Engineered meganucleases specific for recognition sequences in PCSK9 gene

Abstract

To provide a meganuclease engineered to recognize a specific sequence within the human PCSK9 gene.SOLUTION: The present invention encompasses engineered meganucleases which recognize and cleave a recognition sequence within a human PCSK9 gene. The present invention also encompasses methods for using such engineered meganucleases in a pharmaceutical composition and in methods for treating or reducing symptoms of cholesterol-related disorders, such as hypercholesterolemia. Further, the invention encompasses pharmaceutical compositions comprising engineered meganuclease proteins, nucleic acids encoding engineered meganucleases, and use of such compositions for treating cholesterol-related disorders, such as hypercholesterolemia.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the fields of molecular biology and recombinant nucleic acid technology. In particular, the present invention relates to a method for the treatment of PCSK The present invention relates to engineered meganucleases that have specificity for recognition sequences within genes. Engineered meganucleases such as these have been shown to be effective in treating cardiovascular disease and autosomal dominant familial hypercholesterolemia. The present invention is useful in methods for treating hypercholesterolemia, including hypercholesterolemia.

[0002] A reference to a sequence listing submitted as a text file via EFS-WEB This application was submitted in ASCII format via EFS-WEB and is hereby incorporated by reference. The entire text of this document includes the sequence listing, which is incorporated herein by reference. The ASCII copy is named P109070022WO00-SEQTXT-MJT and is 89,660 bytes in size. [Background technology]

[0003] Cardiovascular disease associated with high levels of low-density lipoprotein cholesterol (LDL-C) is the leading cause of death in developed countries, and elevated LDL-C levels are associated with coronary heart disease (CHD). and is a major risk factor for the development of atherosclerotic plaques. H) is associated with high cholesterol levels, especially high levels of LDL-C and early-onset CHD. Heterozygous FH is a genetic disease characterized by the onset of hypercalcaemia. Heterozygous FH patients typically have are treated with lipid-lowering drugs such as statins, which reduce cholesterol levels, specifically LDL-C levels. Indeed, statin treatment has been shown to greatly reduce the risk of cardiovascular disease in certain patient populations. However, some patient populations, particularly those with homozygous FH, may be unable to tolerate high-dose statin-containing treatments. In response, normal LDL-C levels could not be achieved.

[0004] An important advance in the lipid-lowering field is the proprotein convertase subtilisin This was an advance in the treatment of PCSK9 targeting human PCSK9 genes. The gene is located on chromosome 1p32.3 and is 25,378 bp in length. The gene contains 12 exons that code for 692 amino acids. The protein contains a signal peptide, a prodomain, a catalytic domain, and three modules (M1, It contains a C-terminal cysteine-histidine-rich domain consisting of the M domains M1, M2, and M3.

[0005] PCSK9 is synthesized, secreted, and expressed primarily in the liver, but PCSK9 is also expressed in the gastrointestinal tract, kidney, and PCSK9 is expressed at low levels in the central nervous system (CNS) and in the pulmonary artery. Its molecular weight is 75 kDa. After autocatalytic cleavage in the endoplasmic reticulum (ER), the PCSK9 prodomain The prodomain is isolated from the 62 kDa mature PCSK9 protein. It remains non-covalently bound to the mature PCSK9 protein and is involved in the catalytic domain of PCSK9. The PCSK9 complex then forms a prosegment that forces the prosegment into an inactive conformation. The cleaved complex is transported from the ER to the Golgi apparatus and released. Secreted PCSK9 binds to LDL receptors on the hepatic cell membrane. This binding is called PCSK9 The catalytic domain and prodomain of LDL receptor are homologous to the epidermal growth factor-like repeat domain of the LDL receptor. This is mediated by interactions with the main A (EGF-A) and β-propeller domains. do.

[0006] LDL receptors typically bind fatty molecules in the extracellular fluid (including cholesterol) to In the absence of PCSK9, LDL The receptor-ligand complex mediates intracellular transport of LDL and recycling of the LDL receptor to the cell surface. However, PCSK9 binding to the LDL receptor is not possible without a complex This prevents this conformational change in the receptor and guides it to the lysosome for degradation. Fewer LDL receptors are present on the cell surface, and circulating LDL cholesterol levels in the bloodstream In addition to the LDL receptor, PCSK9 also acts on the very low density lipoprotein (VLDL) receptor. ) receptor, apolipoprotein E receptor 2, and LDL receptor-related protein 1. It has been shown to mediate the degradation of other lipid receptors in the mycobacterium.

[0007] PCSK9, along with the genes encoding the LDL receptor and apolipoprotein B, mediates the PCSK9 is the third gene associated with autosomal dominant forms of FH. In autosomal dominant FH, PCSK9 Gain-of-function mutations in the gene further reduce the localization of LDL receptors on the cell surface and inhibit LDL cholesterol. Associated with elevated sterol levels. Interestingly, loss-of-function mutations in PCSK9 In patients with low LDL cholesterol levels, the incidence of cardiovascular events was significantly reduced Furthermore, patients with PCSK9 loss of function were found to have a higher risk of developing statin therapy. has been shown to increase the response to

[0008] Therefore, targeting PCSK9 may be a promising treatment for cardiovascular disease and other conditions with high co-morbidities, including autosomal dominant FH. This is a promising therapeutic approach for the treatment of hypercholesterolemia. Research in this area has been The current focus of the study has been on blocking the interaction of PCSK9 with the LDL receptor using One such inhibitory approach is alirocumab (REGN727 / SAR2 36553), evolocumab (AMG145), and bococizumab (RN316). However, the use of monoclonal antibodies against PCSK9 has not been reported. Roach is concerned not only with the inherent problems of antibody therapy, including hypersensitivity reactions and immunogenicity, but also with the risk of developing inflammatory bowel diseases. The inhibitory approach is based on adnectins and peptide mimetics. Metix has also been studied. The use of molecular inhibitors has been largely unsuccessful.

[0009] Another approach that has been pursued involves inhibition of PCSK9 expression. Such strategies include the use of antisense oligonucleotides to downregulate gene expression. These include the use of nucleotides or siRNAs to permanently alter the PCSK9 gene. Gene editing approaches to knock out protein expression have also been promoted. For example, Ding et al. Using the CRISPR / Cas9 system, the mutation rate of PCSK9 in mouse liver was 3. >50% from day 1 to day 4, PCSK9 levels decreased, and hepatic LDL receptor levels found that the serum cholesterol levels increased by 35% to 40% (Non-Patent Literature Reference 1). Fah with chimeric human liver - / - Rag2 - / - Il2rg - / - (FRG In a further study by Wang et al. using mice with the human PCSK9 gene knockout (KO) Targeted CRISPR-Cas9 therapy demonstrates on-target targeting of PCSK9 This caused high levels of spontaneous mutations, increasing human PCSK9 protein levels in blood by 52%. However, currently, there are no human gene therapy approaches focusing on PCSK9. Modification and / or knock-in of genes specifically in primate liver, including human liver, that are required for therapeutic intervention Evidence that the CRISPR / Cas system can be successfully used to There is a severe lack of it in the field of medicine.

[0010] I-CreI (SEQ ID NO: 1) is a 22-nucleotide sequence encoding a chloroplast chromosome of the alga Chlamydomonas reinhardtii. The homing endonuclease LAGLIDADG( The wild-type I-Cre gene was selected using genetic selection techniques. The preference for I cleavage sites was changed (Non-Patent Documents 3 to 6). I-CreI and other homing enzymes target a wide variety of DNA sites, including the human genome. A monoLAGLIDADG (SEQ ID NO:2) homogenate that can comprehensively redesign endonucleases A method for rationally designing endonuclease has been described (Patent Document 1).

[0011] First, as described in Patent Document 2, I-CreI and its engineered derivatives are generally It is a dimer, but it has a short cleavage domain that connects the C-terminus of the first subunit to the N-terminus of the second subunit. They can be fused to a single polypeptide using a simple peptide linker (Non-Patent Documents 7-8). Thus, a functional "single-chain" meganuclease can be expressed from a single transcript. Cut.

[0012] The use of engineered meganucleases to target the PCSK9 gene and inhibit PCSK9 expression thus reducing circulating LDL levels and total cholesterol in patients with cholesterol-related diseases. This is a promising approach to reducing roll levels. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] International Publication No. WO2007 / 047859 [Patent Document 2] International Publication No. WO2009 / 059195 [Non-patent literature]

[0014] [Non-Patent Document 1] Ding et al. (2014), Circ Res 115(5):488-492 [Non-Patent Document 2] Wang et al. (2016), Arteriosclerosis Thromb Vasc Biol 36:783-786 [Non-Patent Document 3] Sussman et al. (2004), J.Mol.Biol.342:31-41 [Non-Patent Document 4] Chames et al. (2005), Nucleic Acids Res.33:e178 [Non-Patent Document 5] Seligman et al. (2002), Nucleic Acids Res.30:3870-9 [Non-Patent Document 6] Arnould et al. (2006), J.Mol.Biol.355:443-58 [Non-Patent Document 7] Li et al. (2009), Nucleic Acids Res.37:1650-62 [Non-Patent Document 8] Grizot et al. (2009), Nucleic Acids Res.37:5405-19 Summary of the Invention [Problem to be solved by the invention]

[0015] The present invention relates, in part, to a polypeptide that is engineered to recognize a DNA sequence within the PCSK9 gene. The present inventors have developed a novel method to detect PCSK9 using a rare-cutting endonuclease that is specific to the site of PCSK9. The expression and / or activity of the IL-16 polypeptide can be reduced, and subsequently, total cholesterol levels and Researchers identify a specific recognition sequence in the PCSK9 gene that reduces LDL cholesterol levels . [Means for solving the problem]

[0016] Thus, the methods and compositions disclosed herein can be used to treat a cholesterol-related disorder, such as For example, the compounds may be useful in treating or reducing the symptoms of hypercholesterolemia, including autosomal dominant FH. Thus, the present invention provides an additional gene therapy approach for cholesterol-related disorders in primates. This invention fulfills a need in the art for a novel approach.

[0017] The present invention relates to a method for treating a cholesterol-related disorder in a subject, such as hypercholesterolemia, including autosomal dominant FH. The engineered meganucleases of the present invention are useful for treating sterolemia. The meganuclease recognizes and cleaves the recognition sequence (SEQ ID NO: 3) in the PCSK9 gene. Cleavage at such recognition sequences by the engineered meganucleases disclosed herein and inhibiting the expression and / or activity of PCSK9 by non-homologous end joining (NHEJ) at the break site. NHEJ can create insertions, deletions, or interfere with gene expression. This can result in frameshift mutations that disrupt normal gene expression. By so doing, expression and / or activity of PCSK9 can be determined according to the methods disclosed herein. The present invention also provides a method for the detection of a recognition sequence located within the PCSK9 gene. The treatment of cholesterol-related disorders using engineered meganucleases with specificity for The present invention provides pharmaceutical compositions and methods for the treatment of total cholesterol levels and / or or to reduce LDL cholesterol levels and / or to treat cholesterol-related disorders The engineered meganucleases disclosed herein can be used to reduce symptoms associated with The present invention further provides a method of delivering the compound to a subject having a cholesterol-related disorder.

[0018] Thus, in one aspect, the present invention provides an engineered polypeptide that recognizes and cleaves a recognition sequence within the PCSK9 gene. The engineered meganuclease comprises a first subunit, and a second subunit, the first subunit being a first recognition half of the recognition sequence. The second subunit binds to the recognition site and contains the first hypervariable (HVR1) region. It binds to the second recognition half-site of the array and contains the second hypervariable (HVR2) region.

[0019] In some embodiments, the recognition sequence comprises SEQ ID NO:4 (i.e., the PCS7-8 recognition sequence). In some embodiments, the recognition sequence comprises SEQ ID NO:4. for an amino acid sequence corresponding to residues 24 to 79 of any one of SEQ ID NOs: 6 to 14 At least 80%, at least 85%, at least 90%, at least 95%, or Some such embodiments include amino acid sequences having sequence identity of 1 to 100 or more. In one embodiment, the HVR1 region comprises residues 24, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 Supports 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 In certain embodiments, the HVR1 region can include residues corresponding to SEQ ID NO:8. It may further include residues corresponding to groups 48, 50, 71, and 73. In this case, the HVR1 region may comprise residues 24 to 79 of any one of SEQ ID NOs: 6 to 14. can.

[0020] In some such embodiments, the recognition sequence comprises SEQ ID NO:4, the HVR2 region comprises the sequence A small number of amino acid sequences corresponding to residues 215 to 270 of any one of sequence numbers 6 to 14 At least 80%, at least 85%, at least 90%, at least 95%, or more Some such embodiments include amino acid sequences having the above sequence identity. In this embodiment, the HVR2 region comprises residues 215, 217, 218, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 9, 221, 223, 224, 229, 231, 233, 235, 237, 259, 26 In one embodiment, the amino acid sequence may include residues corresponding to 1, 266, and 268. The HVR2 region can further include a residue corresponding to residue 258 of SEQ ID NO:12. In certain embodiments, the HVR2 region comprises residues 215-216 of any one of SEQ ID NOs: 6-14. It may contain 70.

[0021] In such embodiments in which the recognition sequence comprises SEQ ID NO:4, the first subunit comprises SEQ ID NO: At least 80% and at least 85% of residues 7-153 of any one of Nos. 6-14 %, at least 90%, at least 95%, or more sequence identity The second subunit may comprise any one of the residues of SEQ ID NOs: 6 to 14. At least 80%, at least 85%, at least 90%, or at least The amino acid sequence may include an amino acid sequence having at least 95% or more sequence identity. In some embodiments, the first subunit comprises residue 8 of any one of SEQ ID NOs: 6-14. 0 include D, E, Q, N, K, R, and S residues. The second subunit is a position corresponding to residue 271 of any one of SEQ ID NOs: 6 to 14. In certain embodiments, the first subunit comprises D, E, Q, N, K, R, and S residues. The kit contains a residue corresponding to residue 80 of any one of SEQ ID NOs: 6 to 14. In an embodiment, the second subunit is a nucleotide sequence corresponding to residue 271 of any one of SEQ ID NOs: 6-14. In some embodiments, the first subunit comprises a residue corresponding to SEQ ID NO:6-14. Similarly, in some embodiments, the amino acid sequence may include any one of residues 7 to 153 of The second subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 6 to 14. It is possible.

[0022] In certain such embodiments, the recognition sequence comprises SEQ ID NO:4, The ase can include a linker, the linker connecting the first subunit and the second subunit. In certain embodiments, the engineered meganuclease comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6 to 8. The amino acid sequence may include any one of the 14 amino acid sequences.

[0023] In another aspect, the present invention provides a method for the preparation of a nucleic acid encoding any of the engineered meganucleases disclosed herein. In certain embodiments, the polynucleotide comprises a nucleic acid sequence that encodes The peptide may be an mRNA.

[0024] In a further embodiment, the mRNA is comprised of one or more engineered meganucleotides as described herein. In a further embodiment, the present invention provides a method for the preparation of a method for the preparation of a polycistronic mRNA encoding a lyase. The polycistronic mRNA may be expressed by one or more of the engineered meganuclear vectors described herein. and 1) a method for treating cholesterol-related disorders comprising administering to a subject a therapeutically beneficial effect or a number of additional proteins.

[0025] In another aspect, the present invention provides a nucleic acid encoding any of the engineered meganucleases of the present invention. In some embodiments, the recombinant DNA construct comprises a nucleic acid sequence. The construct comprises a promoter and a nucleic acid encoding an engineered meganuclease as described herein. The cassette comprises the sequence.

[0026] In another embodiment, the recombinant DNA construct comprises a promoter and a polycistronic nucleic acid sequence. wherein the promoter drives expression of the polycistronic nucleic acid sequence and the target The cells are caused to produce the polycistronic mRNA described herein.

[0027] In certain embodiments, the recombinant DNA construct is any of the engineered megakaryons disclosed herein. The present invention relates to a viral vector comprising a nucleic acid sequence encoding a nuclease. In embodiments, the viral vector is a retroviral vector, a lentiviral vector, It may be an adenovirus vector or an adeno-associated virus (AAV) vector. In this embodiment, the viral vector may be a recombinant AAV vector.

[0028] In some embodiments, the viral vector comprises a promoter and an operation as described herein. and a nucleic acid sequence encoding the meganuclease. The viral vector comprises two or more cassettes, each cassette comprising a promoter and a and a nucleic acid sequence encoding an engineered meganuclease as described herein.

[0029] In another embodiment, the viral vector comprises a promoter and a polycistronic nucleic acid sequence. a promoter driving expression of the polycistronic nucleic acid sequence; The polycistronic mRNA described herein is produced in the target cell.

[0030] In another aspect, the present invention provides a nucleic acid encoding any of the engineered meganucleases of the present invention. In some embodiments, the viral vector comprises a nucleic acid sequence. is a retroviral vector, a lentiviral vector, an adenoviral vector, or The vector may be an adeno-associated virus (AAV) vector. In certain embodiments, the viral vector The vector can be a recombinant AAV vector. In some embodiments, the viral vector is a promoter and a nucleic acid sequence encoding an engineered meganuclease as described herein; The cassette comprises:

[0031] In a further embodiment, the viral vector comprises a promoter and a polycistronic nucleic acid sequence. wherein the promoter drives expression of the polycistronic nucleic acid sequence. , producing the polycistronic mRNA described herein in the target cell.

[0032] In another aspect, the invention provides a method for the preparation of a compound comprising administering to a subject in need thereof a therapeutically acceptable carrier and (a) an engineered compound as described herein. (b) a nucleic acid encoding an engineered meganuclease as described herein; and a nuclease protein, wherein the engineered meganuclease comprises: It has specificity for a recognition sequence within PCSK9, such as SEQ ID NO:4.

[0033] In one embodiment, a pharmaceutical composition encoding the engineered meganuclease disclosed herein The nucleic acid sequence of the product may be an mRNA as described herein. In this case, the mRNA is expressed by two or more engineered meganucleases as described herein in vivo ( The polycistronic nucleic acid sequences described herein as expressed in target cells (in vivo) It may be mRNA.

[0034] In another embodiment, the pharmaceutical composition comprises an engineered meganuclease disclosed herein. The recombinant DNA constructs described herein include those containing a nucleic acid sequence encoding the In such an embodiment, the recombinant DNA construct comprises a promoter and an engineered meganucleotide of the invention. and a nucleic acid sequence encoding a lyase.

[0035] In another embodiment, the recombinant DNA construct of the pharmaceutical composition comprises a promoter and a polycistronic and a polycistronic nucleic acid sequence, wherein the promoter controls expression of the polycistronic nucleic acid sequence. and expressing two or more engineered meganucleases described herein in a target cell. Thus, the polycistronic mRNA described herein is produced in vivo in target cells.

[0036] In another embodiment, the pharmaceutical composition comprises an engineered meganuclease disclosed herein. In one such embodiment, the viral vector comprises a nucleic acid sequence encoding the The vector can be a retrovirus, lentivirus, adenovirus, or AAV. In certain embodiments, the viral vector may be a recombinant AAV vector.

[0037] In some such embodiments, the viral vector comprises a promoter and a and a nucleic acid sequence encoding the engineered meganuclease as described.

[0038] In other such embodiments, the viral vector comprises a promoter and a polycistronic nucleic acid. and a cassette containing the nucleic acid sequence, the promoter being capable of carrying out two or more of the operations described herein. Expression of the polycistronic nucleic acid sequence such that the selected meganuclease is expressed in the target cell. to produce the polycistronic mRNA described herein in target cells in vivo.

[0039] In one embodiment, the pharmaceutical composition comprises an engineered polypeptide disclosed herein that recognizes and cleaves SEQ ID NO:4. In certain embodiments, the engineered meganuclease can include The ase can comprise any one of the amino acid sequences of SEQ ID NOs:6-14.

[0040] In some embodiments, the pharmaceutical composition is provided herein encapsulated in lipid nanoparticles. In certain embodiments, the pharmaceutical composition may comprise one or more mRNAs described in The lipid nanoparticles can include two or more mRNAs as described herein, each of which is a component of the present invention. In some embodiments, the lipid nanoparticle encodes an engineered meganuclease of The composition enhances delivery and uptake into the liver, particularly into hepatic parenchymal cells.

