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  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• This application incorporates-by-reference nucleotide sequences which are present in the file named “221130_91808-A-PCT_Sequence_Listing_AWG.xml”, which is 133 kilobytes in size, and which was created on November 14, 2022 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the XML file filed November 30, 2022 as part of this application.
• Targeted genome modification is a powerful tool that can be used to reverse the effect of pathogenic genetic variations and therefore has the potential to provide new therapies for human genetic diseases.
• Current genome engineering tools including engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most recently, RNA-guided DNA endonucleases such as CRISPR/Cas, produce sequence-specific DNA breaks in a genome.
• ZFNs zinc finger nucleases
• TALENs transcription activator-like effector nucleases
• CRISPR/Cas RNA-guided DNA endonucleases
• the modification of the genomic sequence occurs at the next step and is the product of the activity of a cellular DNA repair mechanism triggered in response to the newly formed DNA break.
• NHEJ non-homologous end-joining
• HDR homology-directed repair
• CRISPRs Clustered Regularly Interspaced Short Palindromic Repeats
• CRISPR-associated OMNI-79 nucleases with improved activity and their use in genomic engineering, epigenomic engineering, genome targeting, genome editing, and in vitro diagnostics.
• a variant of an OMNI-79 nuclease with increased activity as compared to the wild-type OMNI-79 nuclease as well as methods of using the improved variants.
• the engineered variant OMNI-79 nucleases are active in a CRISPR endonuclease system, and the CRISPR endonuclease system displays increased on-target editing activity relative to a wild-type CRISPR endonuclease system in which a wild-type OMNI-79 nuclease is active.
• an engineered variant OMNI-79 nuclease may display improved nuclease activity at a target region which contains a heterozygous SNP present in only the targeted allele and not present in the non-targeted allele.
• OMNI-79 nuclease protein comprising a sequence that is at least 80% identical to the amino acid sequence of wild-type OMNI-79 nuclease protein (SEQ ID NO: 1).
• a non-natural OMNI-79 nuclease variant having a wild-type OMNI-79 protein sequence (SEQ ID NO: 1) comprising an amino acid substitution in at least one of the following positions: 114, S1005, and E1050.
• a CRISPR system comprising any one of the OMNI-79 nuclease variants disclosed herein complexed with a guide RNA molecule that targets a DNA target site, wherein the CRISPR system displays increased on-target editing activity relative to a wild-type CRISPR system comprising a wild-type OMNI-79 nuclease protein and the guide RNA molecule.
• the OMNI-79 variant nuclease exhibits increased activity at a target site when complexed with a guide RNA targeting the OMNI-79 variant to the target site compared to a wild-type OMNI-79 nuclease (SEQ ID NO: 1).
• a method for gene editing having increased on-target editing activity comprising contacting a DNA target site with an active CRISPR system comprising any one of the OMNI-79 nuclease variant proteins described herein.
• a method for gene editing increased on-target editing activity comprising: contacting a target site with an active CRISPR system comprising a variant OMNI-79 nuclease protein of any one of the variants described herein, wherein the active CRISPR system displays increased on-target editing activity relative to a wild-type CRISPR system having a wild-type OMNI-79 nuclease protein.
• Fig. 1 Wild-type OMNI-79 nuclease and OMNI-79 Variant 5570 editing activity on various hLDLR gene targets. Editing activity was determined by nextgeneration sequencing (NGS) analysis. The average and standard deviation of three replicates is displayed.
• NGS nextgeneration sequencing
• Figs. 2A-2C Editing activity of OMNI-79 single mutants. Single point mutation variants were tested via transfection in HeLa cells targeting three genomic sites: (Fig. 2A) hSERP_gl2R, (Fig. 2B) hLDLR_g46 and (Fig. 2C) hLDLR_g76. Editing activity was determined by NGS analysis. The average and standard deviation of three replicates is displayed.
• Fig. 3 The effect of other substitutions at position 1005 on editing activity.
• the editing activity was tested via transfection in HeLa cells targeting hLDLR_g76. Editing activity was determined by NGS analysis. The average and standard deviation of three replicates is displayed.
• Fig. 4 NGS analysis of editing by OMNI-79 V5570 RNP complexes.
