQALETVLCQAHGLTPEQVVALETVQAHGLCQAHGKKQGQGQGQGQGQGQGQGQGQGQGQGQGQGKQKQALETVQ RllpvlcqahgltqvaiashdggkqaletvqrllpvlcqahgltpeqvaiasngggkqrllpvLCQAHGLTPEQVVALETVLCQAHGLTPEQLTPEQVAIASNI GGKQALETVQRLLPLCQAHGLTPEQVVAIASNGGGKQALLLPVLCQAHGLTPEQVVAIASHDGGKQALLLPVLCQAHGLTVVVQRCQAHGLCQAHGLTPE QVVAIASNGGGKQRLLLPVLCQAHGLTPEQVVVAIASHDGRPAQLSRPALAALACLALALPAPAPAPAPAPALIPAGSHRVAGSQLVKSELEEKS ELRHKLKYVPHEYIEIARNSTQDRILEMKVMEFFMKVYRGKHLGGSRKPDGSPIDYGVIVDTKAYNLPIGQADEMQRNQTRNKHIPSSVTEFKFLFLFLFLFLFLFLFLFLFLFLFLFLFLFLFLFLFLFKFS GNYKAQLTRLNHITNCNGAVLSVELLIGGEMIKAGTLEEVRRRKFNNGEINFRS 26 TALEN 28 right protein MDYKDHDGDYKDHDIDYKDDDDKGGSGPKKKRKVGIHGVPAAVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQ VVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN NGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALE TVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTN RRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHIT NCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS 27 TALEN 33 left protein MDYKDHDGDYKDHDIDYKDDDDKGGSGPKKKRKVGIHGVPAAVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQ VVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYR GKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS 28 TALEN 33 right protein MDYKDHDGDYKDHDIDYKDDDDKGGSGPKKKRKVGIHGVPAAVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQ VVAIAASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN NGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALE TVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLC QAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGG SRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS 29 Forward Primer CCTAGGACCCCTTCTCGTGT 30 reverse primer ACTGAGCCAGGAGAAACGGG 31 SV40 large T antigen NLS PKKKRKV 32 NLS KRPAATKKAGQAKKKK 33 EGL-13 NLS MSRRRKANPTKLSENAKKLAKEVEN 34 c-Myc proto-oncoprotein NLS PAAKRVKLD 35 TUS protein NLS KLKIKRPVK 36 FLAG tag DYKDDDDK 37 FLAG tag EYKEEEEK 38 Six × Histidine HHHHHH 39 myc tag EQKLISEEDL 40 Influenza HA label YPYDVPDYA 41 AcV5 label SWKDASGWS 42 ALFA-label SRLEEELRRLTE 43 Avi Tags GLNDIFEAQKIEWHE 44 E-label GAPVPYPDPLEPR 45 S-tag KETAAAKFERQHMDS 46 Strep-label WSHPQFEK 47 T7 label MASMTGGQQMG 48 Ty1 label EVHTNQDPLD 49 V5 label GKPIPNPLLGLDST 50 VSV-G label YTDIEMNRLGK 51 XPress label DLYDDDDK 52 TALE 28 has an N-terminal left DNA-binding sequence VDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLT PEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIAASHDGG 53 TALE 28 has an N-terminal right DNA binding sequence VDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA HGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGG 54 TALE 33 has an N-terminal left DNA-binding sequence VDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGK QALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLT PEQVVAIASNGGG 55 TALE 33 has an N-terminal right DNA binding sequence VDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA HGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVA IAS NNGG

[0363] It will be readily apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0364] All patents and publications mentioned in this specification are indicative of the level of skill in the art to which this invention pertains.

[0365] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced by either of the other two terms replacement. The terms and expressions which have been employed are terms of description rather than limitation and in the use of such terms and expressions there is no intention to exclude any equivalents to the features shown and described or parts thereof, but it is recognized that in the present invention Various modifications are possible within the scope of the claims. Additionally, where features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is thereby also described in terms of any individual member of the Markush group. Or member subgroups are described. For example, if X is described as being selected from the group consisting of bromine, chlorine, and iodine, fully describe the claims that X is bromine and the claims that X is bromine and chlorine. Other embodiments are within the scope of the following claims. [Simple description of the diagram]

[0041] When reading in conjunction with the accompanying drawings, the foregoing summary of the invention and the following specific embodiments will be better understood. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.