[0041] In another aspect, the present invention provides a method of treatment for decreasing expression or activity of PCSK9 in a subject. Also provided herein are cholesterol-related disorders, such as autosomal dominant FH. to treat hypercholesterolemia or symptoms associated with cholesterol-related disorders, including The method includes administering to a target cell of a subject an effective amount of a target cell antigen. or (b) an effective amount of an engineered meganuclease that is expressed in the cell. and delivering the engineered meganuclease protein. has specificity for the recognition sequence of PCSK9, such as SEQ ID NO:4.

[0042] In some embodiments of the method, the subject has a cholesterol-related disorder. In embodiments, the subject has hypercholesterolemia. Hypercholesterolemia in the body is classified as familial hypercholesterolemia or autosomal dominant familial hypercholesterolemia. In certain embodiments, the subject is a human or non-human primate.

[0043] In certain embodiments of the method, the subject is administered a pharmaceutical composition disclosed herein. The pharmaceutical composition can include a recombinant AAV vector as disclosed herein. In embodiments, the pharmaceutical composition comprises a nucleic acid sequence encoding an engineered meganuclease, as disclosed herein. In certain embodiments, the mRNA is encapsulated in a lipid nanoparticle. It can be made into a system.

[0044] In some embodiments of the method, the engineered meganuclease or engineered meganuclease The nucleic acid encoding the nuclease can be delivered to a target hepatocyte. The target hepatocyte can be a primary hepatic parenchyma. The cells may be hepatocytes, such as hepatocytes.

[0045] In some embodiments of the method, LDL receptors on the cell surface of liver cells of the subject are detected. The display shows increased levels of LDL receptors with treatment compared to baseline levels. The display of LDL receptors on the cell surface is increased relative to baseline LDL receptor levels. When compared, approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% %, 90%, 95%, 100%, 150%, 250%, 500%, 1000%, or any combination thereof It can increase by more than this.

[0046] In certain embodiments of the method, the subject's serum total cholesterol level is reduced by treatment. Serum total cholesterol levels were compared to baseline serum total cholesterol levels. (a) at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, %, 70%, 80%, 90%, 95%, or up to 100%, or (b) 5 to 15 mg / dL, 10~20mg / dL, 10~30mg / dL, 15~30mg / dL, 20~ 30mg / dL, 25~35mg / dL, 25~40mg / dL, 25~50mg / dL , 40-60mg / dL, 50-70mg / dL, 60-80mg / dL, or 70 It can decrease by ~100 mg / dL.

[0047] In certain embodiments, the subject's serum LDL cholesterol level is reduced by treatment. Serum LDL cholesterol levels were compared with baseline serum LDL cholesterol levels. (a) at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to 100%, or (b) 5 to 15 mg / dL, 10~20mg / dL, 10~30mg / dL, 15~30mg / dL, 2 0~30mg / dL, 25~35mg / dL, 25~40mg / dL, 25~50mg / dL, 40-60mg / dL, 50-70mg / dL, or 60-80mg / dL reduction Get a little.

[0048] In certain embodiments of the method, the first recognition sequence can include SEQ ID NO:4. In some such embodiments, the engineered meganuclease recognizes and cleaves SEQ ID NO:4. The meganuclease may be any engineered meganuclease of the invention that cleaves a nucleic acid sequence that is capable of cleaving a nucleic acid sequence that is highly cleavable. The constructed meganuclease comprises any one of the amino acid sequences set forth in SEQ ID NOs: 6 to 14. This can be done.

[0049] In another aspect, the present invention provides an engineered megakaryon comprising the polypeptides described herein for use as a medicament. The present invention provides a nuclease that is useful in treating cholesterol-related disorders, such as autosomal dominant F To treat hypercholesterolemia, including H, or to reduce the activity or expression of PCSK9 or as an agent for reducing symptoms associated with cholesterol-related disorders. Further provided herein are uses of the engineered meganucleases described herein. In certain embodiments, the The invention provides a method for reducing total cholesterol levels and / or LDL cholesterol levels in a subject. The present invention provides a use of an engineered meganuclease as described herein as an agent for the treatment of do.

[0050] In another aspect, the present invention provides an isolated polynucleotide for use as a medicament. The isolated polynucleotides provide the engineered meganucleases disclosed herein. The present invention relates to a method for treating cholesterol-related disorders, such as autosomal dominant To treat hypercholesterolemia, including FH, or to decrease the activity or expression of PCSK9 and isolating it as a pharmaceutical agent for inhibiting or reducing symptoms associated with cholesterol-related disorders. Further provided are uses of the isolated polynucleotides described herein. In certain embodiments, the present invention relates to a method for the production of a method for the production of a subject To reduce a subject's total cholesterol level and / or LDL cholesterol level The isolated polypeptides encoding the engineered meganucleases described herein are useful as therapeutic agents. Uses of oligonucleotides are provided.

[0051] In another aspect, the present invention provides a recombinant AAV vector for use as a medicament, The recombinant AAV vector comprises an isolated polynucleotide. The method comprises the steps of: (a) determining whether a nucleic acid sequence encoding the engineered meganuclease disclosed herein is effective to produce a polypeptide comprising the nucleic acid sequence encoding the engineered meganuclease disclosed herein; The clear focus is on cholesterol-related disorders, such as hypercholesterolemia, including autosomal dominant FH. or to reduce the activity or expression of PCSK9 or to treat cholesterol-related The present invention further provides for the use of recombinant AAV vectors as agents for reducing symptoms associated with the disorder. The recombinant AAV vector comprises an isolated polynucleotide, The nucleic acid sequence encoding the engineered meganuclease disclosed herein is The present invention relates to a method for determining the total cholesterol level and / or LDL cholesterol level of a subject. The present invention further provides a use of a recombinant AAV vector as an agent for reducing The vector comprises an isolated polynucleotide, and the isolated polynucleotide is The present invention includes nucleic acid sequences encoding the engineered meganucleases disclosed herein.

[0052] These and other aspects and embodiments of the invention are described in detail below with reference to the following detailed description and claims. The present invention can be more fully understood by reference to the following embodiments, which are not illustrated in the accompanying drawings for clarity. Certain features of the invention which are described in the context of the present invention may be provided in combination in a single embodiment. All combinations of the embodiments are specifically embraced by the present invention and may be combined as if Each and every combination is disclosed herein as if each and every combination were individually and explicitly disclosed. Conversely, various aspects of the invention that are described in the context of a single embodiment for brevity may be used in the following embodiments. The features may be provided separately or in any suitable subcombination. All subcombinations of the recited features are specifically embraced by the present invention, as if by any combination. Each and every subcombination is expressly disclosed herein as if each and every The embodiments of each aspect of the invention disclosed herein are intended to be illustrative, mutatis mutandis. and apply to each other aspect of the present invention. [Brief description of the drawings]

[0053] [Figure 1] Engineered meganuclease recognition sequence of the human PCSK9 gene. The recognition sequence targeted by the engineered meganuclease of the present invention contains two recognition half-sites. Each recognition half-site contains 9 base pairs, and the two half-sites are separated by a central sequence of 4 base pairs. The PCS7-8 recognition sequence (SEQ ID NO: 4) contains two recognition half-sites, designated PCS7 and PCS8. [Diagram 2]The engineered meganucleases of the invention comprise two subunits, the first subunit comprising an HVR1 region that binds to a first recognition half-site (e.g., PCS7) and the second subunit comprising an HVR2 region that binds to a second recognition half-site (e.g., PCS8). In embodiments where the engineered meganuclease is a single-chain meganuclease, the first subunit comprising the HVR1 region can be located as either the N-terminal or C-terminal subunit. Similarly, the second subunit comprising the HVR2 region can be located as either the N-terminal or C-terminal subunit. [Diagram 3] Overview of reporter assay in CHO (Chinese Hamster Ovary) cells to evaluate engineered meganucleases targeted to the PCS7-8 recognition sequence. For the engineered meganucleases described herein, a CHO cell line was generated and a reporter cassette was stably integrated into the genome of the cells. The reporter cassette contained, in 5' to 3' order, the SV40 early promoter, the 5' 2 / 3 of the GFP gene, the recognition sequence of the engineered meganuclease of the invention (e.g., the PCS7-8 recognition sequence), the recognition sequence of the CHO-23 / 24 meganuclease (WO 2012 / 167192), and the 3' 2 / 3 of the GFP gene. Cells stably transfected with this cassette did not express GFP in the absence of a DNA cleavage inducer. Meganucleases were introduced by transduction of plasmid DNA or mRNA encoding the respective meganuclease. When DNA cleavage is induced at any of the meganuclease recognition sequences, the duplicated regions of the GFP gene recombine with each other to produce functional GFP protein, and the percentage of GFP-expressing cells can then be determined by flow cytometry as an indirect measure of the frequency of genome cleavage by the meganuclease. [Figure 4]Efficiency of engineered meganucleases to recognize and cleave recognition sequences within the PCSK9 gene in a CHO cell reporter assay. Engineered meganucleases set forth in SEQ ID NOs: 6-14 were engineered to target the PCS7-8 recognition sequence (SEQ ID NO: 4) and screened for efficacy in a CHO cell reporter assay. The results shown provide the percentage of GFP expressing cells observed in each assay, indicating the efficacy of each meganuclease to cleave the target recognition sequence or the CHO-23 / 24 recognition sequence. A negative control (bs) was also included in each assay. Figures 4A-C show meganucleases targeted to the PCS7-8 recognition sequence. Figure 4A shows PCS7-8x.88 and PCS7-8x.66. Figure 4B shows PCS7-8L.197, PCS7-8L.204, PCS7-8L.209, PCS7-8L.261, PCS7-8L.262, and PCS7-8L.268. Figure 4C shows PCS7-8L.197 and PCS7-8L.367. [Diagram 5] Efficiency of engineered meganucleases to recognize and cleave recognition sequences within the human PCSK9 gene in a CHO cell reporter assay. Engineered meganucleases set forth in SEQ ID NOs: 6-14 were engineered to target the PCS7-8 recognition sequence (SEQ ID NO: 4) and screened for efficacy in a CHO cell reporter assay at multiple time points over 11 days post-nucleofection. The results shown provide the percentage of GFP expressing cells observed in each assay over the 11 days of analysis, indicating the efficacy of each meganuclease to cleave the target recognition sequence or the CHO-23 / 24 recognition sequence as a function of time. Figure 5A shows PCS7-8x.88 and PCS7-8x.66 meganucleases targeting the PCS7-8 recognition sequence. Figure 5B shows PCS7-8x.88 and PCS7-8L.197 meganucleases targeting the PCS7-8 recognition sequence. FIG. 5C shows the PCS7-8L.367 meganuclease targeting the PCS7-8 recognition sequence. [Figure 6]T7 Endonuclease I (T7E) Assay. A T7E assay was performed to determine whether PCS7-8 meganuclease generated indels at its recognition site in HEK293 cells. In the T7E assay, the PCS7-8 locus was amplified by PCR using primers flanking the PCS7-8 recognition sequence. If there was an indel (unordered insertion or deletion) within the PCS7-8 locus, the resulting PCR product would consist of a mixture of wild-type and mutant alleles. The PCR products were allowed to denaturate and slowly reanneal. Slow reannealing would result in the formation of heteroduplexes consisting of wild-type and mutant alleles, resulting in mismatched bases and / or bulges. The T7E1 enzyme cleaves at the mismatch site, generating cleavage products that can be visualized by gel electrophoresis. PCS7-8x.88 and PCS7-8x.66 meganucleases were evaluated 2 and 5 days after nucleofection. [Figure 7] Serum PCSK9 protein levels in non-human primates. An AAV vector called AAV8.TBG.PI.PCS7-8x.88.WPRE.bGH was prepared and administered at three different doses to three male and one male rhesus macaques. Animal RA1866 (male) received a single dose of 3×10 genome copies (GC) / kg. Animal RA1857 (male) received a single dose of 6×10 GC / kg. Animals RA1829 (female) and RA2334 (male) each received a single dose of 2×10 GC / kg. Blood samples were collected at days −3 and 0, and at multiple time points during the first 168 days (low dose animals) or 280 days (mid and high dose animals) post-dosing for analysis of serum PCSK9 protein levels by ELISA. [Figure 8]Total cholesterol, LDL, HDL, and triglyceride levels were measured on day -3, day 0, and at multiple time points after administration of AAV encoding PCS7-8x.88 meganuclease. Figure 8A shows the LDL levels measured over time for each subject. Figure 8B shows the total cholesterol, HDL, LDL, and triglyceride levels for subject RA1866. Figure 8C shows the total cholesterol, HDL, LDL, and triglyceride levels for subject RA1857. Figure 8D shows the total cholesterol, HDL, LDL, and triglyceride levels for subject RA1829. Figure 8E shows the total cholesterol, HDL, LDL, and triglyceride levels for subject RA2334. [Figure 9] Alanine aminotransferase (ALT) levels were measured in each subject on day -3, day 0, and at multiple time points following administration of AAV encoding PCS7-8x.88 meganuclease. [Figure 10] Frequency of insertions and deletions (indels) observed in vivo in the PCS7-8 recognition sequence in hepatocytes. Liver biopsies were performed 17 days after administration of the AAV8.TBG.PI.PCS7-8x.88.WPRE.bGH vector and examined for the presence of indels in the PCS7-8 recognition sequence. Indels were detected by PCR primers flanking the recognition sequence, amplification of the intervening genomic region, and sequencing of the resulting PCR products. [Figure 11]In situ hybridization to detect PCS7-8 meganuclease mRNA in hepatocytes in vivo. Liver biopsies obtained 17 and 129 days after administration of the AAV8.TBG.PI.PCS7-8x.88.WPRE.bGH vector were examined by in situ hybridization (ISH). Fluorescently labeled oligo probes were designed to bind to PCS7-8x.88 mRNA in biopsied hepatocytes from each subject. Figure 11A shows mock treatment of biopsy cells from another subject, M11657, performed as a control without oligo probe. Figure 11B shows a biopsy sample from subject RA1866. Figure 11C shows a biopsy sample from subject RA1857. Figure 11D shows a biopsy sample from subject RA1829. Figure 11E shows a biopsy sample from subject RA2334. [Figure 12] Serum PCSK9 protein levels and serum LDL in non-human primates. An AAV vector called AAV8.TBG.PI.PCS7-8L.197.WPRE.bGH was prepared and administered at a dose of 6x1012 gene copies / kg to one male and one female rhesus monkey (subjects RA2125 and RA2343). Blood samples were taken pre- and at multiple time points post-dose for analysis of serum PCSK9 protein levels by ELISA. [Figure 13] Total cholesterol, LDL, HDL, and triglyceride levels were measured before administration and at multiple time points after administration of AAV encoding PCS7-8L.197 meganuclease. Figure 13A shows the LDL levels measured over time for each subject. Figure 13B shows the total cholesterol, HDL, LDL, and triglyceride levels for subject RA2125. Figure 13C shows the total cholesterol, HDL, LDL, and triglyceride levels for subject RA2343. [Figure 14] Alanine aminotransferase (ALT) levels were measured in each subject pre-administration and at multiple time points following administration of AAV encoding PCS7-8L.197 meganuclease. [Figure 15]In situ hybridization to detect PCS7-8 meganuclease mRNA in hepatocytes in vivo. Liver biopsies obtained 18 and 128 days after administration of the AAV8.TBG.PI.PCS7-8L.197.WPRE.bGH vector were examined by in situ hybridization (ISH). Fluorescently labeled oligo probes were designed to bind to PCS7-8L.197 mRNA in biopsied hepatocytes from each subject. Figure 15A shows a biopsy sample from subject RA2125. Figure 15B shows a biopsy sample from subject RA2343. [Figure 16] Indel analysis for each subject's rhPCSK9 targeted locus by anchored multiplex PCR (AMP-seq). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] A brief description of the sequence SEQ ID NO:1 defines the amino acid sequence of the wild-type I-CreI meganuclease.

[0055] SEQ ID NO:2 defines the amino acid sequence of LAGLIDADG.

[0056] SEQ ID NO:3 defines the nucleic acid sequence of the human PCSK9 gene.

[0057] SEQ ID NO:4 defines the nucleic acid sequence of the PCS7-8 recognition sequence (sense).

[0058] SEQ ID NO:5 defines the nucleic acid sequence of the PCS7-8 recognition sequence (antisense).

[0059] SEQ ID NO:6 defines the amino acid sequence of the PCS7-8L.197 meganuclease.

[0060] SEQ ID NO:7 sets forth the amino acid sequence of the PCS7-8x.88 meganuclease.

[0061] SEQ ID NO:8 sets forth the amino acid sequence of PCS7-8L.367 meganuclease.

[0062] SEQ ID NO:9 sets forth the amino acid sequence of the PCS7-8L.204 meganuclease.

[0063] SEQ ID NO: 10 defines the amino acid sequence of the PCS7-8L.209 meganuclease. .

[0064] SEQ ID NO: 11 defines the amino acid sequence of PCS7-8L.261 meganuclease .

[0065] SEQ ID NO: 12 defines the amino acid sequence of the PCS7-8L.262 meganuclease. .

[0066] SEQ ID NO: 13 defines the amino acid sequence of PCS7-8L.268 meganuclease .

[0067] SEQ ID NO:14 sets forth the amino acid sequence of the PCS7-8x.66 meganuclease.

[0068] SEQ ID NO: 15 is the PCS7-8L.197 meganuclease PCS7 binding subunit The amino acid sequence of

[0069] SEQ ID NO: 16 is the sequence of the PCS7-8x.88 meganuclease PCS7 binding subunit The amino acid sequence is defined.

[0070] SEQ ID NO: 17 is the PCS7-8L.367 meganuclease PCS7 binding subunit The amino acid sequence of

[0071] SEQ ID NO: 18 is the PCS7-8L.204 meganuclease PCS7 binding subunit The amino acid sequence of

[0072] SEQ ID NO:19 is the PCS7-8L.209 meganuclease PCS7 binding subunit The amino acid sequence of

[0073] SEQ ID NO: 20 is the PCS7-8L.261 meganuclease PCS7 binding subunit The amino acid sequence of

[0074] SEQ ID NO: 21 is the PCS7-8L.262 meganuclease PCS7 binding subunit The amino acid sequence of

[0075] SEQ ID NO: 22 is the PCS7-8L.268 meganuclease PCS7 binding subunit The amino acid sequence of

[0076] SEQ ID NO: 23 is the sequence of the PCS7-8x.66 meganuclease PCS7 binding subunit The amino acid sequence is defined.

[0077] SEQ ID NO:24 is the PCS7-8L.197 meganuclease PCS8 binding subunit The amino acid sequence of

[0078] SEQ ID NO: 25 is the sequence of the PCS7-8x.88 meganuclease PCS8 binding subunit The amino acid sequence is defined.

[0079] SEQ ID NO:26 is the PCS7-8L.367 meganuclease PCS8 binding subunit The amino acid sequence of

[0080] SEQ ID NO:27 is the PCS7-8L.204 meganuclease PCS8 binding subunit The amino acid sequence of

[0081] SEQ ID NO:28 is the PCS7-8L.209 meganuclease PCS8 binding subunit The amino acid sequence of

[0082] SEQ ID NO:29 is the PCS7-8L.261 meganuclease PCS8 binding subunit The amino acid sequence of

[0083] SEQ ID NO:30 is the PCS7-8L.262 meganuclease PCS8 binding subunit The amino acid sequence of

[0084] SEQ ID NO:31 is the PCS7-8L.268 meganuclease PCS8 binding subunit The amino acid sequence of

[0085] SEQ ID NO:32 is the sequence of the PCS7-8x.66 meganuclease PCS8 binding subunit The amino acid sequence is defined.

[0086] SEQ ID NO: 33 shows the in vivo indication following treatment with PCS7-8x.88. The nucleic acid sequence of el is defined.

[0087] SEQ ID NO: 34 represents the in vivo indication following treatment with PCS7-8x.88. The nucleic acid sequence of el is defined.

[0088] SEQ ID NO: 35 shows the in vivo indication following treatment with PCS7-8x.88. The nucleic acid sequence of el is defined.

[0089] SEQ ID NO: 36 shows the in vivo index following treatment with PCS7-8x.88. The nucleic acid sequence of el is defined.

