Engineered high-fidelity OMNI-79 nuclease variants

Abstract

The present invention relates, inter alia, to compositions and methods for genome editing, in particular to a non-naturally occurring OMNI-79 nuclease variant having a wild-type OMNI-79 protein sequence (SEQ ID NO:1) that includes an amino acid substitution in at least one of I14, S1005, and E1050.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 284,858, filed December 1, 2021, the contents of which are incorporated herein by reference.

[0002] Throughout this application, various publications are referenced, including those referenced in parentheses. All publications mentioned in this application are incorporated by reference in their entirety into this specification in order to add context to the art to which this invention pertains and to which the invention may be utilized.

[0003] Reference to sequence listing This application incorporates by reference as part of this application the nucleotide sequence in the file "221130_91808-A-PCT_Sequence_Listing_AWG.xml", 133 kilobytes in size, created on November 14, 2022 in IBM-PC format with operating system compatibility with MS-Windows®, contained in an XML file submitted on November 30, 2022. [Background technology]

[0004] Targeted genome modification is a powerful tool that can be used to reverse the effects of pathogenic genetic alterations and thus may provide novel therapeutic approaches to human genetic diseases. Current genome engineering tools, including genetically modified zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most recently RNA-guided DNA endonucleases such as CRISPR / Cas, generate sequence-specific DNA breaks in the genome. The modification of the genome sequence occurs in the following steps and is the result of the action of the cell's DNA repair mechanisms triggered in response to the newly formed DNA breaks. These mechanisms may include, for example, (1) classical non-homologous end joining (NHEJ), in which the two ends of the break are ligated in a rapid but imprecise manner (i.e., frequent DNA mutations in the form of small insertions or deletions at the break site occur); or (2) homology-directed repair (HDR), in which an intact homologous DNA donor is used to replace the DNA around the break site in a precise manner. Efficient and safe genome editing requires that the initial DNA damage inducer has minimal off-target activity. Summary of the Invention

[0005] Disclosed herein are modified Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-associated OMNI-79 nucleases with improved activity and their uses in genomic engineering, extragenomic engineering, genome targeting, genome editing and in vitro diagnostics.

[0006] In some embodiments, the method of using OMNI-79 nuclease variants and improved variants is provided, which has increased activity compared to wild-type OMNI-79 nuclease.Advantageously, when modified OMNI-79 nuclease variants are active in CRISPR endonuclease system, the CRISPR endonuclease system has increased on-target editing activity compared to wild-type CRISPR endonuclease system in which wild-type OMNI-79 nuclease is active.For example, modified OMNI-79 nuclease variants may show nuclease activity in target region that contains heterozygous SNPs that are only present in target alleles and not present in non-target alleles.

[0007] According to some aspects of the invention, there are provided variants of OMNI-79 nuclease protein that comprise a sequence that is at least 80% identical to the amino acid sequence of the wild-type OMNI-79 nuclease protein (SEQ ID NO:1).

[0008] According to some aspects of the invention, there is provided a non-naturally occurring OMNI-79 nuclease variant having a wild-type OMNI-79 protein sequence (SEQ ID NO:1) that includes an amino acid substitution at at least one of I14, S1005, and E1050.

[0009] According to some aspects of the present invention, there is provided a CRISPR system comprising an OMNI-79 nuclease variant as disclosed herein complexed with a guide RNA molecule targeted to a DNA target site, the CRISPR system having increased on-target editing activity compared to a wild-type CRISPR system comprising a wild-type OMNI-79 nuclease protein and a guide RNA molecule.

[0010] In some embodiments, the OMNI-79 nuclease variant has increased activity at a target site compared to wild-type OMNI-79 nuclease (SEQ ID NO:1) when complexed with a guide RNA that directs the OMNI-79 variant to the target site.

[0011] According to some embodiments of the present invention, there is provided a method of gene editing with increased on-target editing activity comprising contacting a DNA target site with an active CRISPR system comprising an OMNI-79 nuclease variant protein as described herein.

[0012] According to some embodiments, a gene editing method having increased on-target editing activity is provided, comprising contacting a target site with an active CRISPR system comprising an OMNI-79 nuclease protein variant as described herein, wherein the active CRISPR system has increased on-target editing activity compared to a wild-type CRISPR system having a wild-type OMNI-79 nuclease protein.

[0013] Further aspects and the full scope of applicability of the present invention will become apparent from the following detailed description, but it should be understood that the detailed description and examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0014] [Figure 1] Editing activity of wild-type OMNI-79 nuclease and OMNI-79 variant 5570 on various hLDLR gene targets. Editing activity was determined by next generation sequencing (NGS) analysis. The mean and standard deviation of triplicates are shown. [Figure 2A-2C] Editing activity of OMNI-79 single mutants. Single point mutation variants targeting three genomic sites, hSERP_g12R (Figure 2A), hLDLR_g46 (Figure 2B) and hLDLR_g76 (Figure 2C), were tested by transfection into HeLa cells. Editing activity was determined by NGS analysis. The mean and standard deviation of triplicates are shown. [Diagram 3]Effect of other substitutions at position 1005 on editing activity. Editing activity targeting hLDLR_g76 was tested by transfection into HeLa cells. Editing activity was determined by NGS analysis. The mean and standard deviation of triplicates are shown. [Figure 4] NGS analysis of editing by OMNI-79 V5570 RNP complex. Preassembled complexes of purified OMNI-79 V5570 protein (105 pmol) and sgRNA (124 pmol) were electroporated into HepG2 (human hepatic carcinoma) cells using a Lonza Nucleofector® X unit according to the manufacturer's instructions. After recovery, cells were plated in 12-well tissue culture plates and maintained in a 37°C, 5% CO2 incubator. 72 hours after electroporation, cells were dispersed using trypsin and genomic extracts were prepared using QuickExtract solution according to the manufacturer's instructions. Analysis of indel rates was performed by NGS. [Diagram 5] NGS analysis of OMNI-79 V5570 mRNA-induced editing in HepG2 cells. 1 μg of OMNI-79 V5570-encoding mRNA (Trilink) and 124 pmol sgRNA (Agilent) were electroporated into HepG2 cells using a Lonza Nucleofector® X unit according to the manufacturer's instructions. After recovery, cells were plated in 12-well tissue culture plates and maintained in a 37°C, 5% CO2 incubator. 72 hours after electroporation, cells were dispersed using trypsin and genomic extracts were prepared using QuickExtract solution according to the manufacturer's instructions. Analysis of indel rates was performed by NGS. [Figure 6]NGS analysis of OMNI-79 V5570 and gRNA-mediated editing expressed by AAV infection. Adeno-associated virus DJ serotype (AAV-DJ) carrying OMNI-79 V5570 and the corresponding sgRNA sequence was infected into Hepa1-6 (mouse hepatocellular carcinoma) cells and HepG2 cells at a multiplicity of infection (MOI) of 1 × 105 to 3 × 105. Cells were plated in 12-well tissue culture plates and maintained in a 37 °C, 5% CO2 incubator for 16 h, followed by washing off residual virus and incubating with fresh medium for an additional 48 h. Cells were dispersed using trypsin and genomic extracts were prepared using QuickExtract solution according to the manufacturer's instructions. Analysis of indel rates was performed by NGS. [Figure 7] NGS analysis of editing by OMNI-79 V5570 mRNA in HeLa cells. Editing activity targeting the hSERP_g12 and CXCR4_s25 sites was tested by transfection of OMNI-79 V5570-encoding mRNA (in-house IVT) into HeLa cells. Editing activity was determined by NGS analysis. The mean and standard deviation of triplicates are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Detailed Description This specification provides modified OMNI-79 nucleases that exhibit increased activity at target sites compared to wild-type OMNI-79 nuclease (SEQ ID NO: 1). Wild-type OMNI-79 nuclease is disclosed in PCT International Patent Application No. PCT / US2021 / 035928, which is incorporated herein by reference. When the modified OMNI-79 nuclease variant is active in a CRISPR endonuclease system, the CRISPR endonuclease system has increased on-target editing activity compared to a system that includes wild-type OMNI-79 nuclease. In some embodiments, the modified OMNI-79 nuclease is an OMNI-79 nuclease variant that includes at least one amino acid substitution relative to wild-type OMNI-79 nuclease. In some embodiments, the modified OMNI-79 nuclease has multiple amino acid substitutions relative to wild-type OMNI-79 nuclease.

[0016] In some embodiments, OMNI-79 nuclease variants are at least 80%, e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1. By way of non-limiting example, OMNI-79 nuclease variants may differ by up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or 20% of the amino acid residues from the sequence shown in SEQ ID NO: 1. Such sequence differences may be revealed by sequence alignment. OMNI-79 nuclease variants may be generated by substituting at least one amino acid residue of an OMNI-79 wild-type nuclease with another amino acid residue, e.g., with a conservative or non-conservative amino acid substitution, and / or by making an insertion or deletion at an amino acid residue of the OMNI-79 wild-type nuclease. Such mutations, including but not limited to substitutions, insertions or deletions, may be made in addition to or in addition to other mutations described herein to generate an OMNI-79 nuclease variant from an OMNI-79 wild-type nuclease. In some embodiments, an OMNI-79 nuclease variant retains a desired activity of the parent wild-type OMNI-79 nuclease, e.g., the ability to interact with guide RNA and target DNA, and / or nuclease activity (e.g., the ability to cause double-stranded DNA breaks, single-stranded DNA breaks, or lack of nuclease or nickase activity). In some embodiments, the variants retain a desired activity, e.g., nuclease activity, at a level greater than or equal to the activity of the parent. In some embodiments, the variants retain a desired activity of the parent at a level of at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the activity of the parent. In some embodiments, the OMNI-79 nuclease variants have improved on-target efficacy compared to OMNI-79 wild-type nuclease.In some embodiments, variants of OMNI-79 nickases are provided that have increased activity compared to wild-type OMNI-79 nickases. In some embodiments, variants of OMNI-79 inactive nucleases are provided that have increased activity compared to wild-type OMNI-79 inactive nucleases. In some embodiments, the OMNI-79 nuclease variants are nickases with inactivated RuvC or HNH domains and further comprise amino acid substitutions at at least one of S1005, I14, and E1050. In some embodiments, the OMNI-79 nuclease variants are nickases with inactivated RuvC and HNH domains and further comprise amino acid substitutions at at least one of S1005, I14, and E1050.