[0042] The patent or application file contains at least one color drawing. Copies of this Patent or Patent Application Publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0043] Figures 1A-1C show the TALEN 28 and 33 target sites in the HBV DNA gene. Figure 1A: Schematic representation of overlapping open reading frames of the HBV genome and viral proteins (pre-core / core (preC / Core), pol, pre-S1, pre-S2, HBsAg and HBx). TALEN 28 and 33 target a site within the gene encoding HBsAg / pol. Figure 1B: HBV DNA sequences targeted by the left and right TALEN arms of TALEN28 (top, SEQ ID NO: 5) and TALEN33 (bottom, SEQ ID NO: 6). Figure 1C: Schematic representation of TALEN mRNA constructs. The HBV DNA binding domains are located within an array of 15-19 TALE repeats, each of which contains two amino acids called repeat variable diresidues (RVDs) designed to target HBV sequences.

[0044] Figures 2A-2B show the conservation analysis of the HBV TALEN 28 target sequence. Conservation signatures were generated based on the abundance of each targeted nucleotide in the left (FIG. 2A) and right (FIG. 2B) arms of TALEN 28. The higher the conservation, the larger the size of the abundant nucleotide(s).

[0045] Figures 3A-3B show HBV TALEN 33 target sequence conservation analysis. Conservation signatures were generated based on the abundance of each targeted nucleotide in the left (Figure 3A) and right (Figure 3B) arms of TALEN 33. The higher the conservation, the larger the size of the abundant nucleotide(s).

[0046] Figures 4A-4C show TALEN 28 and TALEN 33 targeting broad HBV genotype coverage. The left TALEN of each of TALEN 28 ( FIG. 4A ) and TALEN 33 ( FIG. 4B ) targeted a highly conserved sequence with a single mismatch within the right TALEN arm observed in genotype (GT) A and GT C. Figure 4C shows the results of DNA cleavage of in vitro translated TALEN proteins against HBV DNA isolates of GT A to H.

[0047] Figures 5A-5D show the effects of TALEN 28 and TALEN 33 in HepG2.2.15 cells. Figure 5A: TALEN plasmids were transcribed in vitro and transfected into cells. 24 hours after transfection, nuclei were stained with DAPI, and TALEN expression in the nuclei was confirmed by immunofluorescence. After 6 days, the cells were evaluated for the effect of TALEN activity on HBsAg secreted protein content in cell culture medium ( FIG. 5B ), cell viability ( FIG. 5C ) and HBV DNA gene editing ( FIG. 5D ).

[0048] Figures 6A-6E show the effect of TALEN 28 in primary human hepatocytes (PHH) infected with HBV. Figure 6A: Schematic illustration of HBV infection and TALEN mRNA transfection using PHH. PHH were treated with or without TALEN 28 mRNA pair on day 5 after HBV infection. Figure 6B: TALEN 28 expression was detected by immunofluorescence (red) and nuclei were stained by using DAPI (blue) 6 hours after transfection. Figure 6C: Antiviral effect of TALEN 28-treated cells against HBsAg and HBeAg production. On the 11th day after infection, the contents of HBsAg and HBeAg were measured. Figure 6D: TALEN-induced disruption of targeted cccDNA sequences was examined by T7E1 digestion assay 6 days after TALEN treatment. Asterisks indicate rupture sequences. Figure 6E: Gene-edited cccDNA shows a majority of deletions within the TALEN 28 target sequence. Sequences were analyzed by next-generation sequencing.

[0049] Figures 7A-7D show the role of TALEN 33 in HBV-infected PHH. Figure 7A: Schematic illustration of HBV infection and TALEN mRNA transfection using PHH. On day 5 after HBV infection, PHH were treated with or without TALEN 33 mRNA pair. Figure 7B: TALEN 33 expression was detected by immunofluorescence (green) and nuclei were stained by using DAPI (blue) 6 hours after transfection. Figure 7C: Antiviral effect of TALEN 33-treated cells against HBsAg and HBeAg production. On day 11 post-infection, HBsAg and HBeAg levels in the presence or absence of TALEN 33 were determined. Data were calculated from at least 3 independent studies and accompanying error bars represent means. Figure 7D: TALEN-induced disruption of targeted cccDNA sequences examined by T7E1 digestion assay. Asterisks indicate rupture sequences.