[0090] SEQ ID NO: 37 shows the in vivo indication following treatment with PCS7-8x.88. The nucleic acid sequence of el is defined.

[0091] SEQ ID NO: 38 shows the in vivo index following treatment with PCS7-8x.88. The nucleic acid sequence of el is defined.

[0092] SEQ ID NO: 39 shows the in vivo index following treatment with PCS7-8x.88. The nucleic acid sequence of el is defined.

[0093] SEQ ID NO: 40 shows the in vivo indication following treatment with PCS7-8x.88. The nucleic acid sequence of el is defined.

[0094] 1.1 References and Definitions The patent and scientific literature referred to herein establishes knowledge that is available to those with skill in the art. Any issued U.S. patents, including GenBank database sequences, cited herein, The granted applications, published foreign applications, and references are all incorporated herein by reference as if each were specific and binding. No. 6,333,635, filed on Oct. 13, 2007, and incorporated by reference herein to the same extent as if individually set forth and incorporated by reference. It is being read.

[0095] This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, it is to be construed as an inventive concept, and is not to be construed as limiting the present invention, so that this disclosure will be thorough and complete, and will not be construed as limiting the present invention to those of ordinary skill in the art. These embodiments are provided to fully convey the scope of the present invention. Features illustrated with respect to one embodiment may be incorporated in other embodiments, and may be used with respect to a particular embodiment. In addition, in light of this disclosure, any feature illustrated in any of the above examples may be omitted from the embodiments. Numerous modifications and additions to the embodiments shown herein may be made by those skilled in the art without departing from the invention. is obvious.

[0096] Unless otherwise defined, technical and scientific terms used herein are intended to be understood as meaning those terms which fall within the scope of the present invention. The term "compound" as used herein has the same meaning as commonly understood by those skilled in the art. The terminology used in the description is merely for the purpose of describing the detailed embodiments and is not intended to limit the present invention. It is not intended to.

[0097] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. No. 6,399,433, which is incorporated herein by reference.

[0098] As used herein, "a," "an," or "the" means one or more For example, "a cell" can mean a single cell or multiple cells. good.

[0099] As used herein, unless otherwise stated, the word "or" means "and / or." is used in the inclusive sense of "either / or," and in the exclusive sense of "either / or." It is not.

[0100] As used herein, the terms "nuclease" and "endonuclease" have the same meaning. A naturally occurring enzyme used in the synthesis of phosphodiesterases that cleave phosphodiester bonds in polynucleotide chains. The term refers to an enzyme that has been engineered or isolated.

[0101] As used herein, the term "meganuclease" refers to a nuclease that has a recognition sequence of more than 12 base pairs. Preferably, the meganuclease of the present invention is an endonuclease that binds to double-stranded DNA. The recognition sequence for the enzyme is 22 base pairs. The meganuclease is derived from I-CreI. The endonuclease may be an endonuclease that exhibits, for example, DNA binding specificity, DNA cleavage activity, DNA binding activity, I-CreI modified in terms of binding affinity or dimerization properties compared to the native I-CreI. Such engineered mutants of I-CreI may be referred to as I-CreI- ... Methods for making the same are known in the art (see, for example, International Publication No. WO 2005 / 013361). As used herein, meganucleases are heterodimers. Binds to double-stranded DNA. Meganucleases further contain a pair of DNA-binding domains that bind to peptides. It was a "single-chain meganuclease" linked to a single polypeptide using a linker. The term "homing endonuclease" is synonymous with the term "meganuclease." The meganucleases of the invention have a molecular weight of: No adverse effects on cell viability or significant reduction in meganuclease cleavage activity were observed. They are free of toxic and substantially non-toxic when expressed in cells.

[0102] As used herein, the term "single-chain meganuclease" refers to a single-chain meganuclease linked by a linker. It refers to a polypeptide that contains a pair of nuclease subunits connected together. The enzyme has the structure of N-terminal subunit-linker-C-terminal subunit. The meganuclease subunits of the present invention are generally not identical in amino acid sequence. Therefore, single-stranded meganucleases typically recognize pseudotyped DNA sequences. It cleaves palindromic or non-palindromic recognition sequences. Single-chain meganucleases In fact, even though it is not a dimer, it is called a "single-chain heterodimer" or "single-chain heterodimer For clarity, unless otherwise specified, The term "meganuclease" can refer to dimeric or single-chain meganucleases.

[0103] As used herein, the term "linker" refers to a linker that is used to link two meganuclease subunits. Linker refers to an exogenous peptide sequence used to link the A polypeptide may have a sequence found in a naturally occurring protein or may have a sequence not found in a naturally occurring protein. The linker may be flexible and devoid of secondary structure, or may be an artificial sequence that is not naturally occurring. The linker may have a property of forming a specific three-dimensional structure under physical conditions. , including but not limited to, U.S. Pat. No. 8,445,251 and U.S. Pat. No. 9,434 In some embodiments, the linker has an amino acid sequence including residues 154 to 195 of any one of SEQ ID NOs: 6 to 14 It is possible.

[0104] As used herein with respect to proteins, the terms "recombinant" or "engineered" refer to Genetically engineered nucleic acid that encodes a protein and a cell or organism that expresses that protein It means that the amino acid sequence has been altered as a result of the application of a technique. The terms "recombinant" or "engineered" refer to organisms that have been altered as a result of the application of genetic engineering techniques. Genetic engineering techniques include PCR and DNA cloning. cloning techniques; transfection, transformation and other gene transfer techniques; homologous recombination; site-specific mutagenesis; and gene fusion. By definition, a protein has an amino acid sequence identical to that of a naturally occurring protein, but is expressed in a heterologous host. Proteins produced by cloning and expression of are not considered recombinant.

[0105] As used herein, the term "wild type" refers to a gene that is a member of a population of alleles of the same species. Refers to the most common naturally occurring allele (i.e., polynucleotide sequence) and is the wild type The polypeptide encoded by the allele has its original function. The term "wild type" The term also refers to a polypeptide encoded by a wild-type allele. i.e., polynucleotides and polypeptides that have one or more mutations relative to the wild-type sequence. and / or substitutions. A wild-type allele or polypeptide confers a normal phenotype in an organism. However, mutant or variant alleles or polypeptides may optionally be Wild-type nucleases may confer an altered phenotype. The term "wild type" refers to the wild type allele of a particular gene, or for comparison purposes, Cells, organisms, and / or subjects having cells, organisms, and / or subjects used for It can also refer to a test subject.

[0106] As used herein, the term "genetically modified" refers to a genetic modification of a cell or organism, or an ancestor thereof. The term "cell or organism whose genomic DNA sequence has been deliberately altered by recombinant technology" was used above. As used herein, the term "genetically modified" includes the term "transgenic." Includes.

[0107] As used herein with respect to recombinant proteins, the term "modification" refers to a modification of a reference sequence (e.g., Insertion, deletion, or deletion of amino acid residues in the recombinant sequence relative to the wild-type or native sequence. It means substitution.

[0108] As used herein, the term "recognition sequence" or "recognition site" refers to a recognition sequence that is generated by an endonuclease. In the case of meganucleases, the recognition sequence is It contains a pair of inverted 9-base pair "half sites" separated by 4 base pairs. In the case of ganucleases, the N-terminal domain of the protein contacts the first half-site and The C-terminal domain of the protein contacts the second half-site. The cleavage produces a four base pair 3' "overhang." " is a short fragment that can be produced by endonuclease cleavage of a double-stranded DNA sequence. A single-stranded DNA segment. Meganuclease derived from I-CreI and single-stranded meganuclease In the case of nucleases, the overhang comprises bases 10 to 13 of the 22 base pair recognition sequence.

[0109] As used herein, the term "target site" or "target sequence" refers to a target site for recognition of a nuclease. Refers to the region of a cell's chromosomal DNA that contains the sequence.

[0110] As used herein, the term "DNA binding affinity" or "binding affinity" refers to the binding affinity of a meganuclease. The enzyme tends to non-covalently associate with a reference DNA molecule (e.g., a recognition sequence or any sequence). The binding affinity is expressed as the dissociation constant K d As used herein, If K d is statistically significant relative to the reference nuclease If the binding site is increased or decreased by a significant amount (p<0.05), the nuclease is considered to have "altered" binding sites. It has harmony.

[0111] As used herein, the term "specificity" refers to the ability of a particular base pair sequence, called a recognition sequence. Recognize and cleave double-stranded DNA molecules only in a sequence or at a specific set of recognition sequences. The set of recognition sequences refers to the ability of a meganuclease to recognize specific conserved positions or They share a sequence motif but may be degenerate at one or more positions. A ganucleases can cleave only one or a very few recognition sequences. Specificity is can be determined by any method known in the art. When used, the meganuclease is a reference meganuclease (e.g., a wild-type meganuclease) that is capable of synthesizing a nucleotide sequence similar to that of a reference meganuclease (e.g., a wild-type meganuclease) under physiological conditions. When the enzyme binds to and cleaves a recognition sequence that is not cleaved (type 1), or when the cleavage rate of the recognition sequence is A biologically significant amount (e.g., at least 2-fold or 2-fold) relative to the reference meganuclease If the specificity of the protein is increased or decreased (~10-fold), it has "altered" specificity.

[0112] As used herein, the term "homologous recombination" or "HR" refers to the process by which double-stranded DNA breaks occur in a homologous This refers to the natural cellular process by which DNA is repaired using the same DNA sequence as a repair template (e.g., Cahill et al.(2006),Front.Biosci.11:1958 Homologous DNA sequences may be endogenous chromosomal sequences or exogenous nucleic acids delivered to the cell. That's fine.

[0113] As used herein, the term "non-homologous end joining" or "NHEJ" refers to the process of joining double-stranded DNA A natural cellular process in which a break is repaired by direct joining of two non-homologous DNA segments. (e.g., Cahill et al. (2006), Front. Bioscience (See i.11:1958-1976.) DNA repair by non-homologous end joining is error-prone. This is prone to breakage, and the addition or deletion of non-template DNA sequences at the repair site frequently occurs. In some cases, cleavage at the target recognition sequence results in NHEJ at the target recognition site. Nuclease-induced cleavage of a target site in the coding sequence of a gene, followed by DNA repair by NHEJ, This introduces mutations into the coding sequence, such as frameshift mutations, disrupting gene function. Thus, engineered meganucleases could potentially isolate genes in a cell population. It can be used to effectively knock out enemies.

[0114] As used herein, the term "reduced" refers to a reduction in the symptoms or severity of a cholesterol-related disease. refers to a decrease in severity or a decrease in the protein expression or activity of PCSK9. In some circumstances, such reductions may be as low as 5%, 10%, 20%, 30%, 40%, 50% %, 60%, 70%, 80%, 90%, 95%, or even up to 100%. The term "reduced" refers to a decrease in a pathology, protein expression, protein activity, or binding to the LDL receptor. Both partial and complete reduction in binding of PCSK9 is included.

[0115] As used herein, the term "increased" refers to the display of LDL receptors on the cell surface. Such increases may be up to 5%, 10%, 20%, 30%, 40%, 5 0%, 60%, 70%, 80%, 90%, 95%, 100%, 150%, 250%, 50 0%, 1000%, or more. Increased surface display of LDL receptors Any method can be used to measure

[0116] As used herein, the term "hypercholesterolemia" refers to a condition in which cholesterol levels are In certain embodiments, LDL cholesterol is elevated above a desirable level. The terol level is elevated above a desirable level. In certain embodiments, the serum L DL-cholesterol levels are elevated above desirable levels. In this case, the term "familial hypercholesterolemia" or "FH" refers to the presence or absence of serum low-density lipoprotein This refers to a genetic disease characterized by elevated levels of low-density lipoprotein (LDL) cholesterol. Compared with the LDL cholesterol levels of Patients with combined FH had levels between 350 and 550 mg / dL, whereas patients with homozygous FH had levels between 6 These levels in patients or subjects with FH are Increased LDL cholesterol in the blood is associated with increased cholesterol deposition in tissues and cardiovascular disease in young adults. This leads to increased risk of disease.

[0117] As used herein with respect to both amino acid and nucleic acid sequences, the term "percent identity" refers to ", "sequence identity", "percent similarity", "sequence similarity", etc. are based on aligned amino acid residues or The similarity between two sequences based on aligning the sequences to maximize nucleotide similarity It refers to a measure of the degree of similarity between identical or similar residues or nucleotides in a sequence alignment. as a function of the number of octides, the number of total residues or nucleotides, and the presence and length of gaps. There are various algorithms and methods for determining sequence similarity using standard parameters. As used herein, sequence similarity is defined as the degree of similarity between two sequences. Using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences Both were measured by the National Center for Biotechnology Information. nology Information(www.ncbi.nlm.nih.gov / ) and are available, for example, in Altschul et al. (1990), J .Mol.Biol.215:403?410;Gish and States(19 93),Nature Genet.3:266?272;Madden et al. (1996),Meth.Enzymol.266:131?141;Altschul et al. (1997), Nucleic Acids Res. 25:33 89 ?3402;Zhang et al.(2000),J.Comput.Biol.7 (1-2):203-14. As used herein, the two amino acid sequences The percentage similarity of the strings was calculated using the BLASTp algorithm parameters: word size=3; Gap opening penalty = -11, gap extension penalty = -1, and score RING MATRIX = score based on BLOSUM62. As used herein The percentage similarity of two nucleic acid sequences is determined by the parameters of the BLASTn algorithm, i.e. Word size = 11, Gap opening penalty = -5, Gap extension penalty = =-2, match reward = 1, and mismatch penalty = -3.

[0118] As used herein in reference to modifications of two proteins or amino acid sequences, the term "antibody" refers to a combination of two or more amino acids. "Corresponds to" means that a particular modification in a first protein is the same as a modification in a second protein. The amino acid positions of the modifications in the first protein are the same as those in the first protein. Proteins are aligned by standard sequence alignment (e.g., using the BLASTp program). When provided, the amino acid position of the modification in the second protein corresponds to or is Therefore, in a sequence alignment, residues X and If the residues Y correspond to each other, they are the same as the residues for amino acid "A" in the first protein. The modification of group "X" corresponds to the modification of residue "Y" to amino acid "A" in a second protein. where residues X and Y may be in different numbers.

[0119] As used herein, the terms "recognition half-site," "recognition sequence half-site," and A "half site" is simply a monomer of a homodimeric or heterodimeric meganuclease. or by one subunit of a single-chain meganuclease. It refers to a nucleic acid sequence in a single-stranded DNA molecule.

[0120] As used herein, the term "hypervariable region" refers to amino acids having relatively high variability. Hypervariable regions refer to localized sequences within meganuclease monomers or subunits that contain the acid. , about 50-60 consecutive residues, about 53-57 consecutive residues, or preferably about 56 residues. In some embodiments, the hypervariable region residues can include a group selected from the group consisting of SEQ ID NOs:6-1. The hypervariable region may correspond to positions 24-79 or positions 215-270 of any one of the sequences of It may contain one or more residues that contact a DNA base in the recognition sequence, and may be a monomer or The hypervariable regions can also be modified to change the base selection of the subunits. , one that binds to the DNA backbone when the meganuclease associates with a double-stranded DNA recognition sequence or a plurality of residues. Such residues may be included in the DNA backbone and in the target recognition sequence. The present invention can be modified to change the binding affinity of the meganuclease to the In another embodiment, the hypervariable region may comprise 1 to 20 residues exhibiting variability, They can be modified to affect base selection and / or DNA binding affinity. In some embodiments, the variable residues in the hypervariable region are at any one of positions 6-14. Place 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, In certain embodiments, the hypervariable region Variable residues may also be located at one or more of positions 48, 50, 71, and 73 of SEQ ID NO:8. In another embodiment, the variable residues in the hypervariable region correspond to any one of SEQ ID NOs: 6-14. 215, 217, 219, 221, 223, 224, 229, 231, 233, Corresponding to one or more of 235, 237, 259, 261, 266, and 268. In a further embodiment, the variable residue within the hypervariable region also corresponds to position 258 of SEQ ID NO:12. You may do so.

[0121] The terms "recombinant DNA construct," "recombinant construct," "expression cassette," and "expression construct" The terms "chimeric construct," "construct," and "recombinant DNA fragment" are used interchangeably herein. Recombinant constructs include, but are not limited to, nucleic acid fragments that are not naturally occurring. "Genes" includes the artificial combination of nucleic acid fragments containing regulatory and coding sequences not found in the original nucleic acid sequence. For example, a recombinant DNA construct may contain regulatory and coding sequences derived from different sources, or Regulatory sequences and coding sequences derived from the same source and arranged in a manner different from that found in nature. Such constructs may be used alone or in combination with a vector. It may also be used in combination.

[0122] As used herein, a "vector" or "recombinant DNA vector" refers to a vector capable of expressing a given host cell. The method includes the steps of: (a) providing a method for the transcription and translation of a polypeptide coding sequence in a cell; When a vector is used, the selection of the vector is based on the host cell type well known to those skilled in the art. It depends on the method used to transform the host cell. Vectors include plasmids. Vectors and recombinant AAV vectors or genes encoding meganucleases of the present invention Any other vectors known in the art suitable for delivering the Those skilled in the art can easily use the isolated nucleotides or nucleic acids of the present invention, but are not limited to these. To successfully transform, select, and grow host cells containing any of the nucleic acid sequences, We are fully aware of the genetic factors that must be present on the vector.

[0123] As used herein, "vector" may also refer to a viral vector. As for virus vectors, retrovirus vectors, lentivirus vectors, adenovirus vectors, etc. Examples of vectors that can be used include viral vectors and adeno-associated viral vectors (AAV). Not limited to these.

[0124] As used herein, a "polycistronic" mRNA contains two or more coding sequences (i.e. refers to a single messenger RNA that contains multiple cistrons (protococcal RNA) and codes for multiple proteins. Polycistronic mRNAs contain IRES elements, T2A elements, and P2A elements. elements, E2A elements, and F2A elements, Any of the techniques known in the art that allow for the translation of two or more genes from the same mRNA molecule. It may contain any element.

[0125] As used herein, the term "control" or "control cell" refers to a genetically modified cell. A control cell refers to a cell that provides a reference point for measuring changes in cell type or phenotype. For example, (a) a wild-type cell, i.e., the starting material for genetic modification to produce a genetically modified cell; (b) cells of the same genotype as the genetically modified cells, but with a null construct (i.e., carrying a construct that has no known effect on the trait of interest) ) cells, or (c) cells that are genetically identical to the genetically modified cells but have an altered genotype or expression. This includes cells that have not been exposed to conditions or stimuli that induce expression of the phenotype or to further genetic modification. That's fine.

[0126] As used herein in reference to modifications of two proteins or amino acid sequences, the term "antibody" refers to a combination of two or more amino acids. "Corresponds to" means that a particular modification in a first protein is the same as a modification in a second protein. The amino acid positions of the modifications in the first protein are the same as those in the first protein. Proteins are aligned by standard sequence alignment (e.g., using the BLASTp program). When provided, the amino acid position of the modification in the second protein corresponds to or is Therefore, in a sequence alignment, residues X and If the residues Y correspond to each other, they are the same as the residues for amino acid "A" in the first protein. The modification of group "X" corresponds to the modification of residue "Y" to amino acid "A" in a second protein. where residues X and Y may be in different numbers.

[0127] As used herein, the term "treatment" or "treating a subject" refers to the treatment of a subject according to the present invention. The engineered meganuclease or nucleic acid encoding the engineered meganuclease of the invention is Disorders characterized by elevated levels of fat and / or cholesterol circulating in the blood, e.g. For example, the subject may have a cholesterol-related disorder. , cardiovascular disease, hypercholesterolemia including autosomal dominant FH, hypertriglyceridemia, and other cholesterol-related disorders. Preventing the onset or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of a disease, These include a reduction in the rate of disease progression, improvement or temporary relief of the disease condition, and remission or improvement of prognosis. Thus, treating a disease and / or disorder includes, but is not limited to, treating a disease and / or disorder. and / or altering (e.g., slowing) the progression of the disorder, total cholesterol levels and and / or low-density lipoprotein (LDL) cholesterol levels; Improving Gullar-Najjar syndrome, and regulating iron uptake by hepcidin and / or restoring function in hemochromatosis type 2, restoring bile acid metabolism; Reducing the risk of coronary heart disease in familial hypercholesterolemia, hands and / or feet Hyperkeratotic plaques can be cured by In some embodiments, the engineered meganuclear The enzyme, or a nucleic acid encoding same, is administered during therapy in the form of a pharmaceutical composition of the invention.