[0017] In some embodiments, variants of OMNI-79 nuclease proteins are provided that comprise a sequence at least 80% identical to the amino acid sequence of wild-type OMNI-79 (SEQ ID NO:1) and have at least one amino acid substitution. In some embodiments, the amino acid substitution comprises replacing an amino acid residue with a positive, negative, uncharged, hydrophilic, hydrophobic, polar or non-polar amino acid. In some embodiments, the amino acid substitution is selected from replacing an amino acid with another amino acid selected from the group consisting of R, K, H, D, E, S, T, N, Q, C, U, G, P, A, I, L, M, F, W, Y and V.

[0018] In some embodiments, the OMNI-79 nuclease protein variant comprises an amino acid substitution at least one of I14, S1005 and E1050 of the wild-type OMNI-79 protein (SEQ ID NO: 1). Each possibility is a separate aspect of the invention. In some embodiments, a variant of the OMNI-79 nuclease protein is provided that comprises a sequence that is at least 80% identical to the amino acid sequence of wild-type OMNI-79 (SEQ ID NO: 1) and has at least one amino acid substitution at least one of I14, S1005 and E1050 of the wild-type OMNI-79 protein. In some embodiments, the OMNI-79 nuclease protein variant comprises at least one of the following amino acid substitutions at the following positions of the sequence of the wild-type OMNI-79 protein: I14L, S1005R, S1005K and E1050K. Each possibility is a separate aspect of the invention. In some embodiments, the substitutions correspond to those listed in Table 3.

[0019] In some embodiments, the OMNI-79 nuclease protein variant comprises at least one amino acid substitution at I14 and S1005 of the wild-type OMNI-79 protein. In some embodiments, the OMNI-79 nuclease protein variant comprises amino acid substitutions at I14 and S1005 of the wild-type OMNI-79 protein. In some embodiments, the OMNI-79 nuclease protein variant comprises amino acid substitutions I14L and S1005R in the wild-type OMNI-79 protein.

[0020] In some embodiments, the OMNI-79 nuclease protein variant comprises at least one amino acid substitution at S1005 and E1050 of the wild-type OMNI-79 protein. In some embodiments, the OMNI-79 nuclease protein variant comprises an amino acid substitution at S1005 and E1050 of the wild-type OMNI-79 protein. In some embodiments, the OMNI-79 nuclease protein variant comprises an amino acid substitution at S1005 and E1050 of the wild-type OMNI-79 protein. In some embodiments, the OMNI-79 nuclease protein variant comprises the amino acid substitutions S1005K and E1050K in the wild-type OMNI-79 protein.

[0021] In some embodiments, the OMNI-79 nuclease variant further comprises one or more of a nuclear localization sequence (NLS), a cell penetrating peptide sequence, and / or an affinity tag. In some embodiments, the OMNI-79 nuclease variant comprises one or more nuclear localization sequences that are strong enough to promote accumulation of a CRISPR complex comprising a detectable amount of the CRISPR nuclease in the nucleus of a eukaryotic cell.

[0022] In some embodiments, the OMNI-79 nuclease variant comprises an amino acid substitution selected from those corresponding to the indicated substitutions in Table 3 compared to wild-type OMNI-79.

[0023] According to some aspects, there is provided an isolated OMNI-79 nuclease protein variant comprising one or more substitutions or mutations in a wild-type OMNI-79 nuclease sequence, wherein said isolated OMNI-79 nuclease variant is active in a CRISPR system, wherein said CRISPR system has increased on-target editing activity compared to a wild-type CRISPR system.

[0024] In some embodiments, other mutations to the OMNI-79 nuclease variants described herein may be made. Examples include, but are not limited to, mutations that change the PAM recognition sequence, mutations that change the nuclease activity of the enzyme, and truncation or removal of portions of the nuclease. In some embodiments, the OMNI-79 nuclease variants may be encoded by a nucleotide sequence that produces the desired amino acid sequence of the variant. For example, the nucleotide sequence may be codon-optimized for cells such as bacteria, plants, or mammals.

[0025] In some embodiments of the present invention, the CRISPR nuclease and the targeting molecule form a CRISPR complex that binds to and cleaves a target DNA sequence. The CRISPR nuclease may form a CRISPR complex that includes the CRISPR nuclease and a single guide RNA (sgRNA) molecule. Alternatively, the CRISPR nuclease may form a CRISPR complex that includes the CRISPR nuclease, a crRNA molecule, and a tracrRNA molecule.

[0026] According to some aspects of the invention, there is provided a gene editing method having increased on-target editing activity, the method comprising contacting a target site with an active CRISPR system having an OMNI-79 protein variant complexed with a suitable guide RNA or guide RNA complex, wherein the active CRISPR endonuclease system has enhanced on-target editing activity compared to a wild-type OMNI-79 CRISPR system.

[0027] According to some aspects, a non-naturally occurring OMNI-79 nuclease variant is provided having a wild-type OMNI-79 protein sequence (SEQ ID NO:1) that includes an amino acid substitution at at least one of I14, S1005, and E1050.

[0028] In some embodiments, the amino acid substitution at S1005 and / or E1050 is to an amino acid having a positively charged R group.

[0029] In some embodiments, the amino acid having a positively charged R group is lysine or arginine.

[0030] In some aspects, the amino acid substitution is any one of I14L, S1005R, S1005K, and E1050K.

[0031] In some aspects, the OMNI-79 nuclease variant comprises an amino acid substitution at each of I14 and S1005.

[0032] In some embodiments, the amino acid substitutions are I14L and S1005R.

[0033] In some aspects, the OMNI-79 nuclease variant comprises an amino acid substitution at each of S1005 and E1050.

[0034] In some embodiments, the amino acid substitutions are S1005K and E1050K.

[0035] In some embodiments, the OMNI-79 nuclease variant has the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, or any one of SEQ ID NOs:12-25.

[0036] In some embodiments, the position of the amino acid substitution is I14 and is any one of I14L, I14V, I14F, I14C, I14A, or I14T.

[0037] In some embodiments, the amino acid substitution is I14L.

[0038] In some embodiments, the amino acid substitution at S1005 is to an amino acid having a positively charged R group.

[0039] In some aspects, the position of the amino acid substitution is S1005 and is any one of S1005R, S1005K, S1005Q, S1005I, S1005M, S1005V, S1005T, S1005N, S1005F, S1005A, S1005G or S1005E.

[0040] In some embodiments, the amino acid substitution at S1005 is to an amino acid having a positively charged R group.

[0041] In some embodiments, the amino acid substitution at S1005 is to an amino acid having a polar R group.

[0042] In some embodiments, the amino acid substitution is S1005R.

[0043] In some embodiments, the amino acid substitution is S1005K.

[0044] In some embodiments, the amino acid substitution is S1005T.

[0045] In some embodiments, the amino acid substitution is S1005N.

[0046] In some embodiments, the amino acid substitution is S1005Q.

[0047] In some aspects, the position of the amino acid substitution is E1050 and is any one of E1050K, E1050R, E1050P, E1050A, E1050I, E1050L, E1050V, E1050G, or E1050T.

[0048] In some embodiments, the amino acid substitution is E1050K.

[0049] In some embodiments, the amino acid substitution at E1050 is to an amino acid having a positively charged R group.

[0050] In some embodiments, OMNI-79 nuclease variants have at least 80% sequence identity to the wild-type OMNI-79 protein sequence (SEQ ID NO:1). For example, in some embodiments, OMNI-79 nuclease variants have at least 80% sequence identity to the wild-type OMNI-79 protein sequence (SEQ ID NO:1) and may have any one of the amino acid substitutions described herein. In some embodiments, OMNI-79 nuclease variants may have the amino acid substitutions described herein relative to the sequence shown in SEQ ID NO:1, with the remaining amino acid sequence having at least 80% sequence identity to the wild-type OMNI-79 protein sequence (SEQ ID NO:1).

[0051] In some embodiments, the OMNI-79 nuclease variant further comprises a nuclear localization sequence (NLS).

[0052] In some embodiments, the OMNI-79 nuclease variant, when complexed with a guide RNA molecule that targets the variant to a DNA target site, has increased activity against a DNA target site compared to wild-type OMNI-79 nuclease complexed with the guide RNA molecule.

[0053] According to some aspects of the present invention, there is provided a CRISPR system comprising an OMNI-79 nuclease variant as described herein complexed with a guide RNA molecule that targets a DNA target site, the CRISPR system having increased on-target editing activity compared to a wild-type CRISPR system comprising a wild-type OMNI-79 nuclease protein and a guide RNA molecule.

[0054] According to some embodiments of the present invention, there is provided a method of gene editing with increased on-target editing activity comprising contacting a DNA target site with an active CRISPR system comprising an OMNI-79 nuclease variant protein as described herein.

[0055] In some aspects, gene editing occurs in eukaryotic or prokaryotic cells.

[0056] In some embodiments, the eukaryotic cell is a plant cell or a mammalian cell.

[0057] In some embodiments, the mammalian cell is a human cell.

[0058] In some embodiments, the DNA target site is within or near a pathogenic allele of a gene.

[0059] In some embodiments, the DNA target is repaired with an exogenous donor molecule.

[0060] In some embodiments, the on-target editing activity is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 10-fold, 10-fold, 2Double, 10 3 Double, 10 4 Double, 10 5 Double or 10 6 double increase.