[0050] Figure 8 shows that TALEN 33 edits cccDNA from PHH infected with HBV GT A to D. T7E1 analysis was performed on cccDNA extracted from HBV-infected PHHs treated with mRNA pairs encoding TALEN 33 (+) or not treated with mRNA pairs encoding TALEN 33 (-). Asterisks indicate rupture sequences.

[0051] FIG. 9A-FIG. 9C show that the TALEN 33 mRNA pair (LNP-HBV TALEN) encapsulated into lipid nanoparticles reduces serum HBsAg (FIG. 9A) and serum HBV DNA (FIG. 9B), and shows infection with AAV - Dose-dependent gene editing activity in mice of HBV (Fig. 9C).

[0052] FIGS. 10A-10B show the comparison results of LNP-HBV TALEN and HBV therapeutic agent entecavir (ETV) in mice infected with AAV-HBV. Sustained reductions in plasma HBsAg ( FIG. 10A ) and HBV DNA ( FIG. 10B ) were observed over 91 days with a single dose of LNP-HBV TALENs but not with non-HBV-targeting TTR TALENs. ETV administered QD for 7 days reduced plasma HBV DNA, but viral rebound was observed after treatment cessation.

Sequence Listing

[0366] <![CDATA[<110> Janssen Pharmaceuticals, Inc.]]> Arcturus Therapeutics, Inc. <![CDATA[<120 > Targets transcriptional activator effector nucleases like HBV (TALEN)]]> <![CDATA[<130> 065814.11515 / 45WO1]]> <![CDATA[<140> TW110149158]]> <![CDATA[ <141> 2021-12-28]]> <![CDATA[<150> 63 / 131,145]]> <![CDATA[<151> 2020-12-28]]> <![CDATA[<160> 55 ]]> <![CDATA[<170> PatentIn version 3.5]]> <![CDATA[<210> 1]]> <![CDATA[<211> 20]]> <![CDATA[<212> DNA ]]> <![CDATA[<213> artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> TALE 28 left DNA binding target]]> <![CDATA[< 400> 1]]> tgctgctatg cctcatcttc 20 <![CDATA[<210> 2]]> <![CDATA[<211> 16]]> <![CDATA[<212> DNA]]> <![CDATA[ <213> artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> TALE 28 right DNA binding target]]> <![CDATA[<400> 2]]> ctggactatc aaggta 16 <![CDATA[<210> 3]]> <![CDATA[<211> 19]]> <![CDATA[<212> DNA]]> <![CDATA[<213> artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> TALE 33 left DNA binding target]]> <![CDATA[<400> 3]]> tcctgctgct atgcctcat 19 <![CDATA[<210 > 4]]> <![CDATA[<211> 19]]> <![CDATA[<212> DNA]]> <![CDATA[<213> artificial sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> TALE 33 right DNA binding target]]> <![CDATA[<400> 4]]> cttctggact atcaaggta 19 <![CDATA[<210> 5]]> <![ CDATA[<211> 48]]> <![CDATA[<212> DNA]]> <![CDATA[<213> artificial sequence]]> <![CDATA[<220>]]> <![CDATA[ <223> TALE 28 DNA binding target]]> <![CDATA[<400> 5]]> tgctgctatg cctcatcttc ttgttggttc ttctggacta tcaaggta 48 <![CDATA[<210> 6]]> <![CDATA[<211> 51 ]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> TALE 33 DNA Combine target]]> <![CDATA[<400> 6]]> tcctgctgct atgcctcatc ttcttattgg ttcttctgga ctatcaaggt a 51 <![CDATA[<210> 7]]> <![CDATA[<211> 627]]> <! [CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> TALE 28 Left DNA Binding Sequence]] > <![CDATA[<400> 7]]> Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys 1 5 10 15 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30 His Gly Leu Thr Pro Glu Gln Val Ala Ile Ala Ser His Asp Gly 35 40 45 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 50 55 60 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn 65 70 75 80 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 85 90 95 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Ala Ile Ala 100 105 110 Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 115 120 125 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 130 135 140 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 145 150 155 160 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 165 170 175 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val 180 185 190 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 195 200 205 Gln Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu 210 215 220 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 225 230 235 240 Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala 245 250 255 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 260 265 270 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys 275 280 285 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 290 295 300 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly 305 310 315 320 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 325 330 335 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His 340 345 350 Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 355 360 365 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 370 375 380 Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 385 390 395 400 Pro Val Leu Cys Gln A la His Gly Leu Thr Pro Glu Gln Val Val Ala 405 410 415 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 420 425 430 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 435 440 445 Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val 450 455 460 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 465 470 475 480 Gln Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu 485 490 495 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 500 505 510 Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 515 520 525 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 530 535 540 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys 545 550 555 560 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 565 570 575 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly 580 585590 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 595 600 605 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His 610 615 620 Asp Gly Gly 625 <![CDATA[<210 > 8]]> <![CDATA[<211> 491]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> TALE 28 Right DNA Binding Sequence]]> <![CDATA[<400> 8]]> Leu Thr Pro Glu Gln Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 1 5 10 15 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly 35 40 45 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 50 55 60 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His 65 70 75 80 Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 85 90 95 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Ala Ile Ala 100 105 110 Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 115 120 125 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 130 135 140 Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 145 150 155 160 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 165 170 175 Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val 180 185 190 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 195 200 205 Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu 210 