[0128] As used herein, a "cholesterol-related disease" includes hypercholesterolemia, Heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease Alzheimer's disease, and general dyslipidemia, e.g., high serum total cholesterol, high Cysts that can be manifested by LDL, high triglycerides, VLDL, and / or low HDL The engineered meganucleases disclosed herein include any one or more of the following: Primary and secondary inflammatory diseases that can be treated with Some non-limiting examples of secondary dyslipidemia include metabolic syndrome, Diabetes mellitus, familial combined hyperlipidemia, familial hypertriglyceridemia, heterozygous hypercholesterolemia cholesterolemia, familial hypercholesterolemia including homozygous hypercholesterolemia, family Defective apolipoprotein B-100; Polygenic hypercholesterolemia; Remnant removal Hepatic lipase deficiency; poor diet; hypothyroidism; estrogen and progesterone Following any of the following medications: gestin therapy, beta-blockers, and thiazide diuretics dyslipidemia caused by nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary hepatitis Cirrhosis, glycogen storage disease, liver cancer, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency and alcoholic hypertriglyceridemia. Ganucleases are also useful in treating, for example, coronary heart disease, coronary artery disease, peripheral artery disease, stroke, and other conditions. (ischemic and hemorrhagic), angina, or cerebrovascular disease and acute coronary syndrome, myocardial infarction, etc. They may also be useful in the prevention or treatment of atherosclerosis.

[0129] The term "proprotein convertase subtilisin kexin type 9" or "PCSK9" The human PCSK9 gene (and active PCSK9 polypeptide) as set forth in SEQ ID NO:3 A polypeptide encoded by the PCSK9 gene, such as a PCSK9-coding mutant thereof, or Fragments, as well as allelic, splice, derivative and substitution variants variants, deletion variants, and / or insertion variants (including the addition of an N-terminal methionine) ), fusion polypeptides, and interspecies homologs. In certain embodiments, the PCSK9 polypeptide refers to a leader peptide. sequence residues, target residues, amino-terminal methionine residues, lysine residues, tag residues, and / or fusion residues. These include, but are not limited to, terminal residues such as PCSK9 fusion protein residues. " is a protein that regulates the expression of FH3, NARC1, HCHOLA3, and proprotein convertase subtilisin Also known as kexin type 9 and neuronal apoptosis-regulating convertase 1. PCSK Nine genes belong to the proteinase K subfamily of the secretory subtilase family. The term "PCSK9" refers to a proprotein convertase protein. It refers to both the product produced after autocatalysis of the protein and the proprotein. When only the product is cited, the protein may be expressed as "mature," "cleaved," or "pro PCSK9 can be referred to as "activated" or "active" PCSK9. Only the inactive form is When used, the protein refers to the "inactive," "pro" or "proform" of PCSK9. The term "unprocessed" may be used herein to refer to the "unprocessed" form. "PCSK9" is a naturally occurring mutant of the 9-aminobutyric acid (ACA) protein that inhibits the 9-aminobutyric acid (ACA) mutations D374Y, S127R, and F216L. The term "PCSK9" also includes glycosylated, PEGylated PCSK9 sequences. PCSK9 sequence with its signal sequence cleaved, catalytic domain to its prodomain PCSK9 sequences that are truncated from the PCSK9 domain but not separated from the catalytic domain Also included are PCSK9 molecules that incorporate post-translational modifications of the amino acid sequence.

[0130] The term "PCSK9 activity" includes any biological action of PCSK9. In some embodiments, PCSK9 activity is determined by interacting with a substrate or receptor, or by binding to a substrate or receptor. In some embodiments, PCSK9 activity comprises the ability of PCSK9 to bind to a receptor. , expressed by the ability of PCSK9 to bind to the LDL receptor (LDLR). In embodiments, PCSK9 binds to the LDLR and catalyzes reactions involving the LDLR. For example, PCSK9 activity alters (e.g., decreases) the availability of LDLR. Thus, in some embodiments, PCSK9 activity is measured in a subject In certain embodiments, the PCSK9 activity includes the ability of PCSK9 to increase the amount of LDL in These include the ability of PCSK9 to reduce the amount of LDLR available to bind to LDL. Thus, by decreasing PCSK9 activity, the surface-displayed and In some embodiments, the amount of LDLR that can find LDL is increased. "Activity" includes any biological activity resulting from PCSK9 signaling. Exemplary activities include These include the binding of PCSK9 to LDLR, PCSK9 cleaving LDLR or other proteins, and K9 enzyme activity, binding of PCSK9 to proteins other than LDLR that promote PCSK9 action In this case, PCSK9, which alters APOB secretion (Sun et al. (2005), Hum an Molecular Genetics 14:1161-1169, and Aug. uerram et al. (2004), Arterioscler thromb Vasc Biol.24:1448-1453), in liver regeneration and neuronal differentiation The role of PCSK9 in the regulation of inflammatory bowel disease (Seidah et al., PNAS 100:928-93 3, 2003) and the role of PCSK9 in hepatic glucose metabolism (Costet t al. (2006), J. Biol. Chem. 281(10):6211-18) These include, but are not limited to:

[0131] The terms "effective amount" or "therapeutically effective amount" refer to an amount that induces beneficial or desired biological and / or clinical results. A therapeutically effective amount refers to an amount of a meganuclease combination or composition, therapeutic Depending on the disease and its severity, age, weight, health status, and responsiveness of the subject being treated In certain embodiments, an effective amount of an engineered meganuclease disclosed herein Alternatively, the pharmaceutical composition may be used to increase the total cholesterol or LDL cholesterol (i.e., serum L It reduces the levels of cholesterol and other cholesterol-related cholesterol-related disorders, and / or dyslipidemia, atherosclerosis, and cardiovascular disease (CVD) or coronary heart disease.

[0132] The term "lipid nanoparticles" refers to spherical particles typically with an average diameter of 10 to 1000 nanometers. In some formulations, the lipid nanoparticles are at least one one cationic lipid, at least one non-cationic lipid and at least one complex lipid It is well known in the art that is suitable for encapsulating nucleic acids such as mRNA. Any lipid nanoparticle known in the art is contemplated for use in the present invention.

[0133] As used herein, the recitation of a numerical range for a variable indicates that the invention encompasses any of the values ​​within that range. The intent is to convey that the implementation may be implemented with variables equal to any of the above. For a discrete variable, the variable can be equal to any integer value within the numeric range, including the end points of the range. Similarly, if a variable is inherently continuous, the variable can be expressed as a number within a numerical range, including the end-points of the range. By way of example and not limitation, , may have a value between 0 and 2. A variable marked with "|" can take on values ​​of 0, 1, or 2 if the variable is discrete in nature. and, if the variable is continuous in nature, 0.0, 0.1, 0.01, 0.0 It can be 0, 1, or any other real value between 0 and 2 inclusive.

[0134] 2.1 Principle of the present invention The present invention relates, in part, to a method for reducing the expression of PCSK9 using engineered meganucleases. This can reduce the amount of cholesterol in the blood, thereby increasing the removal of fat and / or cholesterol from the blood. The hypothesis is that decreased expression of PCSK9 can block LDL receptors on the cell surface. This can increase the amount of lipids in the bloodstream, thereby increasing the amount of lipids removed from the bloodstream. To deliver an engineered meganuclease specific to the recognition sequence of the PCSK9 gene. By using the antibody, PCSK9 expression can be reduced, which subsequently reduces the total PCSK9 in the blood of the subject. It is possible to reduce cholesterol (e.g., blood LDL). The disclosed methods and compositions provide an increased ability to inhibit PCSK9 expression levels compared to well-controlled PCSK9 expression levels. of particular use in the treatment of cholesterol-related disorders caused by increased expression of Find a way.

[0135] Thus, the present invention provides an engineered megagene that recognizes and cleaves a recognition sequence within the PCSK9 gene. The present invention relates to pharmaceutical compositions and methods for treating cholesterol-related disorders, such as , and the use of such engineered meganucleases in methods of treating hypercholesterolemia. The present invention also encompasses methods of using engineered meganuclease proteins, or a pharmaceutical composition comprising a nucleic acid encoding an engineered meganuclease; and cholesterol. and the use of such compositions for the treatment of related disorders, such as hypercholesterolemia. Includes.

[0136] 2.2 Meganuclease for recognizing and cleaving the recognition sequence in the PCSK9 gene Site-specific nucleases can be used to introduce breaks into the PCSK9 gene, Repair of such breaks prevents expression of the active PCSK9 gene via NHEJ. Thus, in one embodiment, the present invention provides a method for the production of engineered This can be done using recombinant meganucleases.

[0137] In a preferred embodiment, the nuclease used to practice the present invention is a single-stranded megakaryon The single-chain meganuclease is a nuclease consisting of a nucleotide sequence linked by a linker peptide. It contains an N-terminal subunit and a C-terminal subunit. Each of the two domains has a recognition sequence The recognition site of the DNA is determined by the position of the two subunits. The DNA strand break occurs when the meganuclease breaks the DNA. offset by 4 base pairs so that the fragment generates a 4 base pair 3' single-stranded overhang. will be done.

[0138] In some instances, the engineered meganucleases of the invention comprise the PCS7-8 recognition sequence ( The PCS7-8 recognition sequence is the sequence The PCSK9 gene is located within the PCSK9 gene as defined in SEQ ID NO:3. The nucleases are collectively referred to herein as "PCS7-8 meganucleases." Suitable PCS7-8 meganucleases are provided in SEQ ID NOs:6-14.

[0139] The engineered meganuclease of the present invention comprises a first hypervariable (HVR1) region. and a second subunit comprising a second hypervariable (HVR2) region. The first subunit binds to the first recognition half-site of the recognition sequence (e.g., PCS7 half-site). The first subunit binds to a second recognition half-site of the recognition sequence (e.g., For example, the PCS7 half-site. The engineered meganuclease binds to the single-stranded meganuclease. In an embodiment, the first half-site is a ribozyme that comprises an HVR1 region and binds to the first half-site. The second half of the The first subunit is arranged so that the second subunit that binds to the site is located as the C-terminal subunit. The HVR1 region and the second subunit can be oriented. and a first subunit that binds to the first half-site as a C-terminal subunit. The second subunit, which contains the HVR2 region and binds to the second half-site, is located at the N-terminus Orienting the first and second subunits so that they are positioned as end subunits Exemplary PCS7-8 meganucleases of the invention are shown in Table 1.

[0140] Table 1. Exemplary polypeptides engineered to recognize and cleave the PCS7-8 recognition sequence (SEQ ID NO:4) Engineered meganucleases [Table 1]

[0141] * "PCS7 subunit %" is the percentage of PCS7-binding subunit domains in each meganuclease. Amiability between the PCS7-binding subunit region and the PCS7-8L.197 meganuclease "PCS8 subunit %" indicates the amino acid sequence identity of each meganuclease. 8-binding subunit domain and PCS8-binding subunit of PCS7-8L.197 meganuclease It indicates the amino acid sequence identity with the unit region.

[0142] 2.3 Methods for delivery and expression of endonucleases Disclosed herein are methods for treating hypercholesterolemia and cardiovascular disease in a subject. Similarly, a pharma- ceutically acceptable carrier and the engineered meganuclease ( or a nucleic acid encoding an engineered meganuclease. Methods for reducing symptoms of hypercholesterolemia and cardiovascular disease in a subject are provided, comprising administering to a subject a The engineered meganucleases disclosed herein can be delivered to target cells in a subject. Also provided are methods for reducing expression and / or activity of PCSK9 in a subject, comprising administering to a subject a therapeutically effective amount of PCSK9. In the methods of the invention, the engineered meganucleases disclosed herein induce D The polypeptide may be delivered to and / or expressed from NA / RNA.

[0143] The engineered meganucleases disclosed herein are available in the form of proteins or, preferably, The engineered meganuclease is delivered to the cell as a nucleic acid encoding the engineered meganuclease. The nucleic acid may be DNA (e.g., circular or linear plasmid DNA or PCR products) or RNA. The engineered meganuclease coding sequence may be a DNA fragment (e.g., an mRNA). In the embodiment where the nuclease gene is delivered in a nucleic acid form, the promoter is provided to drive transcription of the nuclease gene. Mammalian promoters suitable for the present invention include constitutive promoters, Thom type promoters, such as the cytomegalovirus early (CMV) promoter sen et al. (1984), Proc Natl Acad Sci USA. 81(3):659-63) or the SV40 early promoter (Benoist and Chambon(1981), Nature 290(5804):304-10) Inducible promoters, such as the tetracycline-inducible promoter (Dingerman et al. ann et al. (1992), Mol Cell Biol. 12(9):403 8-45). The engineered meganucleases of the present invention are linked to synthetic promoters. Synthetic promoters include the JeT promoter (International Publication No. Examples of the specific implementations include, but are not limited to, those disclosed in the US Pat. In embodiments, the nucleic acid sequence encoding the engineered meganuclease disclosed herein is , can be operably linked to a liver-specific promoter or a hepatocyte-specific promoter Examples of liver-specific promoters include, but are not limited to, the human alpha-1 antitrypsin promoter. Motor, a hybrid liver-specific promoter (hepatic locus control region of the ApoE gene) region (ApoE-HCR and liver-specific α1-antitrypsin promoter), human thyroxine The thymine-binding globulin (TBG) promoter and the apolipoprotein A-II promoter - are some examples.

[0144] In certain embodiments, a nucleic acid encoding at least one engineered meganuclease The sequence may be delivered in a recombinant DNA construct or an expression cassette. For example, The present invention relates to a method for producing a meganuclease comprising the steps of: In another embodiment, the nucleic acid sequence may be included in an expression cassette (i.e., a "cassette") that includes the nucleic acid sequence. The recombinant DNA construct comprises two or more cassettes, each cassette comprising a promoter and a and a nucleic acid sequence encoding the engineered meganuclease described herein. In some embodiments, the recombinant DNA construct may include two cassettes, three cassettes, four cassettes, or can include more.

[0145] In another embodiment, the recombinant DNA construct comprises a promoter and a polycistronic nucleic acid sequence. wherein the promoter drives expression of the polycistronic nucleic acid sequence and the target The cells are caused to produce the polycistronic mRNA described herein.

[0146] In some embodiments, the mRNA encoding the engineered meganuclease is expressed in the cell. The gene encoding the engineered meganuclease is delivered to the This reduces the chances that the engineered meganuclear fragment will be integrated into the cell's genome. Such mRNA encoding the enzyme can be prepared by methods known in the art, such as in vitro transcription. In some embodiments, the mRNA can be produced using 7-methylguanosine. Capped using Syn, ARCA, CleanCap or Vaccin In some embodiments, the nucleic acid is capped enzymatically, such as with a capping enzyme. In some cases, the mRNA may be polyadenylated. In order to enhance the expression of meganuclease and / or to increase the stability of the mRNA itself, various The mRNA may contain 5' and 3' untranslated sequence elements such as pseudouridine, 5 -methylcytidine, N6-methyladenosine, 5-methyluridine, or 2-thiouridine The nucleoside analogues may include nucleoside analogues such as riboflavin.

[0147] In certain embodiments, the mRNA encoding the engineered nuclease of the invention is It was a polycistronic mRNA encoding two or more nucleases that were simultaneously expressed in the In some embodiments, the polycistronic mRNA may comprise two or more of the sequences described herein. and at least one further modification that provides a therapeutically beneficial effect in a cell. The polycistronic mRNA of the present invention can encode a protein having an IRES element. element, T2A element, P2A element, E2A element, and F2A element These include, but are not limited to, those that allow translation of two or more genes from the same mRNA molecule. The present invention may include any element known in the art that enables a particular implementation. In this form, the polycistronic mRNA encodes two meganucleases as described herein. A bicistronic mRNA encoding the three meganucleases described herein It is a tricistronic mRNA.

[0148] In another specific embodiment, the nucleic acid encoding the endonuclease of the invention is a single-stranded D The single-stranded DNA can be delivered to target cells as a NA template by engineered meganucleases. 5' and / or 3' AAV inverted terminal repeats (I In another embodiment, the single stranded DNA may further comprise an engineered megagene. The 5' and / or 3' homology arms are located upstream and / or downstream of the nucleic acid encoding sequence. It may further include:

[0149] In another specific embodiment, the gene encoding the endonuclease of the present invention is a linear D In some instances, the endonuclease can be delivered to a target cell as a NA template. The encoding plasmid DNA is linearized from the circular plasmid DNA prior to delivery to the target cell. The nucleic acid may be digested with one or more restriction enzymes so as to

[0150] The purified nuclease protein is delivered to cells to induce cellular cellular responses, as described in further detail herein below. Cleavage of genomic DNA by a variety of different mechanisms known in the art, including It is possible.

[0151] Target tissues for delivery of the engineered meganucleases of the present invention include, but are not limited to, However, hepatocytes, or preferably primary hepatocytes, more preferably human hepatocytes or human Hepatic cells such as primary human hepatocytes, HepG2.2.15 or HepG2-hNTCP cells In certain embodiments, the cells are primate hepatocytes, such as primary primate hepatocytes. As explained, the meganuclease of the present invention is available as a purified protein. or as RNA or DNA encoding the meganuclease. In one embodiment, the protein encoding the meganuclease or endonuclease The mRNA or DNA vectors can be delivered to target cells (e.g. , liver cells). Alternatively, the meganuclease protein, mRNA, or DNA can be delivered systemically via the circulatory system.

[0152] In some embodiments, the endonuclease protein or meganuclease is The DNA / mRNA to be loaded is prepared in a pharma- ceutically acceptable carrier according to known techniques. It can be formulated for systemic administration or for administration to a target tissue. gton,The Science And Practice of Pharmac In the manufacture of the pharmaceutical formulation according to the invention, The protein / RNA / mRNA is typically mixed with a pharma- ceutically acceptable carrier. must, of course, be acceptable in the sense of being compatible with the other ingredients in the formulation. The carrier may be a solid, liquid, or both. , may be formulated with the compound as a unit dose formulation.

[0153] In some embodiments, the meganuclease protein or the meganuclease is The DNA / mRNA to be delivered is bound to a membrane-permeable peptide or a targeting ligand, allowing for cellular uptake. Examples of membrane-permeable peptides known in the art include polyarginine (Jea rawiririyapaisarn et al.(2008)Mol Ther.16: 1624-9), and the TAT peptide derived from the HIV virus (Hudecz et al. 2005), Med.Res.Rev.25:679-736), MPG(Simeon i et al. (2003), Nucleic Acids Res. 31:2717 -2724), Pep-1 (Deshayes et al. (2004), Bioch emistry 43:7698-7706, and HSV-1 VP-22 (Desha yes et al.(2005),Cell Mol Life Sci.62:18 39-49). In an alternative embodiment, the meganuclease protein, or The DNA / mRNA encoding the ganucleases is converted to the meganuclease protein / DNA on the target cell so that the mRNA binds to and is internalized by the target cell. The antibody is covalently or non-covalently linked to an antibody that recognizes a specific cell surface receptor expressed on the Alternatively, meganuclease proteins / DNA / mRNA may be involved in the transcription of such cell surface receptors. It can be covalently or non-covalently bound to the natural ligand (or a portion of the natural ligand) McCall et al. (2014), Tissue Barriers.2(4) :e944449;Dinda et al.(2013),Curr Pharm B iotechnol.14:1264-74;Kang et al.(2014),C urr Pharm Biotechnol.15 (3):220-30; Qian et al.(2014),Expert Opin Drug Metab Tox icol.10(11):1491-508).

[0154] In some embodiments, the meganuclease protein or the meganuclease is The DNA / mRNA to be delivered is delivered via a biodegradable hydrogel for injection or implantation into the desired area of ​​the liver. The hydrogel allows the drug to be delivered to the target tissue without the need for frequent injections. The present invention provides a method for the delivery of therapeutic payloads to a desired area of ​​tissue by providing a sustained and tunable release of the therapeutic payload to a desired area of ​​tissue. Thermoresponsive materials (e.g., temperature-responsive hydrogels and pH-responsive hydrogels) are environmentally or They can be designed to release a payload in response to an exogenously applied cue ( Kang Derwent et al.(2008),Trans Am Ophth almol Soc.106:206-214).