[0061] According to some aspects of the invention there is provided a modified cell obtained by the methods described herein.

[0062] In some embodiments, the cells are capable of engraftment.

[0063] In some embodiments, progeny cells can be generated following engraftment.

[0064] In some embodiments, progeny cells can be generated following autologous transplantation.

[0065] In some embodiments, the cells are capable of giving rise to progeny cells for at least 12 months or at least 24 months after engraftment.

[0066] In some embodiments, the cells are selected from the group consisting of hematopoietic stem cells, progenitor cells, CD34+ hematopoietic stem cells, bone marrow cells, and peripheral mononuclear cells.

[0067] According to some aspects of the invention, there is provided a composition comprising the modified cells described herein and a pharma- ceutically acceptable carrier. According to some aspects of the invention, there is provided a method of producing the composition in vitro or ex vivo, comprising combining the cells with a pharma- ceutically acceptable carrier.

[0068] According to some aspects of the present invention there are provided polynucleotide molecules encoding the OMNI-79 nuclease variant proteins described herein.

[0069] delivery The OMNI-79 variant compositions of the present application may be delivered as a protein, a DNA molecule, an RNA molecule, a ribonucleoprotein (RNP), a nucleic acid vector, or a combination thereof. In some embodiments, the RNA molecule comprises a chemical modification. Non-limiting examples of suitable chemical modifications include 2'-O-methyl (M), 2'-O-methyl-3'-phosphorothioate (MS) or 2'-O-methyl-3'-thioPACE (MSP), pseudouridine, and 1-methylpseudouridine. Each possibility is a separate aspect of this invention.

[0070] The OMNI-79 variants of the present application and / or the polynucleotides encoding same and / or additional molecules such as single guide RNA molecules, crRNA molecules, tracrRNA molecules or nucleotide molecules encoding them may be delivered to the target cell by suitable means. The target cell may be any cell, such as a eukaryotic or prokaryotic cell, isolated or not, in culture, in vitro, ex vivo, in vivo or in planta, in any environment. The target site in the target cell may be within the nucleus of the cell.

[0071] The compositions described herein may be introduced into cells as part of a vector molecule with additional sequences, such as, for example, an origin of replication, a promoter, and a gene encoding antibiotic resistance. Additionally, the compositions may be introduced into cells as naked nucleic acid or protein, or as nucleic acid or protein complexed with or packaged within an agent, such as a liposome, exosome, or poloxamer, or delivered by a recombinant virus (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV)) or virus-like particle. By way of non-limiting example, the compositions may be packaged into an adeno-associated virus (AAV) or into a lentivirus, such as a non-integrating lentivirus or a lentivirus lacking reverse transcription. Additional non-limiting examples include packaging the compositions into liposomes, extracellular vesicles, or exosomes, which may be pseudotyped with vesicular stomatitis glycoprotein (VSVG) or conjugated to a cell membrane-permeable peptide, an antibody, a targeting moiety, or any combination thereof.

[0072] In some embodiments, the composition delivered comprises a nuclease mRNA and a guide RNA. In some embodiments, the composition delivered comprises a nuclease mRNA, a guide RNA and a donor template. In some embodiments, the composition delivered comprises a CRISPR nuclease and a guide RNA. In some embodiments, the composition delivered comprises a CRISPR nuclease, a guide RNA and a donor template for gene editing, e.g., by homology-directed repair. The lentivirus optionally comprises a nuclease mRNA and a guide RNA molecule (e.g., a single guide RNA molecule or a crRNA molecule) and is used to guide the nuclease to a target site. In some embodiments, the composition delivered to the cell comprises a nuclease mRNA, a guide RNA molecule and a donor template molecule. The lentivirus optionally comprises a nuclease protein variant and a guide RNA molecule. The composition delivered to the cell optionally comprises a nuclease protein variant, a guide RNA molecule and / or a donor template for homology-directed repair. The composition delivered to the cell optionally comprises an mRNA of a nuclease variant, a DNA targeting crRNA molecule and a tracrRNA molecule. The composition delivered to the cell optionally comprises an mRNA of a nuclease variant, a DNA targeting crRNA molecule and a tracrRNA molecule, and a donor template molecule. The composition delivered to the cell optionally comprises a nuclease protein variant, a DNA targeting crRNA molecule and a tracrRNA molecule. The composition delivered to the cell optionally comprises a nuclease protein variant, a DNA targeting crRNA molecule and a tracrRNA molecule, and a DNA donor template molecule for homology directed repair.

[0073] Such compositions can be delivered using a suitable viral vector system. Conventional viral and non-viral based gene transfer methods can introduce the nucleic acid and / or OMNI-79 nuclease variant protein into cells (e.g., mammalian cells, plant cells, etc.) and target tissues. Such methods can also be used to administer in vitro encoded nucleic acid and / or OMNI-79 nuclease variant protein to cells. In some embodiments, the nucleic acid and / or OMNI-79 nuclease variant protein is administered for in vivo or ex vivo gene therapy. Non-viral vector delivery systems include naked nucleic acid and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. For reviews of gene therapy procedures, see Anderson, Science 256:808-813 (1992);Nabel & Felgner, TIBTECH 11:211-217 (1993);Mitani & Caskey, TIBTECH 11:162-166 (1993);Dillon, TIBTECH 11:167-175 (1993);Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1988);Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995);Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995);Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and See Bohm (eds.) (1995) and Yu et al., Gene Therapy 1:13-26 (1994).

[0074] Non-viral methods of delivery of nucleic acids and / or proteins include electroporation, lipofection, microinjection, biolistics, particle gun acceleration, virosomes, virus-like particles, exosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, artificial virions, and drug-enhanced nucleic acid uptake, or can be delivered to plant cells by bacteria or viruses (e.g., Agrobacterium, Rhizobium sp. NGR234, Sinorhizoboium meliloti, Mesorhizobium loti, Tobacco Mosaic Virus, Potato Virus X, Cauliflower Mosaic Virus, Cassava Vein Mosaic Virus). See, e.g., Chung et al. Trends Plant Sci. (2006). Sonoporation, for example using the Sonitron 2000 system (Rich-Mar), can also be used to deliver nucleic acids. Cationic lipid-mediated delivery of proteins and / or nucleic acids is also contemplated as an in vivo or in vitro delivery method. See Zuris et al., Nat. Biotechnol. (2015); Coelho et al., N. Engl. J. Med. (2013); Judge et al., Mol. Ther. (2006) and Basha et al., Mol. Ther. (2011).

[0075] Non-viral vectors, such as transposon-based systems (e.g., recombinant Sleeping Beauty transposon system or recombinant PiggyBac transposon system), may also be used to deliver to target cells and transfer the polynucleotide sequences of, or polynucleotide sequences encoding, the molecules of the composition in the target cells.

[0076] Other representative nucleic acid delivery systems include those provided by Amaxa® Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics Inc. (see, e.g., U.S. Pat. No. 6,008,336). Lipofectin is described, for example, in U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam®, Lipofectin®, and Lipofectamine® RNAiMAX). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those disclosed in WO 91 / 17424 and WO 91 / 16024. Delivery to cells (ex vivo administration) or target tissues (in vivo administration) is possible.

[0077] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820). (1992); see U.S. Patent Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028 and 4,946,787.

[0078] Another delivery method involves using packaging of the nucleic acid to be delivered in an EnGeneIC delivery vehicle (EDV). The EDV is delivered specifically to the target tissue using a bispecific antibody, where one arm of the antibody has specificity for the target tissue and the other arm has specificity for the EDV. The antibody carries the EDV to the surface of the target cell, where it is then carried into the cell by endocytosis. Once inside the cell, the contents are released (see MacDiamid et al (2009) Nature Biotechnology 27(7) p. 643).

[0079] The use of RNA or DNA virus-based systems for nucleic acid delivery utilizes highly evolved methods to target viruses to specific cells in the body and transport the viral payload to the nucleus. Viral vectors can be administered directly to the patient (in vivo) or can be used to treat cells in vitro and the modified cells are administered to the patient (ex vivo). RNA or DNA virus-based systems for nucleic acid delivery include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated viral, vaccinia virus and herpes simplex virus vectors for gene transfer. Integration into the host genome is possible using retroviral, lentiviral and adeno-associated viral gene transfer methods, often resulting in long-term expression of the inserted transgene. High transduction efficiency has also been observed in a variety of cells and target tissues. Nucleic acids expressing OMNI-79 variants or variants and related nucleic acids may be delivered by non-integrating lentiviruses. If necessary, lentiviral RNA delivery is utilized. The lentivirus optionally comprises a nuclease mRNA and a guide RNA molecule (e.g., a single guide RNA molecule or a crRNA molecule) used to direct the nuclease to a target site. The lentivirus optionally comprises a nuclease mRNA, a guide RNA molecule, and a donor template molecule. The lentivirus optionally comprises a nuclease protein variant and a guide RNA molecule. The lentivirus optionally comprises a nuclease protein variant, a guide RNA molecule, and / or a donor template molecule for homologous recombination repair. The lentivirus optionally comprises a nuclease variant mRNA, a DNA targeting crRNA molecule, and a tracrRNA molecule. The lentivirus optionally comprises a nuclease variant mRNA, a DNA targeting crRNA molecule, and a tracrRNA molecule, and a donor template molecule. The lentivirus optionally comprises a nuclease protein variant, a DNA targeting crRNA molecule, and a tracrRNA molecule.The lentivirus optionally contains nuclease protein variants, DNA targeting crRNA and tracrRNA molecules, and donor template molecules for homology directed repair.

[0080] As previously described, the compositions of the present application can be delivered to target cells using non-integrating lentiviral particle methods (e.g., the LentiFlash® system). Such methods include: It may be used to deliver mRNA or other RNA to a target cell, where delivery of the RNA to the target cell results in assembly of the composition within the target cell (see also WO 2013 / 014537, WO 2014 / 016690, WO 2016 / 185125, WO 2017 / 194902, WO 2017 / 194903).