215 220 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 225 230 235 240 Pro Glu Gln Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala 245 250 255 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 260 265 270 LEU Thr Pro Gln Val Val Ala Ile Ala Ser asn Ile GLY GLY LYS 275 280 Gln Ala Leu ThR Val Gln ARG Leu Pro Val Leu Cys GLN ALA 290 295 300 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly 305 310 315 320 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 325 330 335 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser ASN 340 345 350 Gly GLY GLY LYS GLN ALA Leu Gl Val Gln ARG Leu Pro Val 355 360 Leu Cys Gln Ala His Glu Gln Val Val Val Val Ala 370 37 37 37 5 380 Ser His asp Gly GLY LYS GLN Ala Leu Glu Thr Val Gln Arg Leu Leu 385 390 395 400 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 405 410 415 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 420 425 430 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 435 440 445 Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val 450 455 460 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 465 470 475 480 Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly 485 490 <![CDATA[<210> 9]]> <![CDATA[<211> 593]]> <![CDATA [<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> TALE 33 Left DNA Binding Sequence]]> < ![CDATA[<400> 9]]> Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys 1 5 10 15 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly 35 40 45 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 50 55 60 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn 65 70 75 80 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 85 90 95 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Ala Ile Ala 100 105 110 Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 115 120 125 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 130 135 140 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 145 150 155 160 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 165 175 Val Ala Ile Ala Sern Gly GLY LYS GLN ALA Leu ThR Val 180 185 190 GL n aRG Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 195 200 205 Gln Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu 210 215 220 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 225 230 235 240 Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 245 250 255 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 260 265 270 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys 275 280 285 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 290 295 300 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly 305 310 315 320 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 325 330 335 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn 340 345 350Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 355 360 365 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 370 375 380 Ser Asn Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val GLN ARG Leu Leu 390 395 400 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Val Val Val Ala 405 415 ILE ALA His ASP GLY LYS GLN Ala THR Val Gln ARG 420 425 430 LEU Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 435 440 445 Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val 450 455 460 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 465 470 475 480 Gln Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu 485 490 495 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 500 505 510 Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 515 520 525 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 530 535 540 Leu Thr Pro Glu Gln Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 545 550 555 56 0 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 565 570 575 His Gly Leu Thr Pro Glu Gln Val Ala Ile Ala Ser Asn Gly Gly 580 585 590 Gly <![CDATA[<210> 10]] > <![CDATA[<211> 593]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> < ![CDATA[<223> TALE 33 right DNA binding sequence]]> <![CDATA[<400> 10]]> Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 1 5 10 15 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly 35 40 45 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 50 55 60 Gln Ala His Gly Leu Thr Pro Glu Gln Val Ala Ile Ala Ser His 65 70 75 80 Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 85 90 95 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 100 105 110 Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 115 120 125 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 130 135 140 Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 145 150 155 160 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 165 170 175 Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val 180 185 190 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 195 200 205 Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu 210 215 220 Thr Val Gl no Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 225 230 235 240 Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala 245 250 255 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 260 265 270 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 275 280 285 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 290 295 300 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly 305 310 315 320 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 325 330 335 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn 340 34 5 350 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 355 360 365 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 370 375 380 Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln ARG Leu Leu 385 390 395 400 Pro Val Leu Cys Gln Ala His GLY Leu Glu Gln Val Val Val Ala 405 415 ILE Ala Ser His GLN Ala Leu Glu ThR Val. GLN ARG 420 425 430 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 435 440 445 Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val 450 455 460 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 465 470 475 480 Gln Val Ala Ile Ala Ser Asn Asn Asn Gly Gly Lys Gln Ala Leu Glu 485 490 495 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 500 505 510 Pro Glu Gln Val Val A la ile ALA Sern Ile Gly GLY GLN GLN ALA 515 520 525 Leu ThR Val Gln ARG Leu Prou ​​Val Leu Cys Gln Ala His GLI 530 540 Leu ThR PRU Glu Gln Val Val Ala Ile Ala Sera Sera Sn Ile Gly Gly Lys 545 550 555 560 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 565 570 575 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly 580 585 590 Gly <![CDATA[<210> 11] ]> <![CDATA[<211> 198]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[&l...