[0155] In some embodiments, the meganuclease protein or the meganuclease is The DNA / mRNA to be encoded is covalently attached to the nanoparticles using methods known in the art. Preferably, the nanoparticles are non-covalently bound or encapsulated within the nanoparticles. arma et al.(2014),Biomed Res Int.2014:32 7950). Nanoparticles are nanoparticles with length scales less than 1 μm, preferably less than 100 nm. Nanoscale delivery systems include nanoparticles made of metals, lipids, polymers, Alternatively, the meganuclease protein may be designed with a core composed of a biopolymer. Multiple copies of proteins, mRNA, or DNA are attached to or encapsulated in the nanoparticle core. This allows the protein / mRNA / DNA copies delivered to each cell to be By increasing the number of targets and thus the intracellular expression of each meganuclease, This maximizes the chance of the array being cleaved. For example, chitosan, cationic polymers, or cationic lipids) to further modify the surface. Form core-shell nanoparticles with additional functionalization for cellular delivery and payload uptake (Jian et al. (2012), Biomaterials 33(30):7621-30). Advantageously, the nanoparticles can be further conjugated to a targeting molecule to The nanoparticles may be targeted to the appropriate cell type and / or may increase the likelihood of cellular uptake. Examples of such targeting molecules include antibodies specific for cell surface receptors and natural targets of cell surface receptors. Ligands (or parts of natural ligands).

[0156] In some embodiments, the meganuclease protein or the meganuclease is The DNA / mRNA to be delivered is encapsulated or complexed in liposomes using cationic lipids. (e.g., LIPOFECTAMINE transfection reagent, Life Technologies Corp., Carlsbad, CA; al.(2015), Nat Biotechnol.33:73-80;Mishra et al. (2011), J Drug Deliv.2011:863734. Liposome and lipoplex formulations protect the payload from degradation and deliver it to the target site. and promotes cellular uptake by fusing with and / or disrupting the plasma membrane of target cells. This can enhance the absorption and delivery efficiency.

[0157] In some embodiments, the meganuclease protein or the meganuclease is The DNA / mRNA to be delivered is encapsulated within a polymer scaffold (e.g., PLGA) or or complexed with cationic polymers (e.g., PEI, PLL) (Tambo li et al. (2011), Ther Deliv.2(4):523-536) Polymeric carriers provide tunable drug release rates through control of polymer erosion and drug diffusion. can be designed to deliver highly efficient drug encapsulation to desired target cells This can provide protection of the therapeutic payload until intracellular delivery to the population.

[0158] In some embodiments, the meganuclease protein or engineered meganuclease The DNA / mRNA encoding the enzyme is combined with amphiphilic molecules that self-assemble into micelles. (Tong et al. (2007), J Gene Med.9(11):95 6-66). Polymer micelles are made of hydrophilic polymers (e.g., polyethylene glycol). A micellar shell may be formed, and the hydrophilic polymer may act to prevent aggregation and to inhibit charge interactions. This can mask the effects of the antibody and reduce non-specific interactions.

[0159] In some embodiments, the meganuclease protein or the meganuclease is The DNA / mRNA to be injected may be formulated in an emulsion or other suitable formulation for administration and / or delivery to target cells. The emulsion is formulated into a nanoemulsion (i.e., an average particle diameter of less than 1 nm). "Polymerization" includes, but is not limited to, oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil. It refers to a dispersion or droplet in which non-polar residues (e.g., long carbon atoms) form when the water-immiscible phase is mixed with the aqueous phase. Hydrophobic forces that move the hydrogen chain away from the water and the polar head group towards the water These other lipid structures include: , unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and Emulsions include, but are not limited to, lamellar phases. Emulsions are composed of an aqueous phase and a lipophilic phase ( Typically, emulsions are made up of one or more liquid phases (usually containing oil and organic solvents). They often contain multiple surfactants. Nanoemulsion formulations are described, for example, in U.S. Pat. Application Nos. 2002 / 0045667 and 2004 / 0043041, and U.S. Pat. Nos. 6,015,832, 6,506,803, 6,635,676, and 6 ,559,189, each of which is incorporated herein by reference. The entirety of which is incorporated herein.

[0160] In some embodiments, the meganuclease protein or the meganuclease is The DNA / mRNA to be encoded is prepared using multifunctional polymer complexes, DNA dendrimers, and polymers. -Covalently or non-covalently attached to the dendrimer (Mastorakos et al.(2015),Nanoscale 7(9):3845-56;Che ng et al. (2008), J Pharm Sci.97(1):123-43 Dendrimer synthesis allows for control of payload capacity and size, resulting in high drug delivery. Further, the display of multiple surface groups can be used to provide payload capacity. , improving stability, reducing non-specific interactions, and enhancing cell-specific targeting and drug release. It is possible.

[0161] In some embodiments, the gene encoding the meganuclease is delivered via a viral vector. Such vectors are known in the art and are used to deliver the vectors to the host. vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viruses Examples of vectors include AAV vectors (Vannucci et al. (2013), N (Reviewed in J. Microbiol. 36:1-22). Alternatively, the viral vector is injected directly into the target tissue (e.g., liver tissue). In the past, viral vectors have been delivered systemically via the circulatory system. It is known that AAV vectors tend to localize in different tissues. Effective transduction of hepatocytes has been demonstrated, for example, with AAV serotypes 2, 8, and 9. (Sands(2011),Methods Mol Biol 807:141-15 7; International Patent Publication No. 2003 / 052051). AAV vectors can also be used to infect host cells. The primers may be self-complementary so as not to require second strand DNA synthesis in the presence of a nucleotide (McCarty et al. (2001), Gene Ther 8:1248-54).

[0162] In one embodiment, the viral vector used for endonuclease gene delivery is Self-limiting viral vectors are vectors that have an engineered Due to the presence of a recognition sequence for the meganuclease, its duration may be limited in a cell or organism. Thus, a self-limiting viral vector can be engineered to contain a promoter, an endonucleaser as described herein. It is possible to provide codes for the endonuclease and the endonuclease recognition site within the ITR. Self-limiting viral vectors can deliver endonuclease genes to cells, tissues, or organisms. Upon delivery to the target organism, the endonuclease is expressed and targets the cell at its endogenous recognition sequence in the genome. The delivered endonuclease also allows the endonuclease to cleave the genome of the host. It finds its target site within the self-limiting viral vector itself and cuts the vector at this target site. Once cleaved, the 5' and 3' ends of the viral genome are digested by exonucleases. When exposed to the virus, it is degraded, thereby killing the virus and stopping the production of the endonuclease. do.

[0163] The endonuclease gene is in DNA form (e.g., a plasmid) and / or is viral When delivered via a vector (e.g., AAV), they are operably linked to a promoter In some embodiments, this may be linked to a viral vector (e.g., lentivirus). viral vector LTR) or the well-known cytomegalovirus early promoter or Viral promoters, such as endogenous promoters derived from the SV40 virus early promoter In a preferred embodiment, the meganuclease gene is The gene is operably linked to a promoter that preferentially drives gene expression. Examples of promoters include, but are not limited to, the human alpha-1 antitrypsin promoter, the hybrid The hepatic locus control region of the ApoE gene (ApoE-HC R) and liver-specific α1-antitrypsin promoter), human thyroxine-binding globulin (TBG) promoter and the apolipoprotein A-II promoter.

[0164] In certain embodiments, the viral vector comprises a promoter and an engineered vector as described herein. and a nucleic acid sequence encoding a meganuclease. It may also contain two or more cassettes, each of which contains a promoter and a manipulation as described herein. and a nucleic acid sequence encoding the engineered meganuclease. The virus vector contains a cassette containing a promoter and a polycistronic nucleic acid sequence. The promoter drives expression of the polycistronic nucleic acid sequence as described herein in the target cell. The polycistronic mRNA may be, for example, a polycistronic mRNA encoding an engineered meganuclease. Produces thrombotic mRNA.

[0165] People with cholesterol-related disorders, such as hypercholesterolemia, including autosomal dominant FH Methods and compositions for delivering the meganucleases disclosed herein to the liver of a subject suffering from In one embodiment, the natural hepatocytes derived from the mammal are treated with engineered megakaryocytes. Alternatively, the subject's natural gene may be transduced with a vector encoding the nuclease. The hepatocytes are then transfected with the engineered meganuclease and / or a molecule that stimulates liver regeneration, e.g., hepatocytes. The adenovirus vector encoding the endotoxin may be transduced ex vivo. Preferably, the hepatotoxin is uPA, which is secreted from hepatocytes when expressed by a viral vector. In another embodiment, the vector is modified to inhibit secretion from hepatocytes. The cells are then extracted from the mammal and the tPA is then inserted into the mammalian host. The transduced hepatocytes can then be transferred back into the mammalian host, where they express the engineered meganucleases. Typically, the transduced hepatocytes are delivered via the spleen or portal system. The drug can be administered to the patient over a period of 1 to 5 days or more. This may be once or multiple times throughout.

[0166] In an in vivo embodiment of the method of the present invention, a subject is administered a The retroviral, pseudotyped or adenoviral associated vector to be administered is constructed. Administration of a vector encoding the meganuclease induces the production of a secretory hepatotoxin, or an antigen encoding tPA that stimulates hepatocyte regeneration without acting as a hepatotoxin. This can be accomplished by administration of a denovovirus vector.

[0167] The appropriate dose depends, among other factors, on the liposomal formulation used and the AAV vector of choice. The specificity of the agent (e.g., serotype, etc.), the route of administration, the subject being treated (i.e., age of the subject, The appropriate dose depends on the patient's age, weight, sex, and general condition, and the method of administration. The appropriate effective amount can be readily determined by one of ordinary skill in the art. Treatment may be a single dose schedule or a multiple dose schedule. The compound may be administered as many times as necessary. Those skilled in the art can readily determine the appropriate dosage frequency. Alternative routes of administration may be considered, or the therapeutic benefits may need to be balanced against side effects. Doses may need to be adjusted to accommodate the increased risk of pulmonary embolism.

[0168] The engineered meganucleases disclosed herein or the engineered meganucleases disclosed herein Delivery of a nucleic acid encoding a nuclease to a subject can be used to treat a cholesterol-related disorder, e.g., To treat or reduce the severity of hypercholesterolemia, including autosomal dominant FH In certain embodiments, the engineered meganucleases disclosed herein can be Alternatively, delivery of a nucleic acid encoding an engineered meganuclease to a subject can be achieved by administering to the subject cholesterol-lowering At least one symptom of hypercholesterolemia, including autosomal dominant FH, In some embodiments, the engineered meganucleases, or The nucleic acid encoding the engineered meganuclease is delivered to the subject in an effective amount. The engineered meganucleases disclosed herein or the engineered meganucleases disclosed herein The subject to which the nucleic acid encoding the lyase is delivered may be any mammal. In embodiments, the subject may be a domesticated animal (e.g., cows, sheep, cats, dogs, and horses), spirit animal, or animal of a similar age. Long-legged primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., In certain embodiments, the subject is a non-human primate or a human. In some embodiments, the subject has a cholesterol-related disorder. Subjects were randomly assigned to receive a 2-item randomized controlled trial for hypercholesterolemia, familial hypercholesterolemia, or autosomal dominant familial hypercholesterolemia. In certain embodiments, the subject may have hypercholesterolemia. / dL or more than 200 mg / dL, e.g., 170-200 mg / dL or 200-25 0mg / dL, 200~300mg / dL, 200~350mg / dL, 200~400 mg / dL, 200-450mg / dL, 200-500mg / dL, or 200-60 In some embodiments, the subject has a total cholesterol level of 0 mg / dL. 110 mg / dL or more than 130 mg / dL, for example, 110 to 120 mg / dL, 110 ~130mg / dL, 130~150mg / dL, 130~180mg / dL, 130~ 200mg / dL, 130~250mg / dL, 130~300mg / dL, 130~3 50mg / dL, 130~400mg / dL, 130~450mg / dL, 130~50 0 mg / dL, or have an LDL cholesterol level between 130 and 600 mg / dL.

[0169] In certain embodiments, the meganucleases disclosed herein or the engineered Delivery of a nucleic acid encoding a specific meganuclease to a subject reduces expression of PCSK9 and / or decrease PCSK9 activity. For example, expression of PCSK9 or the activity is at least about 5% greater than or equal to the baseline PCSK9 activity in a control cell. %, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% %, or up to 100%. In some embodiments, expression of PCSK9 and / or Activity is approximately 5-10%, 5-10%, or 5-10% compared to control cells or baseline PCSK9 activity. 20%, 10-30%, 20-40%, 30-50%, 40-60%, 50-70%, 6 PCSK9 activity can be reduced by 0-80%, 70-90%, or 80-100%. As disclosed elsewhere in this specification, measured by any means known in the art. As used herein, a control cell can be a pharmaceutical composition, an engineered megakaryon, Nucleic acids encoding the nucleases or engineered meganucleases disclosed herein The control may be any suitable control, such as cells from the subject prior to delivery of the antibody. The target cells can be administered a pharmaceutical composition, an engineered meganuclease, or an engineered method as disclosed herein. Hepatocytes or primary hepatocytes of a subject to which a nucleic acid encoding a meganuclease has not been delivered. As used herein, a subject's "baseline" level (e.g., PCSK9 expression or activity or total cholesterol level or LDL cholesterol The baseline level of the level, etc., is measured before administration of the pharmaceutical compositions described herein to the individual. In certain embodiments, baseline refers to the level of The mean or average of two or more measurements taken before administration That's fine.

[0170] In some embodiments, the meganucleases disclosed herein or Delivery of the nucleic acid encoding the engineered meganuclease to a target cell of a subject is achieved by cell-mediated isoformation of the nucleic acid. Increase the display of LDL receptors on the surface or increase the level of LDL receptors. The level of LDL receptors can be determined by measuring the presence or absence of LDL receptors or agonists in the liver of a subject. The level of polypoprotein B (APOB) receptor may be measured by any method known in the art. For example, the display or level of LDL receptors can be measured by When compared to control cells or baseline levels of LDL receptors on the cell surface, or an engineered meganuclease as disclosed herein. The nucleic acid is delivered to the target cell, and the nucleic acid is then depleted by at least about 5%, 10%, 20%, 30%, 40%, or 50% after delivery to the target cell. %, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 150%, 250% , 500%, 1000% or more.

[0171] The pharmaceutical compositions, engineered meganucleases, or engineered meganucleases disclosed herein Delivery of a nucleic acid encoding a leukemia enzyme to a target cell in a subject reduces the total cholesterol level of the subject prior to delivery. The effect of the present invention may be to reduce the total cholesterol level of a subject when compared to the total cholesterol level of a subject. , total cholesterol levels are reduced by at least about 5%, 10%, or 20% compared to subjects before treatment. %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or In certain embodiments, the pharmaceutical compositions disclosed herein can reduce Total cholesterol levels in subjects receiving the drug were monitored for at least 2 weeks after the last dose. The total cholesterol level is measured for at least 1 month, at least 2 months, or at least 3 months prior to delivery of the pharmaceutical composition. maintains a reduction relative to teratogenic levels.

[0172] In certain embodiments, total cholesterol levels are measured in subjects receiving the pharmaceutical compositions disclosed herein. Compared to cholesterol levels before delivery to the body, the levels were approximately 5 mg / dL and 10 mg / dL. L, 15mg / dL, 20mg / dL, 25mg / dL, 30mg / dL, 40mg / d L, 50mg / dL, 60mg / dL, 70mg / dL, 80mg / dL, 90mg / d L, 100mg / dL, 110mg / dL, 5~15mg / dL, 10~20mg / dL , 10~30mg / dL, 15~30mg / dL, 20~30mg / dL, 25~35m g / dL, 25~40mg / dL, 25~50mg / dL, 40~60mg / dL, 50 ~70mg / dL, 60-80mg / dL, 70-100mg / dL, or more decrease In certain embodiments, the subject has a blood glucose level of 140 mg / dL, 150 mg / dL, 160 mg / dL, g / dL, 170mg / dL, 175mg / dL, 180mg / dL, 185mg / dL , 190mg / dL, 195mg / dL, 200mg / dL, 205mg / dL, 210 mg / dL, 215mg / dL, 220mg / dL, 225mg / dL, 230mg / d L, 235mg / dL, 240mg / dL, 250mg / dL, 260mg / dL, 27 0 mg / dL, 280 mg / dL, 290 mg / dL, 300 mg / dL, or higher In some embodiments, the total cholesterol level is The cholesterol level is a serum cholesterol level or a total body cholesterol level.

[0173] The pharmaceutical compositions, engineered meganucleases, or engineered meganucleases disclosed herein Delivery of a nucleic acid encoding a leukase to a target cell in a subject may be achieved by administering to the subject an LDL-collector concentration prior to delivery. It reduces the LDL cholesterol level of a subject when compared to the sterol level. For example, LDL cholesterol levels can be increased or decreased as compared to the subject prior to treatment. At least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% In certain embodiments, the amount of the ion exchange reaction may be reduced by 90%, 95%, or up to 100%. The LDL cholesterol levels of subjects receiving the indicated pharmaceutical compositions increased slightly after the final dose. At least 2 weeks, at least 1 month, at least 2 months, or 3 months, The reduction is maintained compared to pre-delivery total cholesterol levels.

[0174] In certain embodiments, LDL cholesterol levels are measured using the pharmaceutical compositions disclosed herein. When compared to the LDL cholesterol level before delivery to the subject, about 5 mg / dL, 1 0mg / dL, 15mg / dL, 20mg / dL, 25mg / dL, 30mg / dL, 4 0mg / dL, 50mg / dL, 60mg / dL, 70mg / dL, 80mg / dL, 9 0mg / dL, 5~15mg / dL, 10~20mg / dL, 10~30mg / dL, 1 5~30mg / dL, 20~30mg / dL, 25~35mg / dL, 25~40mg / dL, 25~50mg / dL, 40~60mg / dL, 50~70mg / dL, 60~8 In certain embodiments, the blood glucose level is reduced by 0 mg / dL, 70-100 mg / dL, or more. Subjects were assigned to the following levels: 100 mg / dL, 110 mg / dL, 115 mg / dL, and 120 mg / dL. , 125mg / dL, 130mg / dL, 135mg / dL, 140mg / dL, 145 mg / dL, 150mg / dL, 155mg / dL, 160mg / dL, 165mg / d L, 170mg / dL, 175mg / dL, 180mg / dL, 185mg / dL, 19 Baseline LD of 0 mg / dL, 195 mg / dL, 200 mg / dL, or greater In some embodiments, the LDL cholesterol level The level is serum LDL cholesterol level or total body LDL cholesterol level.

[0175] In some embodiments, the compositions and methods described herein comprise the pharmaceutical The size of the atherosclerotic plaque in the subject is reduced when compared to the initial size of the atherosclerotic plaque before the composition is delivered to the subject. Reduce the size of loam spots by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, %, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19 %, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29 %, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39 %, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49 %, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59 %, or 60%, or even more. , about 19%~24%, 14%~29%, 12%~35%, 10~40%, 8%~45%, It may be reduced by 5% to 50%, 2% to 60%, or 1% to 70%.

[0176] 2.4 Pharmaceutical Compositions In some embodiments, the present invention provides a method for the preparation of an engineered membrane comprising administering to a subject a medicament comprising administering to said subject a medicament an engineered membrane ... or a pharma- ceutically acceptable carrier and an engineered meganuclease of the invention. and a polynucleotide comprising a nucleic acid encoding the polypeptide. The present invention relates to a method for the delivery of a pharmaceutical composition to a target tissue, comprising administering to said target tissue a pharmaceutical composition comprising a pharmacopoietin and a medicament for the delivery to said target tissue. and a cell of the invention expressing the engineered meganuclease disclosed herein. The pharmaceutical composition of the present invention is useful for treating cardiovascular disease and hypercholesterolemia including autosomal dominant FH. Useful for treating a subject with PCSK9, or for reducing the expression and / or activity of PCSK9. It could be.