[0081] Retroviral tropism can be altered by incorporating foreign envelope proteins, expanding the potential target cell targeting. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and usually produce high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats that can package up to 6-10 kb of foreign sequences. A minimal number of cis-acting LTRs are sufficient for vector replication and packaging, which are then used to integrate therapeutic genes into target cells and provide permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher Panganiban, J. Virol. (1992); Johann et al., J. Virol. (1992); Sommerfelt et al., Virol. (1990); Wilson et al., J. Virol. (1989); Miller et al., J. Virol. (1991); WO 94 / 26877).

[0082] At least six viral vector approaches are currently available for gene transfer in clinical trials, using methods involving complementation of a defective vector by a gene inserted into a helper cell line to generate the transducing agent.

[0083] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al., Blood (1995);Kohn et al., Nat. Med. (1995);Malech et al., PNAS (1997)). PA317 / pLASN was the first therapeutic vector used in gene therapy (Blaese et al., Science (1995)). Transduction efficiencies of over 50% have been observed with MFG-S packaged vectors (Ellem et al., Immunol Immunother. (1997);Dranoff et al., Hum. Gene Ther. (1997)).

[0084] Packaging cells are used to form viral particles capable of infecting host cells. Such cells include 293 cells, which package adenovirus AAV, and psi.2 or PA317 cells, which package retrovirus. Viral vectors for use in gene therapy are usually obtained by producer cell lines that package nucleic acid vectors into viral particles. The vectors usually contain the minimal viral sequences required for packaging and subsequent integration into the host (if applicable), other viral sequences being replaced by expression cassettes that code for the proteins to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors for use in gene therapy usually only possess the terminal inverted repeat (ITR) sequences of the AAV genome, which are required for packaging and integration into the host genome. The viral DNA is packaged in a cell line that contains a helper plasmid that codes for other AAV genes, namely rep and cap, but lacks the ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus facilitates the replication of the AAV vector and the expression of the AAV genes from the helper plasmid. The helper plasmid is not packaged in large quantities because it lacks ITR sequences. Contamination with adenovirus can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV. Furthermore, AAV can be produced on a clinical scale using the baculovirus system (see U.S. Patent No. 7,479,554).

[0085] In many gene therapies, it is desirable to deliver gene therapy vectors with high specificity to specific tissues. Thus, viral vectors can be modified to have specificity for cells of interest by expressing a ligand on the outer surface of the virus as a fusion protein with the viral coat protein. The ligand is selected to have affinity for a receptor known to be present on the cells of interest. For example, Han et al., Proc. Natl. Acad. Sci. USA 92:9747-9751 (1995) reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing the human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs, where the target cell expresses a receptor and the virus expresses a fusion protein containing a ligand for the cell surface receptor. For example, filamentous phage can be modified to display antibody fragments (e.g., FAB or Fv) with specific binding affinity for virtually any cellular receptor. This description applies primarily to viral vectors, but the same principles can be applied to non-viral vectors. Such vectors can be modified to include uptake sequences that facilitate uptake by specific target cells. Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial injection) or local application, as described below. Alternatively, vectors can be delivered ex vivo to cells transplanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, biopsy tissue) or to cells such as hematopoietic stem cells from a universal donor, which are then re-implanted into the patient, typically after selection of cells that have incorporated the vector.

[0086] Ex vivo cell transfection for diagnostics, research, or gene therapy (e.g., by re-injection of the transfected cells into the host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from the subject organism, transfected with an RNA composition, and re-injected into the subject organism (e.g., patient). A variety of cells suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994) and references cited therein for a discussion of methods for isolating and culturing cells from patients).

[0087] Suitable cells include, but are not limited to, eukaryotic and prokaryotic cells and / or cell lines. Non-limiting examples of such cells or cell lines generated from such cells include COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2 / 0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T) and perC6 cells, any plant cell (differentiated or undifferentiated), as well as insect cells such as Spodoptera fugiperda (Sf), or fungal cells such as Saccharomyces, Pichia and chizosaccharomyces. In one embodiment, the cell line is a CHO-K1, MDCK or HEK293 cell line. Additionally, primary cells may be isolated and treated with a nuclease system (e.g., CRISPR / Cas) before being used ex vivo for reintroduction into the subject. Suitable primary cells include peripheral blood mononuclear cells (PBMCs) and blood cell subsets, such as, but not limited to, CD4+ T cells or CD8+ T cells. Suitable cells also include stem cells, such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neural stem cells and mesenchymal stem cells.

[0088] In one embodiment, stem cells are used in ex vivo procedures for cell transfection and gene therapy. The advantage of using stem cells is that they can be differentiated in vitro into other cell types or introduced into a mammal (such as a cell donor) where they engraft in the bone marrow. Methods are known for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such as GM-CSF, IFNγ, and TNFα (see, for non-limiting examples, Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).

[0089] Stem cells are isolated for transduction and differentiation by known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies that bind to unwanted cells such as CD4+ and CD8+ (T cells), CD45+ (pan B cells), GR-1 (granulocytes), and Iad (differentiated antigen presenting cells) (see, for a non-limiting example, Inaba et al., J. Exp. Med. 176:1693-1702 (1992)). In some embodiments, modified stem cells can also be used.

[0090] In particular, the OMNI-79 variants of the present application may be suitable for genome editing of post-mitotic cells or cells that are not actively dividing (e.g., arrested cells). Examples of post-mitotic cells that may be edited using the OMNI-79 variants of the present invention include, but are not limited to, muscle cells, cardiomyocytes, hepatocytes, bone cells, and neurons.

[0091] Vectors (e.g., retroviruses, liposomes, etc.) containing therapeutic RNA compositions can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked RNA or mRNA can be administered. Administration is by routes including, but not limited to, injection, infusion, topical application, and electroporation, which are commonly used to introduce molecules with eventual contact with blood or tissue cells. Suitable methods for administering such nucleic acids are available and are widely known to those of skill in the art. Multiple routes of administration of a particular composition can be used, although certain routes often result in more rapid and effective responses than others.

[0092] Suitable vectors for introducing transgenes into immune cells (e.g., T cells) include non-integrating lentiviral vectors, see, e.g., U.S. Patent Application Publication No. 2009 / 0117617.

[0093] Pharmaceutically acceptable carriers are determined in part by the composition being administered, as well as by the method used to administer the composition. Thus, there is a wide variety of suitable formulations of pharmaceutical compositions available, see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989.

[0094] DNA repair by homologous recombination In some aspects of the invention, OMNI-79 nuclease variants are used to cleave DNA at target sites to induce cellular repair mechanisms, such as, but not limited to, non-homologous end joining (NHEJ) or homology directed repair (HDR).

[0095] The term "homologous recombination repair" or "HDR" refers to a mechanism that repairs DNA damage in cells, for example, during repair of double-stranded and single-stranded breaks in DNA. HDR requires nucleotide sequence homology and uses a "nucleic acid template" (used interchangeably herein as donor template) to repair the sequence (e.g., DNA target sequence) where the double-stranded or single-stranded break occurred. This results, for example, in the transfer of genetic information from the nucleic acid template to the DNA target sequence. If the nucleic acid template sequence differs from the DNA target sequence and some or all of the nucleic acid template polynucleotide or oligonucleotide is incorporated into the DNA target sequence, HDR can lead to alteration of the DNA target sequence (e.g., insertion, deletion, mutation). In some embodiments, all or a portion of the nucleic acid template polynucleotide, or a copy of the nucleic acid template, is incorporated into the site of the DNA target sequence.

[0096] The terms "nucleic acid template" and "donor" refer to a nucleotide sequence to be inserted or copied into a genome. A nucleic acid template comprises a sequence of, e.g., one or more nucleotides that may be added to a target nucleic acid, template a change in a target nucleic acid, or be used to modify a target sequence. The length of the nucleic acid template sequence may be any length, e.g., 2-10,000 nucleotides (or any integer therebetween or greater), preferably about 100-1,000 nucleotides (or any integer therebetween), more preferably about 200-500 nucleotides. A nucleic acid template may be a single-stranded nucleic acid, a double-stranded nucleic acid. In some embodiments, a nucleic acid template comprises a sequence of, e.g., one or more nucleotides, corresponding to a wild-type sequence of a target nucleic acid, e.g., at a target location. In some embodiments, a nucleic acid template comprises a sequence of, e.g., one or more ribonucleotides, corresponding to a wild-type sequence of a target nucleic acid, e.g., at a target location. In some embodiments, a nucleic acid template comprises modified ribonucleotides.

[0097] Insertion of exogenous sequences (also referred to as "donor sequences", "donor templates" or "donors") can also be performed, for example, to correct a mutant gene or increase expression of a wild-type gene. It is readily apparent that a donor sequence is usually not identical to the genomic sequence in which it is placed. A donor sequence can include a non-homologous sequence flanked by two homologous regions to allow efficient HDR at the target location. Additionally, a donor sequence can include a vector molecule that includes a sequence that is not homologous to a target region in cellular chromatin. A donor molecule can include discontinuous regions that are homologous to cellular chromatin. For example, the sequence can be present in the donor nucleic acid molecule for targeted insertion of a sequence that is not normally present in the target region, and can be flanked by regions that are homologous to the sequence of the target region.

[0098] The donor polynucleotide may be single-stranded and / or double-stranded DNA or RNA, and may be introduced into cells in linear or circular form. See, for example, US Patent Application Publication Nos. 2010 / 0047805; 2011 / 0281361; 2011 / 0207221 and 2019 / 0330620. When introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonuclease degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues are added to the 3' end of the linear molecule, and / or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang and Wilson, Proc. Natl. Acad. Sci. USA (1987); Nehls et al., Science (1996). Other methods of protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino groups and the use of modified internucleotide linkages (e.g., phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues).