Claims

1. Use of an assembly of mRNA molecules for preparing a drug for treating hepatitis B virus (HBV) infection in an individual, the assembly of mRNA molecules comprising: (1) a first mRNA molecule comprising a polynucleotide sequence encoding a type I transcription activator effector nuclease (TALEN) monomer, the first TALEN monomer comprising a first TALE DNA-binding domain and a first nuclease catalytic domain, wherein the first TALE DNA-binding domain is capable of binding to a first half-site sequence of a target nucleic acid sequence within an HBV gene; and (2) a second mRNA molecule comprising a polynucleotide sequence encoding a second TALEN monomer, the second TALEN monomer comprising a second TALE DNA-binding domain and a second nuclease catalytic domain, wherein the second TALE DNA-binding domain is capable of binding to a second half-site sequence of the target nucleic acid sequence; wherein when the first TALE DNA-binding domain binds to the first half-site sequence and the second TALE DNA-binding domain binds to the second half-site sequence, the first TALEN monomer and the second TALEN monomer are capable of forming a dimer that cleaves the target nucleic acid sequence; Wherein, the first nuclease catalytic domain is a first FokI nuclease catalytic domain and the second nuclease catalytic domain is a second FokI nuclease catalytic domain; and wherein: (a) The first half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 1, and the second half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 2; or (b) The first half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 3, and the second half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 4; or (c) The target nucleic acid sequence contains the sequence of SEQ ID NO: 5 or SEQ ID NO:

6.

2. As claimed in claim 1, wherein the first mRNA molecule and the second mRNA molecule each further comprise one or more of a 5' cap, a 5'-UTR, a sequence encoding a nuclear localization signal, a sequence encoding an N-terminal domain, a sequence encoding a C-terminal domain, a 3'-UTR, and a polyadenylated tail.

3. As claimed in claim 1, wherein, in each case, the first TALE DNA binding domain comprises an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, the second TALE DNA binding domain comprises an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprise an amino acid sequence of SEQ ID NO:

11.

4. As requested in item 1, wherein the HBV gene system comprises one or more of the following genotypes: A, B, C, D, E, F, G, H, I, and J.

5. The use as claimed in claim 1, wherein the use includes use in combination with a second therapeutic composition comprising an anti-HBV agent.