• HepG2 human hepatic carcinoma
• sgRNA 124 pmol complexes
• 105 pmol a purified OMNI-79 V5570 protein
• sgRNA 124 pmol complexes
• cells were plated in 12-well tissue culture plates and kept in a 37°C, 5% CO2 incubator. 72 hours after electroporation, cells were dissociated with trypsin and genomic extract was prepared with QuickExtract solution according to manufacturer’s instructions.
• InDeis percentage analysis was performed by NGS.
• Fig. 5 NGS analysis of editing by OMNI-79 V5570 mRNA in HepG2 cells.
• HepG2 cells were electroporated with 1 pg of OMNI-79 V5570-encoding mRNA (Trilink), and 124 pmol sgRNA (Agilent) using a Lonza Nucleofector X unit according to manufacturer’s instructions. Following recovery, cells were plated in 12-well tissue culture plates and kept in 37°C, 5% CO2 incubator. 72 hours after electroporation, cells were dissociated with trypsin and genomic extract was prepared with QuickExtract solution according to manufacturer’s instructions. InDeis percentage analysis was performed by NGS. [0019] Fig.
• NGS analysis of editing by OMNI-79 V5570 and gRNA expressed by AAV infection Hepal-6 (mouse hepatoma) cells and HepG2 cells were infected with Adeno-associated viruses - DJ serotype (AAV-DJ) harboring OMNI-79 V5570 and corresponding sgRNA sequences at a multiplicity of infection (MOI) of IxlO 5 - 3xl0 5 .
• MOI multiplicity of infection
• Cells were plated in 12-well tissue culture plates and kept in a 37°C, 5% CO2 incubator for 16 hours, then washed from remaining viruses and incubated for another 48 hours with fresh medium. Cells were dissociated with trypsin and genomic extract was prepared with QuickExtract solution according to manufacturer’s instructions. InDeis percentage analysis was performed by NGS.
• Fig. 7 NGS analysis of editing by OMNI-79 V5570 mRNA in HeLa cells. The editing activity was tested via transfection of OMNI-79 V5570-encoding mRNA (in-house IVT) in HeLa cells targeting hSERP_gl2 and CXCR4_s25 sites. Editing activity was determined by NGS analysis. The average and standard deviation of three replicates is displayed.
• the present disclosure provides an engineered OMNI-79 nuclease exhibiting increased activity at a target site compared to the wild-type OMNI-79 nuclease (SEQ ID NO: 1).
• the wildtype OMNI-79 nuclease is disclosed in PCT International Application No. PCT/US2021/035928, incorporated herein by reference.
• the engineered OMNI-79 nuclease variant is active in a CRISPR endonuclease system
• the CRISPR endonuclease system displays increased on-target editing activity relative to a CRISPR endonuclease system comprising the wild-type OMNI-79 nuclease.
• the engineered OMNI-79 nuclease is an OMNI-79 nuclease variant comprising at least one amino acid substitution relative to the wild-type OMNI-79 nuclease. In some embodiments, the engineered OMNI-79 nuclease comprises multiple amino acid substitutions compared to wild-type OMNI-79 nuclease.
• OMNI-79 nuclease variant is 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.
• an OMNI-79 nuclease variant may have amino acid sequence differences at up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 20% of its residues relative to SEQ ID NO: 1. Such sequence differences may be revealed by a sequence alignment.
• An OMNI-79 variant nuclease may be generated by replacing 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 inserting or deleting an amino acid residue of the OMNI-79 wild-type nuclease. Any such mutations, including but not limited to substitutions, insertions, or deletions, in addition to any other mutations described herein, or with mutations in addition to the mutations described herein, may be used to generate an OMNI-79 variant nuclease from an OMNI-79 wild-type nuclease.
• the OMNI-79 variant nuclease retains a desired activity of the parent wild-type OMNI-79 nuclease, e.g., the ability to interact with a guide RNA and target DNA and/or the activity of the nuclease (e.g. ability to cause a double-strand DNA break, a single-strand DNA break, or lack of any nuclease or nickase activity).
• the variant retains the desired activity of the parent, e.g. nuclease activity, at a level greater than or equal to the level of activity of the parent.