[0099] Thus, the embodiment of the present invention using a donor template for repair may use DNA or RNA, which is a single-stranded and / or double-stranded donor template that can be introduced into cells in a linear or circular form. In an embodiment of the present invention, the gene editing composition comprises (1) an RNA molecule comprising a guide sequence that makes a double-stranded break in the gene before repair, and (2) a donor RNA template for repair, where the RNA molecule comprising the guide sequence is a first RNA molecule and the donor RNA template is a second RNA molecule. In some embodiments, the guide RNA molecule and the template RNA molecule are linked as part of one molecule.

[0100] The donor sequence may be an oligonucleotide and may be used for gene correction or targeted modification of an endogenous sequence. The oligonucleotide may be introduced into the cell by a vector, electroporated into the cell, or by other methods known in the art. The oligonucleotide may be used to "correct" a mutant sequence in an endogenous gene (e.g., the sickle mutation of beta globin) or may be used to insert a sequence at an endogenous locus for a desired purpose.

[0101] Polynucleotides can be introduced into cells as part of a vector molecule that has additional sequences such as, for example, an origin of replication, a promoter, and genes encoding antibiotic resistance. Additionally, donor polynucleotides can be introduced as naked nucleic acid, complexed with or packaged within agents (e.g., liposomes, exosomes, poloxamers), or delivered by recombinant viruses (e.g., adenoviruses, AAV, herpes viruses, retroviruses, lentiviruses, and integrase-deficient lentiviruses (IDLV)) or virus-like particles.

[0102] The donor is generally inserted such that its expression is driven by the endogenous promoter of the integration site, i.e., the promoter that drives expression of the endogenous gene into which the donor is inserted. However, it will be apparent that the donor may also include a promoter and / or enhancer, e.g., a constitutive promoter or an inducible or tissue-specific promoter.

[0103] The donor molecule may be inserted into an endogenous gene such that all or a portion of the endogenous gene is expressed, or none of the endogenous gene is expressed. For example, a transgene of the present application may be inserted into an endogenous locus such that a portion of the endogenous sequence (the N-terminus and / or C-terminus of the transgene) is expressed, or none of the endogenous sequence is expressed, e.g., as a fusion with the transgene. In other embodiments, the transgene (with or without additional coding sequences, e.g., an endogenous gene) is integrated into any endogenous locus, such as a safe harbor locus (e.g., the CCR5 gene, the CXCR4 gene, the PPP1R12c (also known as AAVS1) gene, the albumin gene, or the Rosa gene). See, e.g., U.S. Patent Nos. 7,951,925 and 8,110,379; U.S. Patent Application Publication Nos. 2008 / 0159996; 20100 / 0218264; 2010 / 0291048; 2012 / 0017290; 2011 / 0265198; 2013 / 0137104; 2013 / 0122591; 2013 / 0177983 and 2013 / 0177960, and U.S. Provisional Application No. 61 / 823,689).

[0104] When an endogenous sequence (part of an endogenous or transgene) is expressed along with a transgene, the endogenous sequence may be a full-length sequence (wild-type or mutant) or a partial sequence. Preferably, the endogenous sequence is functional. Non-limiting examples of functions of these full-length or partial sequences include increasing the half-life of a polypeptide expressed by the transgene (e.g., a therapeutic gene) and / or acting as a carrier.

[0105] In addition, although not essential for expression, the exogenous sequence may also include transcriptional or translational regulatory sequences, such as a promoter, an enhancer, an insulator, an internal ribosome entry site, a sequence encoding a 2A peptide, and / or a polyadenylation signal.

[0106] In one embodiment, the donor molecule comprises a sequence selected from the group consisting of a gene encoding a protein (e.g., a coding sequence encoding a protein that is missing in a cell or individual, or an alternative version of a gene encoding a protein), a regulatory sequence, and / or a sequence encoding a structural nucleic acid such as a microRNA or siRNA.

[0107] DNA-targeting RNA molecules In some embodiments of the present invention, the DNA-targeting RNA molecule comprises a guide sequence portion. The "guide sequence portion" of an RNA molecule refers to a nucleotide sequence that can hybridize with a specific target DNA sequence, e.g., the guide sequence portion is partially or completely complementary to the targeted DNA sequence along its length. In some embodiments, the length of the guide sequence portion is 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, or 50 nucleotides or less. 39, 17-38, 17-37, 17-36, 17-35, 17-34, 17-33, 17-31, 17-30, 17-29, 17-28, 17-27, 17-26, 17-25, 17-24, 17-22, 17-21, 18-25, 18-24, 18-23, 18-22, 18-21, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-22, 18-20, 20-21, 21-22, or 17-20 nucleotides. The entire length of the guide sequence portion is completely complementary to the targeted DNA sequence along its length. The guide sequence portion may be a portion of an RNA molecule that can form a complex with a CRISPR nuclease having a guide sequence portion that functions as the DNA targeting portion of the CRISPR complex. When a DNA molecule having a guide sequence portion is present simultaneously with a CRISPR molecule, the RNA molecule can target the CRISPR nuclease to a specific target DNA sequence. Each possibility is a separate aspect of this invention. The RNA molecule can be specifically designed to target a desired sequence. Thus, a molecule that includes a "guide sequence portion" is a type of targeting molecule. Throughout this application, the terms "guide molecule", "RNA guide molecule", "guide RNA molecule" and "gRNA molecule" are synonymous with a molecule that includes a guide sequence portion, and the term "spacer" is synonymous with "guide sequence portion".

[0108] In some aspects of the invention, the disclosed methods include methods of modifying a nucleotide sequence in a cell-free system or at a target site in the genome of a cell, comprising introducing into a cell a composition as described herein.

[0109] In some embodiments, the cell is a eukaryotic cell, preferably a mammalian cell or a plant cell. In some embodiments, the modification of the genome occurs within the nucleus of the cell.

[0110] In some aspects of the invention, the disclosed methods include the use of the compositions of the present application for the treatment of a subject suffering from a disease associated with a genomic mutation, comprising modifying a nucleotide sequence at a target site in the subject's genome.

[0111] In some aspects of the invention, the disclosed methods include methods of treating a subject having a mutational disorder comprising targeting a composition of the present application to an allele associated with the mutational disorder.

[0112] In some aspects, the mutational disorder is associated with a disease or disorder selected from any of neoplasia, age-related macular degeneration, schizophrenia, neurological disorders, neurodegenerative diseases, movement disorders, fragile X syndrome, secretase-related disorders, prion-related disorders, ALS, addiction, autism, Alzheimer's disease, neutropenia, inflammation-related disorders, Parkinson's disease, blood and coagulation diseases and disorders, beta thalassemia, sickle cell anemia, cellular dysregulation, tumor-related diseases and disorders, inflammation and immune-related diseases and disorders, metabolic, liver, kidney and protein diseases and disorders, muscular and skeletal diseases and disorders, skin diseases and disorders, neurological diseases and disorders, and ophthalmic diseases and disorders.

[0113] Disease and Treatment An embodiment of the present invention targets nucleases to specific loci associated with a disease or disorder as a form of gene editing, treatment or therapeutic method. For example, the novel nucleases disclosed herein may be specifically targeted to pathogenic mutant alleles of genes using specially designed guide RNA molecules to induce gene editing or knockout. It is preferable to design guide RNA molecules by first considering the PAM requirements of the nuclease, which also depends on the system in which gene editing is performed, as shown in the specification. For example, guide RNA molecules designed to target OMNI-79 nuclease to a target site are designed to include a spacer sequence complementary to the DNA strand of the DNA double-stranded region adjacent to the OMNI-79 PAM sequence (e.g., "NGG"). The guide RNA molecule is preferably further designed to include a spacer region (i.e., the region of the guide RNA molecule complementary to the target allele) of sufficient and preferably optimal length to increase the specific activity of the nuclease and reduce off-target effects.

[0114] As a non-limiting example, guide RNA molecules may be designed to target nucleases to specific regions of mutant alleles, for example, near the start codon, such that upon DNA damage by nucleases, non-homologous end joining (NHEJ) pathways are induced, resulting in silencing of the mutant allele by introducing frameshift mutations. This approach to design guide RNA molecules is particularly useful for altering the action of dominant-negative mutations, thereby treating subjects. As another non-limiting example, guide RNA molecules may be designed to target specific pathogenic mutations of mutated alleles, such that upon DNA damage by nucleases, homology-directed repair (HDR) pathways are induced, resulting in template-mediated correction of the mutant allele. This approach to design guide RNA molecules is particularly useful for altering the haploinsufficient action of mutant alleles, thereby treating subjects.

[0115] Non-limiting examples of genes that may be targeted for modification to treat disease or disorders are given below. Disease-associated genes and mutations that induce mutation disorders are described in the literature. Such mutations can be used to design DNA targeting RNA molecules that target the alleles of disease-associated genes, and CRISPR compositions that induce DNA damage to induce DNA repair pathways to modify the alleles, thereby treating the mutation disorder.

[0116] Mutations in ELANE gene are associated with neutropenia.Therefore, the embodiment of the present invention that targets ELANE may be used without restriction in the method of treating subjects suffering from neutropenia.Guide RNA molecules that target ELANE gene and are useful for treating neutropenia are disclosed in PCT International Application No. PCT / US2020 / 059186.

[0117] CXCR4 is a coreceptor in human immunodeficiency virus type 1 (HIV-1) infection. Accordingly, aspects of the invention that target CXCR4 may be used, without limitation, in methods of treating subjects with HIV-1 or conferring resistance to HIV-1 infection in a subject.