6. For any of the uses of requests 1 to 5, wherein the HBV infection is a chronic HBV infection.

7. As requested in item 6, wherein the HBV infection comprises dual infection with HBV and HDV.

8. A composition comprising an assembly of mRNA molecules encapsulated in lipid nanoparticles, comprising: (1) a first mRNA molecule comprising a polynucleotide sequence encoding a first class of transcription activator effector nuclease (TALEN) monomer, the first TALEN monomer comprising a first TALE DNA-binding domain and a first nuclease catalytic domain, wherein the first TALE DNA-binding domain is capable of binding to a first half-site sequence of a target nucleic acid sequence within an HBV gene; and (2) a second mRNA molecule comprising a polynucleotide sequence encoding a second TALEN monomer, the second TALEN monomer comprising a second TALE DNA-binding domain and a second nuclease catalytic domain, wherein the second TALE DNA-binding domain is capable of binding to a second half-site sequence of the target nucleic acid sequence; wherein when the first TALE DNA-binding domain binds to the first half-site sequence and the second TALE DNA-binding domain binds to the second half-site sequence, the first TALEN monomer and the second TALEN monomer are capable of forming a dimer that cleaves the target nucleic acid sequence; Wherein, the first nuclease catalytic domain is a first FokI nuclease catalytic domain and the second nuclease catalytic domain is a second FokI nuclease catalytic domain; and wherein: (a) The first half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 1, and the second half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 2; or (b) The first half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 3, and the second half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 4; or (c) The target nucleic acid sequence contains the sequence of SEQ ID NO: 5 or SEQ ID NO:

6.

9. The composition of claim 8, wherein the first mRNA molecule and the second mRNA molecule each further comprise one or more of a 5' cap, a 5'-UTR, a sequence encoding a nuclear localization signal, a sequence encoding an N-terminal domain, a sequence encoding a C-terminal domain, a 3'-UTR, and a polyadenylated tail.

10. The composition of claim 8, wherein, in each case, the first TALE DNA binding domain comprises an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, the second TALE DNA binding domain comprises an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10, and the first FokI nuclease catalytic domain and the second FokI nuclease catalytic domain each comprise an amino acid sequence of SEQ ID NO:

11.

11. The composition of claim 8, wherein the HBV gene system comprises one or more of genotypes A, B, C, D, E, F, G, H, I and J.

12. The composition of claim 8, wherein the lipid nanoparticles encapsulating the mRNA molecule combination comprise cationic lipids and at least one other lipid selected from the group consisting of anionic lipids, zwitterionic lipids, neutral lipids, steroids, polymer-bound lipids, phospholipids, glycolipids, and combinations thereof.

13. A combination of the following: (1) a first mRNA molecule comprising a polynucleotide sequence encoding a monomer of a class I transcription activator effector nuclease (TALEN), the first TALEN monomer comprising a first TALE DNA-binding domain and a first FokI nuclease catalytic domain, wherein the first TALE DNA-binding domain is capable of binding to a first half-site sequence of a target nucleic acid sequence within an HBV gene, and the first half-site sequence comprises the sequence of SEQ ID NO: 1; and (2) a second mRNA molecule comprising a polynucleotide sequence encoding a monomer of a class II transcription activator effector nuclease (TALEN), the second TALEN monomer comprising a second TALE DNA-binding domain and a second FokI nuclease catalytic domain, wherein the second TALE DNA-binding domain is capable of binding to a second half-site sequence of the target nucleic acid sequence, and the second half-site sequence comprises the sequence of SEQ ID NO: 2; wherein when the first TALE DNA-binding domain binds to the first half-site sequence and the second TALE DNA-binding domain binds to the target nucleic acid sequence, ... binds to the target nucleic acid sequence, the second TALE DNA-binding domain binds to the target nucleic acid sequence, the second TALE DNA-binding domain binds to the target nucleic acid sequence, the second TALE DNA-binding domain binds to the target nucleic acid sequence, the second TALE DNA-binding domain binds to the target nucleic acid sequence, the second TALE DNA-binding domain binds to the target nucleic acid sequence, the second TALE DNA-binding domain binds to the target nucleic acid sequence, the second TALE DNA When the DNA-binding domain binds to the second half-site sequence, the first TALEN monomer and the second TALEN monomer can form a dimer that cleaves the target nucleic acid sequence.

14. A combination of claims 13, wherein the HBV gene system comprises one or more of genotypes A, B, C, D, E, F, G, H, I, and J.