[0177] Such pharmaceutical compositions can be prepared according to known techniques. ington,The Science And Practice of Pharm In the manufacture of the pharmaceutical formulation according to the present invention, Thus, the meganuclease polypeptide (or the DNA / RNA encoding it) is typically In the present invention, the composition is mixed with a pharma- ceutically acceptable carrier and the resulting composition is administered to a subject. must, of course, be acceptable in the sense of being compatible with the other ingredients in the formulation. In some embodiments, the pharmaceutical composition of the present invention The method further comprises administering to the subject one or more additional drugs or biological molecules useful for treating a disease in the subject. Similarly, additional drugs and / or biological molecules may be included in separate compositions. The two agents can be administered simultaneously.

[0178] In certain embodiments of the present invention, the pharmaceutical composition comprises a compound as described elsewhere herein: The lipid nanoparticles may include one or more of the mRNAs described herein encapsulated within the lipid nanoparticles. In certain embodiments, the lipid nanoparticles may comprise one or more multicystic lipid molecules as described herein. Each polycistronic mRNA can contain two or more of the polypeptides of the present invention. In certain embodiments, the lipid nanoparticles encode a meganuclease as described herein. A polycistronic encoding two, three or four engineered meganucleases according to any one of claims 1 to 4. In other particular embodiments, the lipid nanoparticles may comprise a nucleic acid sequence as described herein. Each of the polycistronic mRNAs may comprise two or more of the polycistronic mRNAs described herein, each of which may comprise two or more of the polycistronic mRNAs of the present invention. The present invention encodes an engineered meganuclease.

[0179] Some of the lipid nanoparticles contemplated for use in the present invention include at least one cationic lipid nanoparticle. The lipid composition comprises at least one cationic lipid, at least one non-cationic lipid, and at least one complex lipid. In a further particular example, the lipid nanoparticles comprise from about 40 mol % to about 85 mol % cationic lipid. % to about 49.5 mol % of non-cationic lipids, and It can contain about 10 mol % lipid complexes and have a non-lamellar (i.e., non-bilayer) morphology. It is produced in such a way that

[0180] Cationic lipids include, for example, palmitoyl-oleoyl-nor-arginine (P ONA), MC3, LenMC3, CP-LenMC3, γ-LenMC3, CP-γ- LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 Ami Pan-MC3, Pan-MC4, and Pan MC5, 1,2-dilinoleyloxy Di-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy -N,N-Dimethylaminopropane (DLenDMA), 2,2-Dilinoleyl-4-( 2-Dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA ; "XTC2"), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1 ,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-( 4-Dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA ), 2,2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DL in-K6-DMA), 2,2-Dilinoleyl-4-N-methylpepiazino-[1,3] -Dioxolane (DLin-K-MPZ), 2,2-Dilinoleyl-4-dimethylamino Methyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleyl DLin-C-DAP, 1,2-diaminopropyloxy Linoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-Dilinoleoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoxy-3-morpholinopropane DLinDAP, 1,2-Dilinoleylthio -3-Dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-Linoleoyl DLin-2-DMAP, 1,2-diphenyloxy-3-dimethylaminopropane Noreyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP. Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLi n-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DL inAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane ( DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride DODAC, 1,2-dioleyloxy-N,N-dimethylaminopropane (DO DMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA) ), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylan Ammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium Dioleyl bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N, N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-di Methylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1, 2-Dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl Ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(sperm N,N-dimethyl-1-propaneaminium trifluoride DOSPA, dioctadecylamidoglycylspermine (DOGS), 3-Dimethylamino-2-(cholest-5-en-3-β-oxybutan-4-oxy) -1-(cis,cis-9,12-octadecadieneoxy)propane (CLinDMA) , 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy )-3-Dimethyl-1-(cis,cis-9',1-2'-octadecadieneoxy)propionate Pan (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane Bread (DOcarbDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethyl (DLincarbDAP), or mixtures thereof Examples of the cationic lipid include DLinDMA and DLin-K-C2-DM. A (“XTC2”), MC3, LenMC3, CP-LenMC3, γ-LenMC3, CP-γ-LenMC3, MC3MC, MC2MC, MC3 ether, MC4 ether, MC3 Amide, Pan-MC3, Pan-MC4, Pan MC5, or mixtures thereof may be also possible.

[0181] In various embodiments, the cationic lipid comprises about 40 mol % to about 50 mol % of the total lipid present in the particle. About 90 mol%, about 40 mol% to about 85 mol%, about 40 mol% to about 80 mol%, Approx. 40mol%~Approx. 75mol%, Approx. 40mol%~Approx. 70mol%, Approx. 40mol%~ It may account for about 65 mol %, or from about 40 mol % to about 60 mol %.

[0182] Non-cationic lipids include, for example, one or more anionic lipids and / or neutral lipids. In a preferred embodiment, the non-cationic lipid is a neutral lipid component, i.e., ) cholesterol or its derivatives; (2) phospholipids, or (3) phospholipids and cholesterol Examples of cholesterol derivatives include: For example, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl -2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof. Phospholipids include, but are not limited to, dipalmitoyl Diphosphate phosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC ), dioleoylphosphatidylethanolamine (DOPE), palmitoyl oleoyl Palmitoyl-oleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine nolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (P OPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl Distearoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidyl Ethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethy Diethyl-phosphatidylethanolamine, Diethyl-phosphatidylethanolamine DEPE, Stearoyloleoyl-phosphatidylethanolamine (SOPE) ), egg phosphatidylcholine, and mixtures thereof. In certain preferred embodiments, the phospholipid is DPPC, D SPC, or a mixture thereof.

[0183] In some embodiments, a non-cationic lipid (e.g., one or more phospholipids and / or Cholesterol accounts for approximately 10 mol% to 60 mol% of the total lipids present in the particles. 15mol%~about 60mol%, about 20mol%~about 60mol%, about 25mol%~about 60mol%, about 30mol% to about 60mol%, about 10mol% to about 55mol%, about 15mol%~about 55mol%, about 20mol%~about 55mol%, about 25mol%~about 55mol%, about 30mol% to about 55mol%, about 13mol% to about 50mol%, about It may comprise from about 15 mol % to about 50 mol %, or from about 20 mol % to about 50 mol %. The non-cationic lipid is a mixture of phospholipid and cholesterol or a cholesterol derivative. In this case, the mixture may comprise up to about 40, 50, or 60 mol of the total lipid present in the particle. % may be accounted for.

[0184] Conjugated lipids that inhibit particle aggregation include, for example, polyethylene glycol (PEG)-lipids. Complexes, polyamide (ATTA)-lipid complexes, cationic polymer-lipid complexes (CP L), or mixtures thereof. In the present study, the nucleic acid-lipid particles contained either PEG-lipid conjugates or ATTA-lipid conjugates. In certain embodiments, the PEG-lipid conjugate or the ATTA-lipid conjugate may be coupled to a CPL. The conjugated lipids that inhibit particle aggregation are, for example, PEG-diacylglycerolipids. DAG, PEG dialkyloxypropyl (DAA), PEG-phospholipid, PE It may also contain PEG-lipids such as G-ceramide (Cer), or mixtures thereof. EG-DAA conjugates are PEG-dilauryloxypropyl (C12), PEG-dimyri Styryloxypropyl (C14), PEG-dipalmityloxypropyl (C16), P EG-distearyloxypropyl (C18), or a mixture thereof.

[0185] Additional PEG-lipid conjugates suitable for use in the present invention include mPEG2000-1, 2-Di-O-alkyl-sn3-carbomoyl glyceride (PEG-C-DOMG) is one example. The synthesis of PEG-C-DOMG can be carried out in accordance with, but not limited to, PCT Application No. PCT 2003 / 013666. Further additional PE's suitable for use in the present invention are described in US Pat. The G-lipid complex is 1-[8'-(1,2-dimyristoyl-3-propanoxy) -Carboxamido-3',6'-dioxaoctanyl]carbamoyl-ω-methyl-poly (ethylene glycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in US Pat. No. 7,404,969.

[0186] In some cases, conjugated lipids that inhibit particle aggregation (e.g., PEG-lipid conjugates) can be About 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol% of the total lipids present in the particles ol%, about 1mol% to about 2mol%, about 0.6mol% to about 1.9mol%, about 0.7 mol%~about 1.8mol%, about 0.8mol%~about 1.7mol%, about 1mol%~about 1.8mol%, about 1.2mol%~about 1.8mol%, about 1.2mol%~about 1.7m ol%, about 1.3mol% to about 1.6mol%, about 1.4mol% to about 1.5mol%, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 %, or 2 mol % (or any percentage or range therein). Typically, in such cases, the PEG moiety has an average molecular weight of about 2,000 daltons. In other cases, conjugated lipids (e.g., PEG-lipid conjugates) that inhibit particle aggregation are added to the particles. About 5.0 mol% to about 10 mol%, about 5 mol% to about 9 mol% of the total lipids present in About 5 mol% to about 8 mol%, about 6 mol% to about 9 mol%, about 6 mol% to about 8 mol %, or about 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, or 10 mol 5 vol % (or any percentage or range therein). Typically, In such cases, the PEG moiety has an average molecular weight of about 750 daltons.

[0187] In some embodiments, lipid nanoparticles are capable of being delivered and taken up in the liver, particularly in hepatic parenchymal cells. It has a composition that specifically enhances penetration.

[0188] In some embodiments, the pharmaceutical composition comprises a pharma- ceutical acceptable carrier and an engineered A recombinant DNA construct as described herein comprising a nucleic acid sequence encoding a meganuclease. In certain embodiments, such recombinant DNA constructs are incorporated into lipid nanoparticles. The vesicles may be encapsulated or otherwise suitable for delivery to target cells (e.g., liver cells, particularly hepatocytes). It may also be packaged in other delivery vehicles known in the art.

[0189] In certain embodiments, a pharmaceutical composition comprises a pharma- ceutical acceptable carrier and an engineered and a viral vector as described herein comprising a nucleic acid sequence encoding the meganuclease. In certain embodiments, the viral vector is capable of infecting target cells, particularly liver cells such as hepatocytes. Such an AAV vector may be an AAV vector suitable for delivery to a cell. , AAV8, AAV2, AAV9 capsid, or other liver-targeting vectors known in the art. In certain embodiments, the AAV capsid is an AAV8 capsid. which is a 5' inverted terminal repeat, liver-specific human thyroxine-binding globulin (TBG ) a promoter, an intron, a coding sequence for the engineered meganuclease of the present invention, Contains the WHP posttranscriptional regulatory element and 3' inverted terminal repeat. Includes cassettes containing

[0190] In certain embodiments, a therapeutically effective amount of the pharmaceutical compositions disclosed herein is administered in combination with another therapeutic agent. 2. A method for treating a cholesterol-related disorder, such as hypercholesterolemia, comprising administering to In certain embodiments, the pharmaceutical compositions disclosed herein are administered alone. .

[0191] The pharmaceutical compositions of the present invention can be administered in combination therapy, i.e. in combination with other drugs. In some embodiments, the pharmaceutical compositions disclosed herein contain at least one other therapeutic agent. The pharmaceutical compositions disclosed herein are delivered prior to administration of at least one other therapeutic agent. The drug may be delivered simultaneously with administration of the drug or after administration of at least one other therapeutic agent. In certain embodiments, the combination therapy comprises at least one pharmaceutical composition disclosed herein. The drug may be synthetically produced in vitro. The prepared chemical compositions, antibodies, antigen-binding regions, and combinations and conjugates thereof are In certain embodiments, the agent includes, but is not limited to, an agonist, They can act as antagonists, allosteric modulators, or toxins. In embodiments, the agent acts to inhibit or stimulate its target (e.g., a receptor or enzyme). activation or inhibition of the protein, thereby promoting increased expression of LDLR or expression of PCSK9. Reduces current or serum cholesterol levels.

[0192] The therapeutic agent (other than the pharmaceutical composition disclosed herein) may include at least one other cholesterol-lowering agent. cholesterol-lowering (serum and / or total body cholesterol) drugs or agents, In some embodiments, the agent increases the expression of LDLR and reduces serum HDL. Increase L levels, decrease LDL levels, or decrease triglyceride levels Exemplary drugs include statins (atorvastatin, cerivastatin, etc.) statin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin , Rosuvastatin, Simvastatin), Nicotinic Acid (Niacin) (NIACOR, N IASPAN (sustained-release niacin), SLO-NIACIN (sustained-release niacin), Fibric acid (LOPID (gemfibrozil), TRICOR (fenofibrate), Bile acid sequestrants (QUESTRAN (cholestyramine), colesevelam (WELCHOL) , COLESTID (colestipol), cholesterol absorption inhibitors (ZETIA (Ethyl Zetimibe), combination of nicotinic acid and statins (ADVICOR (LOVASTA TIN and NIASPAN), combination of statins and antiresorptive agents (VYTORIN ( ZOCOR and ZETIA), and / or lipid modifying agents. In some embodiments, the pharmaceutical compositions disclosed herein are PPARγ agonists. , PPARα / γ agonists, squalene synthase inhibitors, CETP inhibitors, antihypertensive agents, Antidiabetic drugs (sulfonylureas, insulin, GLP-1 analogues, DDPIV inhibitors, etc.), It is combined with an ApoB modulator, an MTP inhibitor, and / or an arteriosclerosis obliterans treatment. In some embodiments, the pharmaceutical compositions disclosed herein comprise a statin, oncostatin M certain cytokines, estrogens, and / or certain botanicals such as berberine In combination with an agent that increases the level of LDLR protein in a subject, such as In some embodiments, the pharmaceutical compositions disclosed herein comprise at least one antipsychotic drug, Certain HIV protease inhibitors, high fructose, sucrose, cholesterol or Dietary factors such as certain fatty acids and certain nuclear factors affecting RXR, RAR, LXR, and FXR receptor agonists and antagonists, etc., to increase serum cholesterol levels in a subject. In some embodiments, the pharmaceutical compositions disclosed herein are combined with an agent that: an agent that increases the level of PCSK9 in a subject, such as a statin and / or insulin; The combination of the two is intended to prevent the undesirable side effects of the other drug as disclosed herein. It may be possible to alleviate the symptoms by using a pharmaceutical composition of the present invention.

[0193] In certain embodiments, the pharmaceutical compositions disclosed herein are intended to reduce cholesterol (serum and and / or total cholesterol) drugs. In certain embodiments, the pharmaceutical compositions disclosed herein are used to treat hypercholesterolemia, cardiac To prevent or reduce the onset of any of the following diseases: diabetes, and / or cholesterol-related disorders In certain embodiments, the pharmaceutical compositions disclosed herein can be administered prophylactically to The composition can be administered to treat an existing hypercholesterolemia condition. In some embodiments, the pharmaceutical compositions disclosed herein are useful for treating disorders associated with cholesterol-related disorders. and / or delay the onset of symptoms. The composition is provided to a subject who does not have any symptoms of any one of the cholesterol-related disorders. .

[0194] 2.5 Methods for producing recombinant AAV vectors In some embodiments, the present invention provides a recombinant AA Recombinant AAV vectors are typically expressed in mammalian cells, such as HEK-293. It is produced in an animal cell line. The viral cap and rep genes are required for its autonomous replication. This prevents the therapeutic gene (e.g., meganuclease gene) from being delivered. It is removed from the vector to allow for the expression of the ribozyme in trans in the packaging cell line. In addition, it is necessary to have "helpers" necessary to support replication (e.g., adenovirus). (Cots D, Bosch A, Chillon M(2013)Curr.Gene Ther.13(5):370-81). For example, a recombinant AAV vector may comprise a first plasmid encoding a "helper" component and a second plasmid encoding a ca A second plasmid containing the p and rep genes is packaged into the virus. The cell line was transformed with a third plasmid containing the viral ITRs containing the intervening DNA sequence. triple transfection The genome (the desired ITRs and intermediate genes) is produced using the encapsidated vector. The viral particles are then purified by freeze-thaw cycles, sonication, detergents, or other techniques. The cells are then isolated by cesium chloride density gradient centrifugation or other means known in the art. The particles are purified using centrifugation or affinity chromatography and then subsequently purified from cells, tissues, or human tissue. The gene is delivered to a target gene in an organism such as a patient.

[0195] Recombinant AAV particles are typically produced in cells, allowing the creation of site-specific megagenes. The present invention is carried out to ensure that cleavage enzymes are not expressed in the packaging cells. The viral genome of the present invention is a novel method for the preparation of meganucleases. Since it contains a recognition sequence, any meganuclease expressed in the packaging cell line will be able to bind to the virus. It is possible that the virion cleaves the viral genome before it is packaged into a viral particle. This may be due to reduced packaging efficiency and / or packaging of fragmented genomes. Several approaches can be used to deliver the GFP gene to packaging cells, including: It is possible to prevent meganuclease expression in the 1. The meganuclease is conjugated to a tissue-specific promoter that is not active in the packaging cells. For example, the meganuclease gene can be delivered to muscle tissue. When developing viral vectors for the purpose of inducing IL-1 expression, muscle-specific promoters can be used. An example of a muscle-specific promoter is C5-12 (Liu et al. (2004) ), Hum Gene Ther.15:783-92), muscle-specific creatine kinase (MCK) promoter (Yuasa, et al. (2002), Gene Ther 9:1576-88), or the smooth muscle 22 (SM22) promoter (Haase et al. al.(2013), BMC Biotechnol.13:49-54) Examples of CNS (neuron) specific promoters include NSE, synapsin, and The MeCP2 promoter is an example (Lentz et al. (2012), Neu Robiol Dis. 48:179-88). Examples of liver-specific promoters include: Albumin promoter (Palb, etc.), human α1-antitrypsin (Pa1AT, etc.), and and hemopexin (Phpx, etc.) (Kramer et al. (2003) , Mol. Therapy 7:375-85). Examples of eye-specific promoters include: opsin, and the corneal epithelium-specific K12 promoter (Martin et al. al.(2002),Methods(28):267-75)(Tong et al. (2007), J Gene Med, 9:956-66). Other tissue-specific promoters known in the art are highly expressed in HEK-293 cells. Therefore, when incorporated into the viral vector of the present invention, It is not expected that the results will result in significant levels of meganuclease gene expression in stimulating cells. Similarly, the viral vectors of the present invention do not contain incompatible tissue-specific promoters (i.e. Using a known HeLa cell line (human epithelial cells) and the liver-specific hemopexin promoter It is contemplated that other cell lines may be used in conjunction with the use of tissue-specific promoters. Examples of synovial sarcoma include PDZD4 (cerebellum), C6 (liver), ASB5 (muscle), and PPP 1R12B (heart), SLC5A12 (kidney), cholesterol-regulating APOM (liver), ADPRHL1 (heart), and the monogenic malformation syndrome TP73L (muscle). Jacox E et al. (2010), PLoS One 5(8):e1227 4). 2. Alternatively, the vector can be derived from a different species in which the meganuclease is poorly expressed. For example, the viral particles can be packaged in a non-mammalian packaged cell. The well-known cytomegalovirus early promoter or Using mammalian promoters such as the SV40 virus early promoter, In a preferred embodiment, the method of Gao et al. Viral particles were produced in insect cells using the baculovirus system as described. (Gao et al. (2007), J.Biotechnol.131(2):1 38-43). Meganucleases under the control of mammalian promoters have been shown to be able to express It is unlikely to be expressed in the er.21(4):739-49). Furthermore, insect cells express different mRNAs from mammalian cells. A splicing motif. Therefore, human growth hormone (HGH) insert Introns such as the SV40 large T antigen intron or mammalian introns are used as the introns of meganucleases. These introns can be integrated into the coding sequence of pre-mRNA in insect cells. Insect cells cannot produce functional meganucleases because the transcript is not efficiently spliced. In contrast, the resulting recombinant AAV particles do not express and package the full-length genome. The resulting mammalian cells are able to properly splice the pre-mRNA and produce functional meganuclear Haifeng Chen expressed HGH and SV40 large T protein. The antigen intron was used to express the toxic proteins barnase and The expression of diphtheria toxin fragment A was attenuated, and recombinant AAV vectors carrying these toxin genes were Chen (2012), Mol Ther Nucleic Acids.1(11):e57). 3. The meganuclease gene requires a small molecule inducer for meganuclease expression The inducible promoter may be operably linked to an inducible promoter, as described above. Examples include the Tet-On system (Clontech; Chen et al. (2013) 15), BMC Biotechnol.15(1):4)) and RheoSwitch System (Intrexon; Sowa et al. (2011), Spine 36 (10):E623-8). Both systems and similar systems known in the art The systems respond to small molecule activators (doxycycline or ecdysone, respectively). In response to these conditions, ligand-inducible transcription factors (Tet repressor and exon, respectively) activate transcription. Using such a ligand-inducible transcriptional activator, Practicing the invention in accordance with the present invention involves: 1) placing a gene under the control of a promoter responsive to the corresponding transcription factor; A meganuclease gene is arranged, the meganuclease gene being (a) transcribed 2) have a binding site for the transcription factor in the packaged viral genome The transcription activator is not provided to the same cell. In other cases, meganucleases are not expressed in target cells or tissues after recombinant AAV delivery. The latter step is necessary because the transcriptional activator then binds to its cognate small molecule activator. The meganuclease gene expression is induced only in cells or tissues treated with the activator. This approach allows for selective delivery of small molecule inducers to specific tissues and tissues. This is advantageous because it allows spatiotemporal regulation of meganuclease gene expression. However, the need to include inducers in the viral genome, whose carrying capacity is severely restricted, creates drawbacks to this approach. 4. In another preferred embodiment, the recombinant AAV particles prevent expression of the meganuclease. The transcriptional repressor is produced in a mammalian cell line expressing a transcriptional repressor that is Repressors known in the art include the Tet repressor, Lac repressor, Cro repressor, and Lambda repressor. Many nuclear hormones, such as the ecdysone receptor Receptors act as transcriptional repressors in the absence of their cognate hormone ligands To practice the present invention, the packaging cells are designed to encode a transcriptional repressor. The virus is transfected / transduced with a vector that encodes the meganucleotide in the viral genome. The ase gene (packaging vector) is operably linked to a promoter, A promoter is a transcription factor that regulates the transcription of a gene, just as a repressor silences a promoter. The gene encoding the transcriptional repressor has been modified to contain a binding site for The gene can be located in a variety of locations. It can be encoded by a separate vector. It can be integrated into the packaging vector outside the ITR sequence, and the cap / r It can be incorporated into an ep vector or an adenovirus helper vector, or most preferably Alternatively, the gene may be stably integrated into the genome of the packaging cell so that it is constitutively expressed. Modification of common mammalian promoters to incorporate transcriptional repressor sites Methods for this purpose are known in the art. n expresses the strong constitutive CMV and RSV promoters as Lac repressor operators. wherein the gene expression from the modified promoter expresses a repressor. (Chang and Roninson (1996), Gen e 183:137-42). The use of non-human transcriptional repressors has been shown to be useful in the synthesis of meganuclease genes. Transcription of the gene is suppressed only in packaging cells expressing the repressor, resulting in that the expression of the AAV vector is not suppressed in target cells or tissues transduced with the recombinant AAV vector Ensure that:

[0196] 2.6 Engineered meganuclease variants Embodiments of the present invention include the engineered meganucleases and variants thereof described herein. Further embodiments of the present invention include a method for the preparation of a nucleic acid encoding a meganuclease as described herein. Isolated polynucleotides comprising nucleic acid sequences and variants of such polynucleotides are provided. Includes.