[0118] Disruption of programmed cell death protein 1 (PD-1) promotes CAR-T cell killing of tumor cells, making PD-1 a potential target for cancer therapy. Thus, embodiments of the present invention that target PD-1 may be used without restriction in methods of treating subjects suffering from cancer. In one embodiment, the treatment is CAR-T cell therapy with T cells modified according to the present invention to be PD-1 deficient.

[0119] In addition, BCL11A is a gene involved in the suppression of hemoglobin production. Inhibiting BCL11A may increase globin production and treat diseases such as thalassemia and sickle cell anemia. See, for example, WO 2017 / 077394, U.S. Patent Application Publication No. 2011 / 0182867; Humbert et al. Sci. Transl. Med. (2019) and Canver et al. Nature (2015). Thus, aspects of the invention that target enhancers of BCL11A may be used without restriction in methods of treating subjects suffering from β-thalassemia or sickle cell anemia.

[0120] The present invention may be used to target disease-associated genes in the study, modification or treatment of diseases or disorders listed below in Table A or Table B. Indeed, disease-associated genes having a genetic locus may be studied, modified or treated by using the nucleases disclosed herein to target the appropriate disease-associated gene, such as those listed in US Patent Application Publication No. 2018 / 0282762 and EP Patent No. 3079726 (B1).

[0121] [Table A]

[0122] [Table B-1]

[0123] [Table B-2]

[0124] [Table B-3]

[0125] Unless otherwise defined, all technical and / or scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this invention, representative methods and / or materials are described below. In case of conflict, the specification, including definitions, will control. Additionally, the materials, methods, and examples are merely illustrative and are not intended to be necessarily limiting.

[0126] Unless otherwise stated in the discussion, adjectives such as "substantially" and "about" that modify the state or relationship of features of an embodiment of the invention are understood to mean that the state or relationship is defined within an acceptable range for operation of the embodiment for its intended use. Unless otherwise indicated, the term "or" in the specification and claims is considered an inclusive "or" rather than an exclusive "or" and indicates at least one or any combination of the items it conjugates.

[0127] The term "a" or "an" as used herein should be understood to refer to "one or more" of the listed components. It will be apparent to one of ordinary skill in the art that the use of the singular includes the plural unless otherwise specified. Thus, the terms "a" and "at least one" have the same meaning in this application.

[0128] To better understand the teachings and in no way limit the scope of the teachings, unless otherwise specified, all numbers indicating quantities, percentages or ratios, and other numerical values ​​used in the specification and claims should be understood to be modified in all instances by the term "about." Thus, unless indicated to the contrary, the numerical values ​​set forth in the specification and claims are approximations that may vary depending on the desired properties sought to be obtained. At the very least, each numerical value should be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0129] In this specification and claims, the verbs "contain," "include," and "have" and their conjugations are used to indicate that the object of the verb is not necessarily an exhaustive list of components, elements, or parts of the subject of the verb. Other terms used herein have meanings well known in the art.

[0130] In this specification, the term "targeting sequence" or "targeting molecule" refers to a nucleotide sequence or molecule that includes a nucleotide sequence that can hybridize to a specific target sequence, e.g., a targeting sequence has a nucleotide sequence that is at least partially complementary to the sequence to be targeted along its length. A targeting sequence or targeting molecule may be a portion of an RNA molecule that can form a complex with a CRISPR nuclease, alone or in combination with other RNA molecules, that has a targeting sequence that functions as the targeting portion of the CRISPR complex. When a molecule with a targeting sequence is present simultaneously with a CRISPR molecule, the RNA molecule, alone or in combination with additional RNA molecules (e.g., tracrRNA molecules), can target the CRISPR nuclease to a specific target sequence. As a non-limiting example, the guide sequence portion of a CRISPR RNA molecule or a single guide RNA molecule may function as a targeting molecule. Each possibility is a separate aspect of this invention. A targeting sequence can be specifically designed to target a desired sequence.

[0131] In this specification, the term "target" refers to the targeting sequence of targeting molecule preferentially hybridizes with the nucleic acid having the target nucleotide sequence.It is understood that the term "target" encompasses variable hybridization, such that there is preferential targeting of the nucleic acid having the target nucleotide sequence, but in addition to on-target hybridization, unintended off-target hybridization may also occur.When RNA molecule targets sequence, it is understood that the complex of RNA molecule and CRISPR nuclease molecule targets sequence by nuclease activity.

[0132] As used herein, the term "wild type" refers to a representative form of an organism, strain, gene, or trait that exists in nature, as distinguished from a variant or a variant, as understood by those skilled in the art. Thus, as used herein, when an amino acid or nucleotide sequence refers to a wild type sequence, a variant refers to a variant of that sequence, including, for example, a substitution, deletion, or insertion. In some embodiments of the invention, the modified CRISPR nuclease is a variant of a CRISPR nuclease that includes at least one amino acid modification, also referred to as a "mutation," (e.g., a substitution, deletion, and / or insertion) relative to the wild type OMNI-79 nuclease of SEQ ID NO:1.

[0133] The terms "non-natural," "non-naturally occurring," or "modified" are used interchangeably and refer to human modification. When used with respect to a nucleic acid molecule or polypeptide, the term may mean that the nucleic acid molecule or polypeptide is at least substantially free from at least one component with which it is naturally associated in nature and found in nature.

[0134] The terms "mutant" or "variant" are used interchangeably and refer to a molecule that is not naturally occurring or that has been altered.

[0135] As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0136] As used herein, "genomic DNA" refers to a sequence of linear and / or chromosomal DNA and / or plasmid or other extrachromosomal DNA present in a cell or cells of interest. In some embodiments, the cell of interest is a eukaryotic cell. In some embodiments, the cell of interest is a prokaryotic cell. In some embodiments, the method creates a double-strand break (DSB) at a predetermined target site in the genomic DNA sequence, resulting in a mutation, insertion and / or deletion of the DNA sequence at the target site in the genome.

[0137] "Eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells.

[0138] In this specification, the term "modified cell" or "modified cell" refers to a cell that has undergone a double-strand break by a complex of an RNA molecule and a CRISPR nuclease as a result of hybridization with a target sequence, i.e., on-target hybridization. The term "modified cell" may further include a cell in which a mutation has been repaired or corrected after a double-strand break by a variant. A modified cell may be any cell, such as a eukaryotic or prokaryotic cell, in any environment, isolated or not, or maintained in culture, in vitro, ex vivo, in vivo, or in planta.

[0139] The present invention provides modified cells obtained by the variants or methods described herein. In some embodiments, these modified cells can give rise to progeny cells. In some embodiments, these modified cells can give rise to progeny cells after engraftment. By way of non-limiting example, the modified cells can be hematopoietic stem cells (HSCs) or cells suitable for allogeneic or autologous cell transplantation. The variants and methods described herein can also be utilized to generate chimeric antigen receptor T (CAR-T) cells.

[0140] The invention also provides compositions comprising these modified cells and a pharma- ceutically acceptable carrier, as well as in vitro or ex vivo methods of preparing the same, comprising combining the cells with a pharma- ceutically acceptable carrier.

[0141] As used herein, the term "nuclease" refers to an enzyme capable of cleaving phosphodiester bonds between nucleotide subunits of nucleic acids. Nucleases may be isolated or derived from natural sources. The natural source may be any organism. Alternatively, nucleases may be modified or synthetic proteins that have phosphodiester bond cleavage activity.

[0142] As used herein, the term "protospacer adjacent motif" or "PAM" refers to a nucleotide sequence of a target DNA that is located adjacent to the target DNA sequence and is recognized by a CRISPR nuclease. The PAM sequence may vary depending on the nuclease. For example, wild-type Streptococcus pyogenes Cas9 recognizes the PAM sequence "NGG". Those skilled in the art will appreciate that a single guide RNA molecule or a crRNA:tracrRNA complex can form a complex with a CRISPR nuclease to bind to a target genomic DNA sequence of interest adjacent to a protospacer adjacent motif (PAM). The nuclease then generates a double-stranded break in the protospacer via cleavage of the target DNA.

[0143] As used herein, a sequence or molecule has X% "sequence identity" with respect to another sequence or molecule if X% of the bases or amino acids between the sequences of the molecules are the same and in the same relative positions. For example, a first nucleotide sequence that has at least 95% sequence identity with a second nucleotide sequence has at least 95% of the bases in the same relative positions as the other sequence.

[0144] The terms "nuclear localization sequence" and "NLS" are used interchangeably to refer to an amino acid sequence / peptide that directs the transport of an associated protein from the cytoplasm across the nuclear membrane barrier. The term "NLS" is intended to encompass not only a specific peptide nuclear localization sequence, but also derivatives thereof that can direct the translocation of cytoplasmic polypeptides across the nuclear membrane barrier. An NLS can direct nuclear translocation of a polypeptide when attached to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the polypeptide. Additionally, polypeptides with an NLS linked at the N-terminus or C-terminus to the side chain of an amino acid randomly located in the amino acid sequence of the polypeptide are translocated. An NLS is typically composed of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, although other types of NLS are known. Non-limiting examples of NLSs include NLS sequences derived from SV40 virus large T antigen, nucleoplasmin, c-myc, hRNPA1 M9 NLS, the IBB domain from importin alpha, sarcoma T protein, human p53, mouse c-abl IV, influenza virus NS1, hepatitis virus delta antigen, mouse Mx1 protein, human poly(ADP-ribose) polymerase and steroid hormone receptor (human) glucocorticoid.

[0145] The term "CRISPR system" refers to a CRISPR endonuclease system that includes a CRISPR nuclease protein, such as a mutant or variant described herein, and an appropriate guide RNA molecule or guide RNA complex (e.g., a single guide RNA or crRNA:tracrRNA complex) to direct the CRISPR nuclease protein to a desired target DNA sequence based on complementarity between a portion of the guide RNA molecule or guide RNA complex and the target DNA sequence. The term "wild-type CRISPR endonuclease system" refers to a CRISPR endonuclease system that includes a wild-type CRISPR protein and an appropriate guide RNA molecule or guide RNA complex (e.g., a single guide RNA or crRNA:tracrRNA complex) to direct the wild-type CRISPR nuclease protein to a desired target DNA sequence based on complementarity between a portion of the guide RNA molecule or guide RNA complex and the target DNA sequence.