15. A composition comprising the combination of claim 13, wherein the first nucleic acid and the second nucleic acid are separately or jointly encapsulated in lipid nanoparticles.

16. A nucleic acid molecule encoding at least one of a first mRNA molecule and a second mRNA molecule as described in claim 13.

17. An isolated host cell comprising nucleic acid molecules as claimed in claim 16.

18. The isolated host cells as claimed in claim 17, wherein the host cells are hepatocytes.

19. A pharmaceutical composition comprising a composition of any one of claims 8 to 12 or 15, a combination of claims 13 or 14, a nucleic acid molecule of claim 16, or an isolated host cell of claim 17, and a pharmaceutically acceptable carrier.

20. A method for producing TALEN, comprising transcribing a nucleic acid molecule as claimed in claim 16 in vitro.

21. As claimed in any of claims 1 to 5, wherein the first and second mRNA molecules are encapsulated in lipid nanoparticles.

22. As claimed in claim 21, wherein the lipid nanoparticles encapsulating the mRNA molecule comprise cationic lipids and at least one other lipid selected from the group consisting of anionic lipids, zwitterionic lipids, neutral lipids, steroids, polymer-bound lipids, phospholipids, glycolipids, and combinations thereof.

23. Use of a composition for preparing a medicament for treating hepatitis B virus (HBV) infection in an individual, the composition comprising: a first mRNA molecule comprising a polynucleotide sequence encoding a type-1 transcription activator effector nuclease (TALEN) monomer, the first TALEN monomer comprising a first TALE DNA-binding domain and a first nuclease catalytic domain, wherein the first TALE DNA-binding domain is capable of binding to a first half-site sequence of a target nucleic acid sequence within an HBV genome, wherein the composition is administered in combination with a second mRNA molecule comprising a polynucleotide sequence encoding a second TALEN monomer, the second TALEN monomer comprising a second TALE DNA-binding domain and a second nuclease catalytic domain, wherein the second TALE DNA-binding domain is capable of binding to a second half-site sequence of the target nucleic acid sequence, wherein when the first TALE DNA-binding domain binds to the first half-site sequence and the second TALE DNA-binding domain binds to the second half-site sequence, the first TALEN monomer and the second TALEN monomer are capable of forming a dimer that cleaves the target nucleic acid sequence; Wherein, the first nuclease catalytic domain is a first FokI nuclease catalytic domain and the second nuclease catalytic domain is a second FokI nuclease catalytic domain; and wherein: (a) The first half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 1, and the second half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 2; or (b) The first half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 3, and the second half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 4; or (c) The target nucleic acid sequence contains the sequence of SEQ ID NO: 5 or SEQ ID NO:

6.

24. Use of a composition for preparing a medicament for treating hepatitis B virus (HBV) infection in an individual, the composition comprising: a second mRNA molecule comprising a polynucleotide sequence encoding a type II transcription activator effector nuclease (TALEN) monomer, the second TALEN monomer comprising a second TALE DNA-binding domain and a second nuclease catalytic domain, wherein the second TALE DNA-binding domain is capable of binding to a second half-site sequence of a target nucleic acid sequence, wherein the composition is administered in combination with a first mRNA molecule comprising a polynucleotide sequence encoding a type I transcription activator effector nuclease (TALEN) monomer, the first TALEN monomer comprising a first TALE DNA-binding domain and a first nuclease catalytic domain, wherein the first TALE DNA-binding domain is capable of binding to a first half-site sequence of the target nucleic acid sequence within an HBV genome, wherein when the first TALE DNA-binding domain binds to the first half-site sequence and the second TALE DNA-binding domain binds to the second half-site sequence, the first TALEN monomer and the second TALEN monomer are capable of forming a dimer that cleaves the target nucleic acid sequence; Wherein, the first nuclease catalytic domain is a first FokI nuclease catalytic domain and the second nuclease catalytic domain is a second FokI nuclease catalytic domain; and wherein: (a) The first half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 1, and the second half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 2; or (b) The first half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 3, and the second half-site sequence of the target nucleic acid sequence contains the sequence of SEQ ID NO: 4; or (c) The target nucleic acid sequence contains the sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

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