[0197] As used herein, "variant" is intended to mean a substantially similar sequence. A "mutant" polypeptide is one that has a mutation at one or more internal sites of the native protein. Deletion or addition of one or more amino acids and / or modification of one of the naturally occurring polypeptides. or a "natural" polypeptide by substitution of one or more amino acids at multiple sites. As used herein, "naturally occurring" is intended to mean a polypeptide that is derived from a plant. A polynucleotide or polypeptide includes the parent sequence from which the variant is derived. The variant polypeptides encompassed herein are biologically active, i.e., they have the same structure as the native protein. The desired biological activity of the protein, i.e., for example, a polypeptide that contains the PCS7-8 recognition sequence (SEQ ID NO: 4) Such mutations retain the ability to recognize and cleave the recognition sequence in the PCSK9 gene. Variants may result, for example, from human manipulation. A suitable variant (e.g., SEQ ID NOs: 6-14), or a variant that binds the subunits described herein. Biologically active mutants of the recognition half-sites are described elsewhere herein. As determined by sequence alignment programs and parameters, the naturally occurring polypeptides At least about 40%, about 45%, about 50%, approx. 55%, approx. 60%, approx. 65%, approx. 70%, approx. 75%, approx. 80%, approx. 85%, approx. 90%, approx. 91%, approx. 92%, approx. 93%, approx. 94%, approx. 95%, approx. 96%, approx. 97%, approx. 98%, or about 99% sequence identity. A biologically active variant of the polypeptide or subunit of amino acid residues, only about 1-20, only about 1-10, only about 5, only 4, Only 3, 2 or even 1 amino acid residue may differ.

[0198] Polypeptides of the embodiments may be modified in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are well known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are known in the art. It is well known. For example, see Kunkel (1985), Proc. Natl. Acad. Sc i.USA 82:488-492; Kunkel et al. (1987), Met hods in Enzymol. 154:367-382; U.S. Patent No. 4,873,1 Issue 92;Walker and Gaastra (1983) Techniques in Molecular Biology(MacMillan Publishing) See, for example, the 1990s American Medical Association (American Medical Association, New York) and the references cited therein. Regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest. Guidance is provided by Dayhoff et al., incorporated herein by reference. 1978) Atlas of Protein Sequence and Struc ture(Natl.Biomed.Res.Found.,Washington,D C.) by substituting one amino acid for another with similar properties. Conservative substitutions such as replacing the amino acid sequence with a nucleotide sequence similar to that of the amino acid sequence may be optimal.

[0199] In some embodiments, the engineered meganucleases of the present invention comprise the The parent HVR regions can include mutations in the HVR1 and HVR2 regions. Residues 24-79 or residues 215-270 of the engineered meganuclease. Therefore, the mutated HVR regions can enhance the biological activity (i.e., recognition) of the engineered meganuclease. The engineered meganucleotides exemplified herein are designed to maintain the ability to bind and cleave recognition sequences. The amino acid sequence corresponding to residues 24-79 or 215-270 of the enzyme at least 80%, at least 85%, at least 90%, at least 95% or more In addition, some embodiments of the present invention may include amino acid sequences having sequence identity of In this embodiment, the mutated HVR1 or HVR2 region is found at a specific position within the parent HVR. In this context, "corresponding" refers to a mutation that may be present in a gene. The amino acid residues of the HVRs are located in the same relative positions (i.e., relative to the remaining amino acids in the parent sequence) as the parent amino acid residues. This refers to the same amino acid residues (i.e., distinct identical residues) present in the HVR sequence. For example, if a parent HVR sequence contains a serine residue at position 26, then The "residue-containing" mutant HVR also contains a serine at a position relative to parent position 26.

[0200] A significant number of amines for the DNA recognition domain of the wild-type I-CreI meganuclease were identified. The acid modification has been previously identified (e.g., U.S. Pat. No. 8,021,867), which Alone or in combination, specificity is provided for individual bases within the DNA recognition sequence half-sites. By generating an engineered recombinant meganuclease, the resulting rationally designed meganuclease The cleavage enzymes thus have half-site specificities that differ from the wild-type enzyme. Based on the bases present at each half-site position (-1 to -9) of the recognition half-site, This can be done with engineered meganuclease monomers or subunits to enhance activity. This provides a potential replacement for

[0201] [Table 2]

[0202] The bolded items are wild-type contact residues and do not constitute "alterations" as used herein. An asterisk indicates that the residue contacts a base on the antisense strand.

[0203] With respect to a polynucleotide, a "variant" refers to one or more variants within a naturally occurring polynucleotide. The deletion and / or addition of one or more nucleotides at the site. Recognizing that variants of the sequence are constructed such that the open reading frame is maintained In the case of polynucleotides, conservative variants include those that are practically identical due to the degeneracy of the genetic code. The mutant polypeptides include sequences that code for one of the amino acid sequences of the polypeptides of the present invention. Nucleotides can be generated, for example, using site-directed mutagenesis, but in some embodiments The synthetically derived polynucleoside still encodes the engineered meganuclease of In general, variants of specific polynucleotides of the embodiments are described herein. Determined by sequence alignment programs and parameters described elsewhere. For that particular polypeptide, at least about 40%, about 45%, about 50%, About 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, Approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, The specific polynucleotide variants of the present invention have about 99% or more sequence identity. A variant (i.e., a reference polynucleotide) is encoded by a variant polynucleotide. Sequence identity between the polypeptide and the polypeptide encoded by the reference polynucleotide. It can also be assessed by comparing the homogeneity rates.

[0204] Deletions, insertions, and substitutions in the protein sequences encompassed herein are intended to reduce the characteristics of the polypeptide. However, the positive effects of substitutions, deletions, or insertions are not expected to result in radical changes in the In cases where it is difficult to predict the exact effect in advance, those skilled in the art may Screening polypeptides for their ability to preferentially recognize and cleave a recognition sequence within The participants will understand that their effectiveness will be evaluated by EXAMPLES

[0205] The present invention will be further illustrated by the following examples, which are not intended to limit the invention. Those skilled in the art will be able to, using routine experimentation, adapt the methods described herein to the intended purpose and should not be construed as limiting the scope of the invention. Numerous equivalents to specific substances and procedures may be recognized or identified. Such equivalents are intended to be encompassed in the scope of the claims following the examples below. can be.

[0206] Example 1 Characterization of a meganuclease that recognizes and cleaves the PCSK9 recognition sequence 1. Meganuclease that recognizes and cleaves the PCS7-8 recognition sequence The engineered meganucleases, collectively referred to herein as "PCS7-8 meganucleases," The enzyme (SEQ ID NO: 6 to 14) recognizes and cleaves the PCS1-2 recognition sequence (SEQ ID NO: 4). The PCS1-2 recognition sequence (SEQ ID NO: 4) is located within the PCSK9 gene. Each of the engineered PCS7-8 meganucleases contains an N-terminal nucleotide sequence derived from SV40. a nuclease localization signal, a first meganuclease subunit, and a linker sequence. , and the second meganuclease subunit. One subunit binds to the PCS7 recognition half-site of SEQ ID NO:4, and the second subunit The ribozyme binds to the PCS8 recognition half-site (see Figure 2).

[0207] The PCS7-binding subunit and the PCS8-binding subunit are HVR1 and HVR2, respectively. It contains a 56 base pair hypervariable region called VR2. The PCS7 binding subunit is Similarly, the PCS8 binding subunit is highly conserved outside of the HVR2 region. The PCS7 binding regions of SEQ ID NOs: 6 to 14 are highly conserved outside the region. SEQ ID NOs: 15 to 23 are provided. Each of SEQ ID NOs: 15 to 23 is a meganuclease PCS7-8L.197 (SEQ ID NO:6) PCS7-binding region SEQ ID NO:15 The PCS8 binding regions of SEQ ID NOs: 6 to 14 share 90% sequence identity with each other. SEQ ID NOs: 24 to 32 are provided. Each of SEQ ID NOs: 24 to 32 is a meganuclease PCS7-8L.197 (SEQ ID NO:6) PCS8 binding region SEQ ID NO:24 Both share 90% sequence identity.

[0208] 2. Cleavage of the PCSK9 recognition sequence in CHO cell reporter assay PCS7-8 meganuclease can recognize and cleave its corresponding recognition sequence (SEQ ID NO: 4). To determine whether the expression of β-lactamase in CHO cells can be mediated by the CHO cell reporter assay described previously, Each engineered meganuclease was evaluated (see WO 2012 / 167192 and Figure 1). To perform the assay, we used a nonfunctional green fluorescent protein integrated into the genome of cells. A CHO cell reporter strain carrying a protein (GFP) gene expression cassette was generated. The GFP gene of each cell line was cleaved intracellularly by meganuclease at one of the recognition sequences. A pair of recognition sequences stimulates a homologous recombination event that results in a functional GFP gene. was interrupted.

[0209] In the CHO reporter cell line developed for this study, One of the recognition sequences was the PCS7-8 recognition sequence (SEQ ID NO: 4). The second recognition sequence inserted was found to be in a control meganuclease called "CHO-23 / 24." The CHO-23 / 24 recognition sequence was recognized and cleaved by PCS7-8. and CHO reporter cells containing the CHO-23 / 24 recognition sequence were designated "PCS7-8 cells." Call.

[0210] CHO reporter cells were transfected with plasmids encoding the corresponding engineered meganucleases. Transfect PCS7-8 cells with smid DNA (e.g., PCS7-8 cells were transfected with transfected with plasmid DNA encoding the ganucleases), or CHO- The plasmids encoding the 23 / 34 meganucleases were transfected into each of the assays. In B, 4e 5 Lipofect CHO reporter cells according to manufacturer's instructions. 5 μl of 50 ... 0 ng of plasmid DNA. 48 hours after transfection , cells were assessed by flow cytometry and untransfected negative control The percentage of GFP-positive cells was determined compared to that of PCS bs. Thus, all PCS7-8 meganucleases were negative in cell lines containing the corresponding recognition sequences. It was found to produce GFP-positive cells at frequencies significantly greater than controls.

[0211] After transfection of the meganuclease into CHO reporter cells, PCS7-8 meganuclease The efficacy of the enzyme was also measured in a time-dependent manner. Using Gene Pulser Xcell, PCS7-8 cells (1.0 × 10 6 1 × 10 cells per 6 Electroporation of the copied meganuclease mRNA At the indicated time points after transfection, cells were analyzed by flow cytometry. The percentage of GFP-positive cells was determined. was also included at each time point as a positive control.

[0212] As shown in Figures 5A to 5C, the proteins produced by the different PCS7-8 meganucleases The %GFP expression was relatively consistent across the time course of each study, indicating sustained expression in cells. It demonstrates a lack of cleavage activity and substantial toxicity.

[0213] 3. Conclusion These studies demonstrate that the PCS7-8 meganuclease encompassed by the present invention inhibits the expression of corresponding nucleases in cells. The enzyme was able to efficiently target and cleave the recognition sequence that corresponds to the enzyme, and this effect was consistent over time. They demonstrated that the nuclease is non-toxic to cells.

[0214] Example 2 Cleavage of the PCS7-8 recognition sequence in HEK293 cells 1. Experimental Protocol and T7E Assay This study demonstrated that the PCS7-8 meganuclease encompassed by the present invention can be expressed in HEK293 cells. It was demonstrated that this enzyme can cleave the PCS7-8 recognition sequence.

[0215] 2e 6 293 cells were transfected using a BioRad Gene Pulser according to the manufacturer's instructions. Electroporate 2.7ug of a given PCS meganuclease mRNA using Xcell. Two and five days after transfection, genomic DNA (g DNA) was harvested from cells and subjected to a T7 endonuclease I (T7E) assay to detect endogenous Genetic modifications in the PCS7-8 recognition sequence were evaluated (Figure 6). The PCS7-8 locus was identified by PCR using primers flanking the PCS7-8 recognition sequence. If there was an indel (random insertion or deletion) in the PCS7-8 locus, The PCR products obtained consist of a mixture of wild-type and mutant alleles. The product can be denatured and slowly reannealed. Heteroduplexes consisting of wild type and mutant alleles are formed, resulting in mismatched bases and / or The T7E1 enzyme cleaves at the site of the mismatch and is visualized by gel electrophoresis. This results in cleavage products that can be cleaved.

[0216] 2.Results Two and five days after transfection, low molecular weight DNA fragments were identified in the PCS7-8x. 66 and PCS7-8x.88, but not the GFP control. Mock-transfected or transfected cells were analyzed by full-length PCR. These low molecular weight DNA fragments were the products of meganucleotide synthesis at the PCS recognition site (Figure 6). T7 endonuclease I cleavage at mismatched DNA caused by the activity of the endonuclease It is produced by

[0217] 3. Conclusion T7 endonuclease I assay was performed on PCS7-8 meganuclease-treated H Presence of indels around the PCS7-8 meganuclease recognition site in EK293 cells It detects the presence of target sites and amplifies the error-prone nature of those sites by cleavage and non-homologous end joining (NHEJ). Shows a repair.

[0218] Example 3 Deep sequencing to observe indels in the PCS7-8 recognition sequence 1. Deep Sequencing Protocol To directly observe insertions or deletions at the desired PCS7-8 meganuclease target site, To identify the genomic DNA, a deep sequencing protocol was used. 6 Production in HEK293 cells Using the BioRad Gene Pulser Xcell according to the manufacturer's instructions, g of PCS7-8 meganuclease mRNA was electroporated. Troporation was also performed without mRNA. 48 hours after transfection, The PCS7-8 locus was identified as a genomic DNA (gDNA) clone containing the PCS7-8 recognition sequence. The amplicons were amplified by PCR using flanking primers. To confirm this, agarose gel was used. Extraction was performed using the spin Gel and PCR Clean-up kit. England Biolabs' NEBNext Ultra for Illumina Prepare sequencing libraries using the II DNA Library Kit The paired-end sequencing library was prepared using Miseq D (Illumina). The sequencing data were analyzed using custom scripts. Reads containing a start or end point not within 25 bp of the full-length amplicon were removed. The proportion of del-containing reads was 1.0, which incorporated at least one of the middle 8 bp of the recognition sequence. The indel count was calculated by dividing the number of full-length reads with indels by the total number of full-length reads.

[0219] 2.Results As shown in Table 3, the PCS7-8 meganucleobases were assessed by deep sequencing. Each of the ases showed a significant reduction in the proportion of indels at its desired recognition site compared to mock treatment. There was at least a 100-fold increase.

[0220] [Table 3]

[0221] 3. Conclusion These experiments demonstrated that the PCS7-8 meganuclease of the present invention inhibits its activity in HEK293 cells. Cleave the desired target site (i.e., PCS7-8 recognition sequence) and eliminate errors due to non-homologous end joining. These results clearly demonstrate the ability to induce the emergence of indels via prone-to-repair.

[0222] Example 4 In vivo gene editing in primate liver using PCS7-8 meganuclease 1. Methods and Materials Experiments were carried out to demonstrate that PCS7-8 meganuclease can infect liver cells of a non-human primate model. The ability to edit the PCS7-8 recognition site in vivo was evaluated and correlated with serum levels of PCSK9 in subjects. The effect of such edits on the

[0223] The PCS7-8x.88 meganuclease was delivered via a recombinant AAV vector. AAV vectors have an AAV8 capsid, 5' to 3', 5' inverted terminal repeats, Liver-specific human thyroxine-binding globulin (TBG) promoter, intron, PCS7 -8x.88 meganuclease coding sequence, Woodchuck hepatitis virus (WHP) transduction This vector contained posttranscriptional regulatory elements, and 3' inverted terminal repeats. It is called TBG.PI.PCS7-8x.88.WPRE.bGH.

[0224] The AAV vectors were prepared in a pharmaceutical composition and administered to 4 mice weighing approximately 6.5 kg each on day 0. RA1866 was administered as a single injection to different rhesus monkeys at 3 × 10 13 Patients received a single dose of GC / kg, the highest dose evaluated in these studies. The animal RA1857 (male) had a 6 × 10 12 Single dose of genome copies (GC) / kg Animal RA1829 (female) and animal RA2334 (male) received 2 × 1 0 12 Patients received a single dose of 100 genome copies (GC) / kg. Blood samples were analyzed by ELISA. Analysis of serum PCSK9 protein levels, total cholesterol, HDL, LDL, and Analysis of triglycerides and alanine aminotransferase (ALT) levels For analysis, days -3 and 0, and for 168 days (low dose animals) or 280 days post-dose In addition, the PCS7-8 recognition sequence was PCR analysis of insertions and deletions (indels) in the array and in situ hybridization Characterization of PCS7-8x.88 meganuclease expression in hepatocytes by in situ hybridization (ISH) For analysis, liver biopsy samples were obtained 17 days after administration.

[0225] 2. Changes in serum PCSK9 protein levels Serum PCSK9 protein levels were measured on days -3, 0, and after administration of AAV vectors. High- and mid-dose animals were measured at multiple time points, 280 days post-dose. The low-dose animals were followed for 168 days, whereas the low-dose animals were followed for 170 days.