[0146] In this invention, "maintains on-target editing activity" refers to the ability of the OMNI-79 variant to target a DNA target site targeted by a guide RNA molecule associated with and thereby programmed to the OMNI-79 variant. In some embodiments, the OMNI-79 variant maintains on-target editing activity on a DNA target at or above the editing rate of wild-type OMNI-79 nuclease. In some embodiments, the OMNI-79 variant maintains on-target editing activity on a DNA target at at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40% or 30% of the editing rate of wild-type OMNI-79 nuclease.

[0147] It is intended that the embodiments described above are applicable to each other, for example, it is understood that an RNA molecule or composition of the invention may be utilized in a method of the invention.

[0148] In this specification, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The content of each section is equally applicable to all sections.

[0149] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various aspects and aspects of the present invention as described above and as claimed in the appended claims is experimentally supported in the following examples.

[0150] It will be understood that features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately, in any suitable subcombination, or in any other embodiment of the invention, as appropriate. Certain features described in the context of various embodiments should not be construed as essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0151] Generally, the nomenclature used herein and the laboratory procedures utilized in this invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully described in the literature. See, e.g., Sambrook et al., "Molecular Cloning: A laboratory Manual" (1989); Ausubel, RM (Ed.), "Current Protocols in Molecular Biology" Volumes I-III (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (Eds.), "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. (1998); the methods set forth in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; Cellis, JE (Ed.), "Cell Biology: A Laboratory Handbook", Volumes I-III (1994); Freshney, "Culture of Animal Cells - A Manual of Basic Technique" Third Edition, Wiley-Liss, NY (1994); Coligan JE (Ed.), "Current Protocols in Immunology" Volumes I-III (1994); Stites et al. (Eds.), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994);Mishell and Shiigi (Eds.), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996);Clokie and Kropinski (Eds.), "Bacteriophage Methods and Protocols", Volume 1: Isolation, Characterization, and Interactions (2009), all of which are incorporated by reference. Other general references are provided throughout this specification.

[0152] In order to facilitate a more complete understanding of the present invention, the following examples are provided to illustrate representative modes of making and practicing the present invention. However, the scope of the invention is not limited to the specific embodiments disclosed in these examples, which are intended for illustrative purposes only. EXAMPLES

[0153] Example 1 OMNI-79 CRISPR nuclease variant library The wild-type OMNI-79 CRIPSR nuclease open reading frame was codon-optimized for human cell line expression (SEQ ID NO: 1) and cloned into a dual expression plasmid (pShuttle) allowing both bacterial and mammalian expression using the T7 or CMV promoters, respectively. A complete gene library containing combinatorial random mutations along the full-length OMNI-79 open reading frame (ORF) was constructed using integrated oligos with NNK degenerate codons at each position in the OMNI-79 sequence, resulting in a library with an average of 2.6 amino acid substitutions per ORF.

[0154] Bacteria-based positive selection system To isolate OMNI-79 variants with enhanced activity, a positive selection bacterial system was designed. In this system, the positive selection plasmid was electroporated into E. coli BW25141 strain (1DE3) to generate a positive selection bacterial strain. The positive selection plasmid contains a T7-expressing single guide RNA (sgRNA) and an embedded on-target site. The sequence of the target site, which is a sequence within exon 5 of the human Serpina gene, as well as the spacer and scaffold sequences of the guide RNA molecule, are shown in Table 1. The positive selection plasmid also contains a chloramphenicol resistance cassette and expresses the E. coli toxin gene CcdB under the control of the araBAD promoter. Thus, when the OMNI-79 complete gene library containing the OMNI-79 variants is electroporated into the positive selection bacterial strain, only bacterial colonies expressing active OMNI-79 variants that cleave the positive selection plasmid and neutralize the toxin can survive on selection plates containing arabinose.

[0155] After a 10 minute recovery time in TB medium after electroporation, the transformed bacteria are plated onto selective TB plates containing carbenicillin and 15 mM arabinose and incubated overnight at 37° C. The following morning, the surviving pool is harvested, plasmids are isolated and retransformed into the positively selected bacterial strain for the next round of selection. Seven rounds of positive selection were performed and after the final round single bacterial colonies were randomly picked and fully sequenced.

[0156] [Table 1]

[0157] To test the activity of OMNI-79 nuclease variants after bacterial selection, positively selected clones were isolated and their pShuttle plasmids were individually transfected into HeLa cells together with sgRNAs targeting the endogenous Serpina gene (Table 2). After 72 h, cells were harvested and lysed, and their genomic DNA content was used in a PCR reaction to amplify the putative genomic target site sequences. Next-generation sequencing (NGS) of the amplicons was then performed, and the resulting sequences were used to calculate the percentage of editing at the target site. Short insertions or deletions (indels) around the cleavage site are a typical outcome of repair of DNA ends after nuclease-induced DNA cleavage. Therefore, the calculation of the editing rate was estimated from the percentage of indel-containing sequences within each amplicon.

[0158] Selected OMNI-79 nuclease variants were further analyzed in Neuro-2a mouse neuroblastoma (Mn2A) cells with additional guide RNA molecules targeting the mouse SARM1 target site (see Table 2).

[0159] [Table 2]

[0160] result Two OMNI-79 CRISPR nuclease variants were isolated that were highly enriched after bacterial selection, and both showed increased activity compared to wild-type OMNI-79 CRISPR nuclease in HeLa cells, as shown in Table 3. OMNI-79 variant 5570 also showed high activity when tested in Neuro-2a mouse neuroblastoma (mN2A) cells using the mSARM1 target (see Table 3).

[0161] [Table 3]

[0162] Example 2 result V5570 and V5603 are highly active variants of CRISPR OMNI-79 nuclease (see Table 3). The editing activity of variant V5570 was tested at several target hLDLR gene sites by DNA transfection into HeLa cells (Table 6). In all cases, OMNI-79 variant V5570 showed higher editing activity compared to wild-type OMNI-79 (Figure 1, Table 8).

[0163] OMNI-79 variants V5570 and V5603 each contain two mutations. To investigate the effect and contribution of each mutation to activity, single mutation variants were generated and their activities were tested. Specifically, the activities of four single mutation variants were tested by DNA transfection into HeLa cells for three target genomic sites, hSERP_g12R, hLDLR_g46, and hLDLR_g76 (Table 6). Only variants containing either the S1005R or S1005K mutation showed higher editing activity compared to wild-type OMNI-79 (Figures 2A-2C, Table 8). These results demonstrate that the mutation at position 1005 is responsible for the relatively higher activity observed in variants V5570 and V5603.

[0164] Once it was demonstrated that S1005R and S1005K were responsible for the observed activity of OMNI-79 V5570 and V5603, the effect of other substitutions at position 1005 was tested. Ten further variants were generated, each containing a different amino acid substitution representing a different physicochemical group (Table 4). The effect of each single mutation on the editing activity of the variants was tested as previously described for the hLDLR_g76 target (Figure 3, Table 6). The results showed that positively charged amino acids such as arginine (R) and lysine (K) at position 1005 all supported high editing activity (Figure 3, Table 8). Furthermore, polar amino acids such as threonine (T), asparagine (N), and glutamine (Q) at position 1005 also supported high editing activity (Figure 3, Table 8).

[0165] Materials and Methods DNA transfection into HeLa cells Nuclease activity of endogenous contents in mammalian cells: OMNI-79 nuclease and its variant nucleases were expressed in mammalian cell line (HeLa) by DNA transfection together with sgRNA expression plasmids. Each sgRNA is composed of a tracrRNA portion and a spacer portion. The spacer 3' genomic sequence contains the predicted PAM for OMNI-79 nuclease. All assays were repeated three times. "OMNI nuclease alone" (i.e., no guide) transfected cells served as negative control. Cell lysates were used for site-specific genomic DNA amplification and NGS analysis.

[0166] NGS analysis Cells were harvested 72 hours after transfection and their genomic DNA content was used in PCR reactions to amplify the corresponding putative genomic targets. The amplicons were subjected to NGS and the resulting sequences were subsequently used to calculate the percentage of editing at each target site. Short insertions or deletions (indels) around the cleavage site are a typical result of DNA repair after nuclease-induced DNA cleavage. Therefore, the calculation of the editing rate was estimated from the percentage of indel-containing sequences within each amplicon.

[0167] Example 3 result To test the potential for genome editing with OMNI-79 V5570, several different delivery methods were used. OMNI-79 V5570 was tested in HepG2 cells as part of a ribonucleoprotein (RNP) complex, targeting multiple sites in the LDLR gene. In all cases, editing calculated from NGS was high, ranging from 58.5% up to 84.3% (Figure 4, Table 6). OMNI-79 V5570 was also tested in transfection and electroporation experiments by delivering mRNA molecules encoding variant nucleases. Again, in all cases, high levels of editing were observed in three different genes (Figures 5 and 7, Table 7). Finally, OMNI-79 V5570 was delivered by viral transduction (Figure 6, Table 8).