• the variant retains the desired activity of the parent at a level of at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% the level of activity of the parent.
• the OMNI-79 variant nuclease displays increased on-target effects relative to OMNI-79 wild-type nuclease.
• the OMNI- 79 nuclease variant is a nickase having an inactivated RuvC or HNH domain and further comprises an amino acid substitution in at least one of the following positions: S1005, 114 and E1050. In some embodiments, the OMNI-79 nuclease variant is a dead nuclease having an inactivated RuvC and HNH domains and further comprises an amino acid substitution in at least one of the following positions: S1005, 114 and E1050.
• a variant of OMNI-79 nuclease protein comprising a sequence that is at least 80% identical to the amino acid sequence of wild-type OMNI-79 (SEQ ID NO: 1) and having at least one amino acid substitution.
• the amino acid substitution comprises an amino acid residue replacement to a positive, negative, uncharged, hydrophilic, hydrophobic, polar, or non-polar amino acid.
• the amino acid substitution is selected from replacement of an amino acid to any one of a different 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.
• a variant OMNI-79 nuclease protein contains an amino acid substitution in at least one of the following positions in the wild-type OMNI-79 protein sequence (SEQ ID NO: 1): 114, S1005, and E1050.
• SEQ ID NO: 1 wild-type OMNI-79 protein sequence
• SEQ ID NO: 1 wild-type OMNI-79 protein sequence
• the variant OMNI-79 nuclease protein comprises at least one of the following amino acid substitutions in the following positions in the wild-type OMNI-79 protein sequence: H4L, S1005R, S1005K, and E1050K. Each possibility represents a separate embodiment of the present disclosure. In some embodiments, the substitution corresponds to those listed in Table 3.
• the variant OMNI-79 nuclease protein comprises at least one amino acid substitution in the following positions in the wild-type OMNI-79 protein sequence: 114 and SI 005. In some embodiments, the variant OMNI-79 nuclease protein comprises amino acid substitutions in the following positions in the wild-type OMNI-79 protein sequence: 114 and SI 005. In some embodiments, the variant OMNI-79 nuclease protein comprises the following amino acid substitutions from the wild-type OMNI-79 protein sequence: I14L and S1005R.
• the variant OMNI-79 nuclease protein comprises at least one amino acid substitution in the following positions in the wild-type OMNI-79 protein sequence: SI 005 and El 050. In some embodiments, the variant OMNI-79 nuclease protein comprises amino acid substitutions in the following positions in the wild-type OMNI-79 protein sequence: SI 005 and El 050. In some embodiments, the variant OMNI-79 nuclease protein comprises the following amino acid substitutions from the wild-type OMNI-79 protein sequence: S1005K and E1050K.
• the OMNI-79 variant nuclease further comprises one or more of a nuclear localization sequence (NLS), cell penetrating peptide sequence, and/or affinity tag.
• the OMNI-79 variant nuclease comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of a CRISPR complex comprising the CRISPR nuclease in a detectable amount in the nucleus of a eukaryotic cell.
• the OMNI-79 variant nuclease comprises amino acid substitutions selected from amino acid substitutions corresponding to the substitutions displayed relative to wild-type OMNI-79 in Table 3.
• an isolated OMNI-79 variant nuclease protein comprising one or more substitutions or mutations relative to the wild-type OMNI-79 nuclease sequence, wherein the isolated variant OMNI-79 variant nuclease is active in a CRISPR system, wherein the CRISPR system displays increased on-target editing activity relative to a wild-type CRISPR system.
• additional mutations to the OMNI-79 variant nuclease described herein may be implemented. Examples include, but are not limited to, mutations which alter the PAM recognition sequence, alter the nuclease activity of the enzyme, and truncations or removal of portions of the nuclease.
• the variant OMNI-79 variant nuclease may be encoded by any nucleic acid sequence which produces the desired amino acid sequence of the variant.
• the nucleic acid sequence may be codon-optimized for a cell, such as a bacterial cell, plant cell, or mammalian cell.
• a CRISPR nuclease and a targeting molecule form a CRISPR complex that binds to a target DNA sequence to effect cleavage of the target DNA sequence.