[0226] As shown in Figure 7, a single dose of meganuclease AAV on day 0 resulted in 100% survival by day 7. In studies RA1866, RA1857, and RA1829, all groups The high dose of 3×1 caused a dramatic dose-dependent increase in serum PCSK9 levels. 0 13 Subjects receiving GC / kg RA1866 had an approximately 84% reduction by day 7 and 20 By the end of the study, there was a 91% reduction in the number of patients with 6×10 12 Subjects receiving a dose of GC / kg RA 1857 showed approximately 46% reduction by day 7 and approximately 77% reduction by day 20. 2×10 12 Subjects receiving GC / kg RA1829 experienced a reduction of approximately 35% by day 7; By day 20, there was a reduction of approximately 70%. 12 Subjects receiving GC / kg RA2334 showed a reduction of approximately 35% by day 14, but was back to baseline by day 20. Returning to the Inn area, a decrease of about 10% was observed.

[0227] Additional time points were evaluated to assess the consistency of protein inhibition throughout the duration of the study. A dose-dependent decrease in serum PCSK9 levels continued through the final time points measured. In both low-dose animals (RA1829 and RA2334), there was a reduction of approximately 25% over the first 12 months. This continued to be observed on day 68. In the medium dose animals (RA1857), there was a reduction of approximately 50%. Small amounts continued to be observed at 280 days. In high-dose animals (RA1866), approximately 85% A % decrease was still observed at day 280. Overall, the serum P The decrease in CSK9 levels appeared to be dose-dependent and continued for each subject until the end of the study. It continued in.

[0228] 3. Serum cholesterol levels, LDL levels, HDL levels, and triglyceride levels Changes in the Serum cholesterol levels, LDL levels, HDL levels, and triglyceride levels The effect of PCS7-8 meganuclease treatment on AAV vectors was evaluated at days -3, 0, and 4. The results were also measured at multiple time points after administration of the drug.

[0229] Importantly, total LDL levels were significantly reduced by treatment with PCS7-8 meganuclease. All four animals had serum LD The animals showed a significant reduction in L, which persisted throughout the duration of the study in a dose-dependent manner. A2334 showed a decrease of about 25% up to day 168, whereas the other low dose animal, RA182 9 showed a larger decrease of about 35% at the same time point. A1866 (high dose) each showed approximately a 70% reduction when measured on study day 268. did.

[0230] Notably, a transient increase in LDL levels was observed in each animal on days 28 and 42 of the study. This was consistent with the changes in ALT levels observed at the same time points (Figure 9), and This may result from the activity of T cells against nucleases or vector capsids.

[0231] In Figures 8B to 8E, the total cholesterol, HDL, and The changes in total cholesterol levels, LDL, and triglycerides are shown. The levels of β-lactamase were significantly decreased in high-dose animals (RA1866) and in medium-dose animals (RA1857). However, a more modest effect was observed in the low-dose animals. HDL was also somewhat elevated in the high-dose animals. The triglycerides in each animal decreased slightly, while the other three remained relatively stable. Transient changes in levels were observed over the course of the study, but were near baseline at the final time point measured. It remained at line level.

[0232] 4. Meganuclease Expression and In Vivo Gene Editing of Hepatocytes On day 17, a liver biopsy was performed to confirm the presence of an insertion or deletion (indel) in the PCS7-8 recognition sequence. Indels were detected by PCR primers flanking the recognition sequence, genomic The detection was performed by amplifying the intervening region of the chromosome and sequencing the resulting PCR product. ndel, i.e., both insertions and deletions of different lengths, produce various sequences in the PCS7-8 recognition sequence. Figure 10 shows that the most frequent The eight observed indels and their corresponding frequencies are shown. These eight indels were 73% of indels observed in subject RA1857 and 73% of indels observed in subject RA1866 It accounted for 66% of ndel.

[0233] Was the PCS7-8x.88 meganuclease actually expressed in vivo in hepatocytes? To confirm this, liver biopsies obtained on days 17 and 129 were analyzed by in situ hybridization. Fluorescently labeled oligo probes were designed and analyzed using the biopsy samples from each subject. As a control, the M11 Mock treatment of biopsy cells from 657 was performed without the oligo probe. As shown in FIG. No fluorescent signal was observed in mock-treated cells (Figure 11A). Considerable fluorescence was observed in the test liver cells (Figures 11B to 11E) on day 17. , PCS7-8x.88 meganuclease mRNA was observed to have indel formation, and serum Reductions in PCSK9 and serum lipids were detected early in treated subjects It is clear that the expression of meganuclease mRNA was confirmed after the second round. It further shows that this was no longer observed by day 129 of the study in which liver biopsies were obtained.

[0234] 5. Treatment with PCS7-8L.197 Meganuclease Further studies were carried out to identify the optimized second generation PCS7-8L.197 meganuclease. The study protocol was similar to that previously described. PCS7-8L. The 197 meganuclease was delivered via a recombinant AAV vector. AAV8 capsid, 5' to 3', 5' inverted terminal repeat, liver-specific human tyrosine Toxin-binding globulin (TBG) promoter, intron, PCS7-8L.197 The coding sequence of ganucleases, a posttranscriptional regulatory element of woodchuck hepatitis virus (WHP) The AAV vector contained a 3′ inverted terminal repeat and a 3′ inverted terminal repeat. On day 0, 6 × 10 mice were administered to one male and one female rhesus monkey, each weighing approximately 6.5 kg. 12 The drug was administered as a single injection at a single dose of 100 genome copies (GC) / kg. The pool included analysis of serum PCSK9 protein levels by ELISA and total LDL levels. Samples were taken for analysis on day 0 and day 7 post-dose.

[0235] As shown in FIG. 12, PCSK9 serum levels increased rapidly in both subjects by day 7 of the study. In subject RA2125, PCSK9 levels were reduced rapidly and dramatically from approximately 180% on day 0. ng / mL on day 7 to approximately 55 ng / mL on day 7 (a decrease of approximately 70%). In 343, PCSK9 levels ranged from approximately 245 ng / mL on day 0 to approximately 125 ng / mL on day 7. / mL (a decrease of about 49%). The decrease in PCSK9 levels was This continued through day 182 after AV administration, with each subject continuing to show a reduction of approximately 60%.

[0236] Figure 13A shows that the reduction in serum PCSK9 levels correlated favorably with the serum LDL levels within 7 days of treatment. In subject RA2125, serum LDL levels were significantly decreased on day 0. The blood glucose level decreased from approximately 40 mg / dL to approximately 25 mg / dL on day 7 (a decrease of approximately 38%). With RA2343, serum LDL levels increased from approximately 75 mg / dL on day 0 to approximately 55 mg / dL on day 7. The serum LDL level continued to decrease from 21 to 28 g / dL (a decrease of about 27%). The decrease in serum LDL-cholesterol continued through the first day, reaching a decrease of approximately 65% ​​compared to baseline. The study was continued through study day 168, at which point subjects RA2125 and RA2343 each showed a significant and stable decrease of approximately 35% and 50%.

[0237] In Figures 13B and 13C, total cholesterol, H Changes in total cholesterol, LDL, and triglycerides are shown. The levels of RA decreased modestly throughout the study in each subject, but HDL and triglyceride levels were decreased in each subject. The results were essentially unchanged throughout the study period. Similar to the administration of RA2125, there was a transient decrease in serum lipids between days 28 and 42. A significant increase was observed, which was consistent with the elevation of ALT (Figure 14), and was not observed with either meganuclease or AA. This may be due to T cell activation in response to the V capsid.

[0238] We confirmed that the PCS7-8L.197 meganuclease was expressed in vivo in hepatocytes. To confirm this, liver biopsies obtained on days 18 and 129 were analyzed by in situ hybridization. Fluorescently labeled oligo probes were designed and used to examine the endothelial cell lineages of the biopsies from each subject. Binds to PCS7-8L.197 mRNA in hepatocytes. As shown in Figure 15, Significant fluorescence was observed in hepatocytes from subjects receiving only PCS7-8L at day 18. .197 Meganuclease mRNA was detected to reduce serum PCSK9 and serum lipids. Strong expression in treated subjects at early time points is evident. Expression of ganucleases mRNA was observed at day 128 of the study when a second liver biopsy was obtained. This further indicates that the

[0239] 6. Molecular evaluation of on-target genome editing DNA isolated from liver biopsy samples was subjected to anchored multiplex PCR sequencing. Deep sequencing of amplicons generated by AMP-seq The rhPCSK9 exon 7 target site was characterized for on-target editing. AMP amplicons are generated using nested locus-specific primers, but The forward primer is a fixed site 50 bp upstream of the targeted meganuclease cleavage site. The 3' end of the amplicon hybridizes to the random sequence of the genomic DNA prior to amplification. It is defined by the length of the DNA fragment after pairing.

[0240] In all liver samples, short insertions and The frequency of deletions was measured as observed in negative controls (i.e., PBMC samples from naive animals). The PCS7-8x.88 meganuclease was found to have a higher affinity for nuclease activity than the PCS7-8x.88 meganuclease (Figure 16). At day 129, the frequency of indels detected by AMP-seq was 46% at the high dose. %, and at the lower dose, it decreased dose-proportionally to 13% (mean, n=2). By using the TBG promoter and the GFP-type, only primary hepatocytes were selectively edited. Considering this, AMP-seq is a promising tool to detect the presence of unedited contaminating non-parenchymal cells in liver biopsy samples. Therefore, it is likely that the total frequency of on-target genome editing is underestimated. The total number of events increased with increasing high dose between the two time points (i.e., days 17 and 129). The total number of insertions exceeded the total number of deletions in each sample, but The frequency of deletions varied over time in both high- and mid-dose animals. It was found that the α-amyloides increased over time in the high-dose animals and decreased over time in the low-dose animals, but Insertions consistently decreased over time for all 14.1 cells treated with PCS7-8L.197 AAV. Similar on-target molecular analysis of samples from subjects receiving a medium dose of AAV showed The spectrum and frequency of indels as observed in subject RA1857 are shown.

[0241] 7. Conclusion These studies demonstrated that PCS7-8 meganuclease is a primate It was successfully expressed in vivo in hepatoid cells and subsequently cleaved into the PCS7-8 recognition sequence. It was found that error-prone non-homologous end joining at the cleavage site Subsequently, multiple indels were observed in PCR and AMP-seq analyses of the region. This results in inhibition of PCSK9 protein expression as observed by ELISA. These studies are the first reports of gene editing in primate liver known to the inventors. Moreover, this is the first reported observation of gene editing of the PCSK9 gene in a primate. This observation was made by measuring serum PCSK9 protein levels, serum LDL levels, and total cholesterol levels. The present inventors have found that the administration of Acknowledging the relatively high doses of AAV administered, both efficacy and safety profiles remain to be determined. Further experiments with additional PCS7-8 meganucleases were performed to determine Total cholesterol levels, serum LDL levels, and hepatocyte LDL levels Further analysis will also be performed for preclinical validation of this method of reducing cholesterol.

Claims

1. comprising a first subunit and a second subunit, The first subunit binds to a first recognition half-site of the recognition sequence, The first subunit is (a) a sequence that has at least 80% sequence identity to residues 7-153 of any one of SEQ ID NOs: 6-14; an amino acid sequence having sequence identity; (b) a first hypervariable (HVR1) region; and Including, The second subunit binds to a second recognition half-site of the recognition sequence, The second subunit is (i) at least 80% to residues 198-344 of any one of SEQ ID NOs: 6-14 and an amino acid sequence having a sequence identity of (ii) a second hypervariable (HVR2) region; and An engineered meganuclease that recognizes and cleaves a recognition sequence comprising SEQ ID NO:4, comprising:

2. The HVR1 region comprises an amino acid sequence corresponding to residues 24 to 79 of any one of SEQ ID NOs: 6 to 14. The amino acid sequence has at least 80% sequence identity to the amino acid sequence of claim 1.

2. An engineered meganuclease according to claim 1.

3. The HVR1 region comprises residues 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 , 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77.

3. The engineered meganuclease of claim 1 or 2, comprising a residue.

4. The HVR1 region comprises residues corresponding to residues 48, 50, 71, and 73 of SEQ ID NO:

8. The engineered meganuclease of any one of claims 1 to 3, further comprising:

5. The HVR1 region comprises residues 24 to 79 of any one of SEQ ID NOs: 6 to 14. Item 5. The engineered meganuclease of any one of items 1 to 4.

6. The HVR2 region corresponds to residues 215 to 270 of any one of SEQ ID NOs: 6 to 14. The amino acid sequence has at least 80% sequence identity to the amino acid sequence of 6. The engineered meganuclease of any one of claims 1 to 5.

7. The HVR2 region comprises residues 215, 217, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240 、２２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６１ 7. The method of claim 1, further comprising the steps of: Meganucleases.

8. 7. The HVR2 region further comprises a residue corresponding to residue 258 of SEQ ID NO:

12. Or the engineered meganuclease of claim 7.

9. the HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 6-14; The engineered meganuclease of any one of claims 1 to 8.

10. The first subunit has a residue corresponding to residue 80 of any one of SEQ ID NOs: 6 to 14.

10. The engineered meganuclease of any one of claims 1 to 9, comprising a group.

11. The second subunit corresponds to residue 271 of any one of SEQ ID NOs: 6 to 14. The engineered meganuclease of any one of claims 1 to 10, comprising a residue.

12. The first subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 6 to 14. The engineered meganuclease of any one of claims 1 to 11.

13. The second subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 6 to 14.

13. The engineered meganuclease of any one of claims 1 to 12, comprising:

14. The engineered meganuclease comprises a linker, the linker being linked to the first subunit. The method according to any one of claims 1 to 13, wherein the second subunit is covalently bonded to the unit. of engineered meganucleases.

15. The engineered meganuclease has the amino acid sequence of any one of SEQ ID NOs: 6 to 14.

15. The engineered meganuclease of any one of claims 1 to 14, comprising:

16. A nucleic acid encoding the engineered meganuclease according to any one of claims 1 to 15. A polynucleotide comprising a nucleic acid sequence.

17. The polynucleotide of claim 16, wherein the polynucleotide is an mRNA.

18. The mRNA is the engineered meganuclear vector according to any one of claims 1 to 15. and at least one further polypeptide. The polynucleotide of claim 17.

19. A nucleic acid encoding the engineered meganuclease according to any one of claims 1 to 15. A recombinant DNA construct comprising a nucleic acid sequence.

20. The recombinant DNA construct comprises a promoter and a nucleic acid sequence according to any one of claims 1 to 15. A polypeptide encoding the engineered meganuclease and at least one further polypeptide. and a polycistronic nucleic acid sequence, said promoter being coupled to said polycistronic nucleic acid sequence.

1. The method of claim 1, wherein the nucleic acid sequence of claim 1 is a nucleic acid sequence that drives expression of a polycistronic nucleic acid sequence to produce a polycistronic mRNA in a target cell.

10. The recombinant DNA construct according to claim 9.

21. The recombinant DNA construct comprises the engineered chromosome of any one of claims 1 to 15. A viral vector comprising the nucleic acid sequence encoding a ganucleases, comprising:

20. The recombinant DNA construct according to 19.

22. The viral vector may be a retroviral vector, an adenoviral vector, a lentiviral vector, or a 2. A viral vector or a recombinant adeno-associated virus (AAV) vector.

1. The recombinant DNA construct according to claim 1.

23. The method according to claim 21 or 22, wherein the viral vector is a recombinant AAV vector. Recombinant DNA constructs.

24. A nucleic acid encoding the engineered meganuclease according to any one of claims 1 to 15. A viral vector comprising a nucleic acid sequence.

25. The viral vector may be a retroviral vector, an adenoviral vector, a lentiviral vector, or a The viral vector of claim 24 which is a viral vector or an AAV vector.

26. The method according to claim 24 or 25, wherein the viral vector is a recombinant AAV vector. Illus Vector.

27. The viral vector comprises a promoter and the vector according to any one of claims 1 to 15. A polynucleotide encoding the engineered meganuclease and at least one further polypeptide is provided. and a cassette comprising a polycistronic nucleic acid sequence, said promoter comprising 25. The method of claim 24, wherein the nucleic acid sequence of the present invention is a nucleic acid sequence that drives expression of a polycistronic nucleic acid sequence to produce a polycistronic mRNA in a target cell.

27. A viral vector according to any one of claims 1 to 26.

28. A pharma- ceutically acceptable carrier; (a) encoding the engineered meganuclease of any one of claims 1 to 15; a nucleic acid that (b) the engineered meganuclease of any one of claims 1 to 15; 23. A pharmaceutical composition comprising:

29. The nucleic acid encoding the engineered meganuclease is as described in claim 17 or 18.

29. The pharmaceutical composition of claim 28, wherein the mRNA is

30. The pharmaceutical composition comprises the recombinant DNA construct according to any one of claims 19 to 23.

29. The pharmaceutical composition of claim 28, comprising:

31. The pharmaceutical composition comprises the viral vector according to any one of claims 24 to 27.

30. The pharmaceutical composition of claim 28, comprising:

32. The pharmaceutical composition comprises the engineered meganucleotide of any one of claims 1 to 15. The pharmaceutical composition of claim 28 comprising an ase.

33. The pharmaceutical composition according to claim 17 or 18, encapsulated in lipid nanoparticles.

30. The pharmaceutical composition according to claim 28 or 29, comprising the mRNA.

34. 1. A method for reducing expression of PCSK9 in a subject, the method comprising administering to the subject a target To the cells, (a) an effective amount of the engineered meganuclear molecule of any one of claims 1 to 15; a nucleic acid encoding an engineered meganuclease, wherein the engineered meganuclease is expressed in the target cell. nucleic acid, or (b) an effective amount of the engineered meganuclear molecule of any one of claims 1 to 15. Z wherein the engineered meganuclease comprises delivering to the target cell a sequence identical to SEQ ID NO:

4. and reducing the expression of PCSK9 compared to control cells. Law.

35. 35. The method of claim 34, wherein the subject has hypercholesterolemia.

36. The method of claim 34 or 35, wherein the subject has familial hypercholesterolemia. Law.

37. Claims 34 to 36, wherein the subject has autosomal dominant familial hypercholesterolemia.

2. The method according to claim 1 .

38. The method comprises administering to the subject the pharmaceutical composition according to any one of claims 28 to 33. The method of any one of claims 34 to 37, comprising administering

39. 38. The pharmaceutical composition comprising the recombinant AAV vector of claim 26. The method described above.

40. The pharmaceutical composition according to claim 17 or 18, encapsulated in lipid nanoparticles. The method of claim 38, comprising the above-mentioned mRNA.

41. The method of any one of claims 34 to 40, wherein the target cell is a hepatocyte.

42. 42. The method of claim 41, wherein the liver cells are hepatocytes.

43. The method according to any one of claims 34 to 42, wherein the subject is a human or a non-human primate. How to.

44. The display of LDL receptors on the cell surface increases baseline LDL receptor levels. The method according to any one of claims 34 to 43, wherein the amount of the IL-1 receptor is increased in hepatocytes compared to cells. How to.

45. The display of LDL receptors on the cell surface of hepatocytes is indicative of the basal expression of the hepatocytes. At least about 5%, 10%, 20%, 30%, or more when compared to normal LDL receptor levels %, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to 100% increase The method of claim 44 .

46. Serum total cholesterol levels are decreased in the subject after the expression of PCSK9 is decreased. The method according to any one of claims 34 to 45,

47. The serum total cholesterol level is compared to the baseline serum total cholesterol level. If you do, (a) at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% , 80%, 90%, 95%, or up to 100%, or (b) 5-15mg / dL, 10-20mg / dL, 10-30mg / dL, 15-3 0mg / dL, 20-30mg / dL, 25-35mg / dL, 25-40mg / dL, 25-50mg / dL, 40-60mg / dL, 50-70mg / dL, 60-80mg 47. The method of claim 46, wherein the blood glucose level is reduced by 70-100 mg / dL or by 70-100 mg / dL.

48. 3. The method of claim 2, wherein serum LDL cholesterol levels are decreased in the subject after treatment.

48. The method according to any one of claims 4 to 47.

49. The serum LDL cholesterol level is determined by comparing the baseline serum LDL cholesterol level with the baseline serum LDL cholesterol level. When compared to the (a) at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% , 80%, 90%, 95%, or up to 100%, or (b) 5-15mg / dL, 10-20mg / dL, 10-30mg / dL, 15-3 0mg / dL, 20-30mg / dL, 25-35mg / dL, 25-40mg / dL, 25-50 mg / dL, 40-60 mg / dL, 50-70 mg / dL, or 60- 49. The method of claim 48, wherein the reduction is 80 mg / dL.

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