[0168] Materials and Methods Purification of OMNI-79 protein The expression method for protein production and synthetic guide production to assemble the RNP is as follows: Briefly, the OMNI-79 nuclease open reading frame was codon-optimized for bacteria (Table 1) and cloned into the pNNC plasmid containing the following elements: SV40 NLS-OMNI-79 ORF bacterial optimized-HA tag-SV40 NLS-8His tag (Table 4). The OMNI-79 construct was expressed in KRX cells (PROMEGA). Cells were grown in TB, 0.4% glycerol supplemented with 6.66 mM rhamnose, 0.05% glucose and carb antibiotic. Induction was performed in mid-log phase 4 hours after lowering the temperature to 18°C. Cells were lysed by chemical lysis and the clarified lysate was purified on Ni-NTA resin. Then, purification on CEX (SO3 fractogel) resin followed by SEC purification on Superdex® 200 Increase 16 / 600, AKTA Pure (GE Healthcare Life Sciences) was performed. Fractions containing OMNI-79 protein were pooled in high salt, concentrated to a 22 mg / mL stock, flash frozen in liquid nitrogen, and stored at -80°C.

[0169] In vitro transcription (IVT) and transfection OMNI-79 V5570-encoding mRNA was generated using the HiScribe T7 High Yield RNA Synthesis Kit (NEB# E2040S) with N1-methylpseudouridine-5'-triphosphate and CleanCap according to the manufacturer's instructions. The yield was approximately 150 μg mRNA. HeLa cells were seeded to be 70-90% confluent at the time of transfection. IVT mRNA was transfected with synthetic gRNA using Lipofectamine® 3000 according to the manufacturer's instructions. 72 hours after transfection, cells were dispersed using trypsin and genomic extracts were prepared using QuickExtract solution according to the manufacturer's instructions. Indel rates were analyzed by NGS.

[0170] RNP and mRNA electroporation To deliver V5570 in the form of RNP, 105 pmol of purified protein was mixed with 124 pmol of sgRNA and 100 μM Cas9 electroporation enhancer (IDT) to assemble the complex. After incubation at 25 °C for 10 min, the RNP complex was diluted to 4 × 10 5 The 4D-Nucleofector®X kit was mixed with 100000 pre-washed HepG2 cells and electroporated using the Lonza SF Cell Line 4D-Nucleofector®X kit with the DS-123 program for HepG2 cells according to the manufacturer's instructions. The cells were replated in 12-well tissue culture plates and incubated in a TC incubator (37°C, 5% CO2). 72 hours after electroporation, the cells were harvested. Cell lysis and genomic DNA extraction were performed using Quick extract (Lucigen), and endogenous genomic regions were amplified using specific primers to measure on-target activity by NGS (Figure 4, Table 3).

[0171] To deliver OMNI-79 V5570 in mRNA form, a solution of 1 μg of purified V5570 mRNA (Trilink) was mixed with 124 pmol of sgRNA and 100 μM of Cas9 electroporation enhancer (IDT). 5 The cells were mixed with 10 ...

[0172] Viral transduction To deliver OMNI-79 V5570 in an AAV format, AAV-DJ virus carrying OMNI-79 V5570 and the corresponding sgRNA molecule was prepared and purified (VectorBuilder). HepG2 and Hepa1-6 cells were transfected with OMNI-79 V5570 at an MOI of 1 × 10 5 and 3 x 10 5 The cells were infected with AAV-DJ carrying the corresponding sgRNA at 100°C. The cells with added viral particles were incubated overnight in a TC incubator (37°C, 5% CO2). The next day, the cells were washed and incubated with fresh medium for another 48 h. The cells were harvested 72 h postinfection. Cell lysis and genomic DNA extraction were performed using Quick extract (Lucigen), and endogenous genomic regions were amplified with specific primers to measure on-target activity by NGS (Figure 6, Table 3).

[0173] [Table 4]

[0174] [Table 5]

[0175]

Table 6

[0176]

Table 7

[0177]

Table 8-1

[0178]

Table 8-2

[0179]

Table 9

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Claims

1. A non-naturally occurring OMNI-79 nuclease variant having a wild-type OMNI-79 protein sequence (SEQ ID NO: 1) that includes an amino acid substitution in at least one of S1005, I14, and E1050.

2. The OMNI-79 nuclease variant of claim 1, wherein the amino acid substitution at S1005 and / or E1050 is a substitution with an amino acid having a positively charged R group.

3. The OMNI-79 nuclease variant of claim 2, wherein the amino acid having a positively charged R group is lysine or arginine.

4. The OMNI-79 nuclease variant of claim 1, wherein the amino acid substitution is any one of S1005R, S1005K, I14L, and E1050K.

5. containing an amino acid substitution at each of I14 and S1005; Optionally, the amino acid substitutions are I14L and S1005R. An OMNI-79 nuclease variant according to any one of claims 1 to 4.

6. The OMNI-79 nuclease variant of claim 1 or 2, comprising an amino acid substitution at each of S1005 and E1050.

7. The OMNI-79 nuclease variant of claim 1 or 2, wherein the amino acid substitutions are S1005K and E1050K. (i) having any one of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NOs: 12 to 25, (ii) the position of the amino acid substitution is I14 and is any one of I14L, I14V, I14F, I14C, I14A, or I14T; (iii) the amino acid substitution is I14L; (iv) the position of the amino acid substitution is I14 and the amino acid has an aromatic or hydrophobic R group; (v) the position of the amino acid substitution is S1005 and is any one of S1005R, S1005K, S1005Q, S1005I, S1005M, S1005V, S1005T, S1005N, S1005F, S1005A, S1005G, or S1005E; (vi) the amino acid substitution at S1005 is a substitution with an amino acid having a positively charged R group; (vii) the amino acid substitution at S1005 is a substitution with an amino acid having a polar R group; (viii) the amino acid substitution is S1005R; (ix) the amino acid substitution is S1005K; (x) the amino acid substitution is S1005T; (xi) the amino acid substitution is S1005N; (xii) the amino acid substitution is S1005Q; (xiii) the position of the amino acid substitution is E1050 and is any one of E1050K, E1050R, E1050P, E1050A, E1050I, E1050L, E1050V, E1050G, or E1050T; (xiv) the amino acid substitution is E1050K, or (xv) the amino acid substitution at E1050 is a substitution with an amino acid having a positively charged R group; The OMNI-79 nuclease variant of claim 1.

9. has at least 80% sequence identity with the wild-type OMNI-79 protein sequence (SEQ ID NO: 1); and / or and / or further comprising a nuclear localization sequence (NLS); the variant exhibits increased activity at the DNA target site when complexed with a guide RNA molecule compared to wild-type OMNI-79 nuclease complexed with a guide RNA molecule that targets the variant to the DNA target site. An OMNI-79 nuclease variant according to any one of claims 1 to 4 or 8.

10. 10. A CRISPR system comprising the OMNI-79 nuclease variant of any one of claims 1 to 4 or 8 complexed with a guide RNA molecule that targets a DNA target site, wherein the CRISPR system has increased on-target editing activity compared to a wild-type CRISPR system comprising a wild-type OMNI-79 nuclease protein and the guide RNA molecule.

11. 10. An in vitro or ex vivo method of gene editing with increased on-target editing activity, comprising contacting a DNA target site with an active CRISPR system comprising an OMNI-79 nuclease variant protein of any one of claims 1 to 4 or 8.

12. An in vitro or ex vivo gene editing method with increased on-target editing activity, comprising contacting a DNA target site with an active CRISPR system comprising an OMNI-79 nuclease variant protein according to any one of claims 1 to 4 or 8, wherein the gene editing occurs in a eukaryotic or prokaryotic cell; Optionally, the eukaryotic cell is a plant cell or a mammalian cell. Optionally, the mammalian cell is a human cell.

13. An in vitro or ex vivo gene editing method with increased on-target editing activity, comprising contacting a DNA target site with an active CRISPR system comprising an OMNI-79 nuclease variant protein described in any one of claims 1 to 4 or 8, wherein the DNA target site is within or near a pathogenic allele of a gene.

14. An in vitro or ex vivo gene editing method with increased on-target editing activity, comprising contacting a DNA target site with an active CRISPR system comprising an OMNI-79 nuclease variant protein described in any one of claims 1 to 4 or 8, wherein the DNA target is repaired with an exogenous donor molecule.

15. An in vitro or ex vivo gene editing method having increased on-target editing activity, comprising contacting a DNA target site with an active CRISPR system comprising the OMNI-79 nuclease variant protein of any one of claims 1 to 4 or 8, wherein the on-target editing activity is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 10 ... ２ double, 10 ３ double, 10 ４ double, 10 ５ Double or 10 ６ Doubled increase, way.

16. A modified cell obtained by an in vitro or ex vivo method of contacting a DNA target site with an active CRISPR system comprising an OMNI-79 nuclease variant protein described in any one of claims 1 to 4 or 8, wherein the method increases on-target editing activity.

17. Modified cells obtained by an in vitro or ex vivo method of contacting a DNA target site with an active CRISPR system comprising an OMNI-79 nuclease variant protein according to any one of claims 1 to 4 or 8, wherein the method increases on-target editing activity and the cells are capable of engraftment; and / or the cells are capable of giving rise to progeny cells after engraftment; and / or the cells are capable of giving rise to progeny cells after autologous transplantation; and / or the cells are capable of giving rise to progeny cells for at least 12 months or at least 24 months after engraftment; and / or the cells are selected from the group consisting of hematopoietic stem cells, progenitor cells, CD34+ hematopoietic stem cells, bone marrow cells, and peripheral mononuclear cells; Modified cells.

18. A composition comprising a modified cell obtained by an in vitro or ex vivo method of contacting a DNA target site with an active CRISPR system comprising an OMNI-79 nuclease variant protein described in any one of claims 1 to 4 or 8, said method increasing on-target editing activity, and a pharmaceutically acceptable carrier.

19. An in vitro or ex vivo method for producing a composition, comprising: a modified cell obtained by an in vitro or ex vivo method of contacting a DNA target site with an active CRISPR system comprising an OMNI-79 nuclease variant protein described in any one of claims 1 to 4 or 8, said method increasing on-target editing activity; and mixing the modified cell with a pharmaceutically acceptable carrier.

20. A polynucleotide molecule encoding the OMNI-79 nuclease variant of any one of claims 1 to 4 or 8.

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