• a CRISPR nuclease may form a CRISPR complex comprising the CRISPR nuclease and a single-guide RNA (sgRNA) molecule.
• sgRNA single-guide RNA
• a CRISPR nucleases may form a CRISPR complex comprising the CRISPR nuclease, a crRNA molecule, and a tracrRNA molecule.
• a method of gene editing having increased on-target editing activity comprising: contacting a target site with an active CRISPR endonuclease system having a variant OMNI-79 protein complexed with a suitable guide RNA or guide RNA complex, wherein the active CRISPR endonuclease system displays increased on-target editing activity relative to a wild-type OMNI-79 CRISPR system.
• a non-naturally occurring OMNI-79 nuclease variant having a wild-type OMNI-79 protein sequence (SEQ ID NO: 1) comprising an amino acid substitution in at least one of the following positions: 114, S1005, and E1050.
• the amino acid substitution at SI 005 and/or El 050 are to an amino acid having a positively charged R-group.
• the amino acid having a positively charged R-group is lysine or arginine.
• the amino acid substitution is any one of the following substitutions: I14L, S1005R, S1005K, and E1050K.
• the OMNI-79 nuclease variant comprises an amino acid substitution at each of positions 114 and S1005.
• amino acid substitutions are I14L and S1005R.
• the OMNI-79 nuclease variant comprises an amino acid substitution at each of positions S1005 and E1050.
• amino acid substitutions are S1005K and E1050K.
• the OMNI-79 nuclease variant has an amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NOs: 12-25.
• the amino acid substitution is at 114 and is any one of the following substitutions: I14L, I14V, I14F, I14C, I14A, or I14T.
• the amino acid substitution is I14L.
• the amino acid substitution is at 114 and the amino acid has an aromatic or hydrophobic R-group.
• the amino acid substitution is at SI 005 and is any one of the following substitutions: S1005R, S1005K, S1005Q, SI 0051, S1005M, SI 005V, S1005T, S1005N, S1005F, S1005A, S1005G, or S1005E.
• the amino acid substitution at SI 005 is to an amino acid having a positively charged R-group.
• the amino acid substitution at SI 005 is to an amino acid having a polar R-group.
• the amino acid substitution is S1005R.
• the amino acid substitution is S1005K.
• amino acid substitution is S1005T
• the amino acid substitution is S1005N.
• the amino acid substitution is S1005Q.
• the amino acid substitution is at E1050 and is any one of the following substitutions: E1050K, E1050R, E1050P, E1050A, E1050I, E1050L, E1050V, E1050G, or E1050T.
• the amino acid substitution is E1050K.
• the amino acid substitution at E1050 is to an amino acid having a positively charged R-group.
• the OMNI-79 nuclease variant has at least 80% sequence identity to the wild-type OMNI-79 protein sequence (SEQ ID NO: 1).
• the OMNI-79 nuclease variant may have at least 80% sequence identity to the wild-type OMNI- 79 protein sequence (SEQ ID NO: 1) and any one of the amino acid substitutions provided herein.
• the OMNI-79 nuclease variant may comprise any one of the amino acid substitutions provided herein relative to 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).
• the OMNI-79 nuclease variant further comprises a nuclear localization sequence (NLS).
• NLS nuclear localization sequence
• the OMNI-79 nuclease variant exhibits increased activity toward a DNA target site when complexed with a guide RNA molecule that targets the variant to the said DNA target site relative to a wild-type OMNI-79 nuclease complexed with the guide RNA molecule.
• a CRISPR system comprising any one of the OMNI-79 nuclease variants described herein complexed with a guide RNA molecule that targets a DNA target site, wherein the CRISPR system displays increased on-target editing activity relative to a wild-type CRISPR system comprising a wild-type OMNI-79 nuclease protein and the guide RNA molecule.
• a method for gene editing having increased on-target editing activity comprising contacting a DNA target site with an active CRISPR system comprising any one of the OMNI-79 nuclease variant proteins described herein.
• the gene editing occurs in a eukaryotic cell or prokaryotic cell.
• the eukaryotic cell is a plant cell or mammalian cell.
• the mammalian cell is a human cell.
• the DNA target site is located within or in proximity to a pathogenic allele of a gene.
• the DNA target is repaired with an exogenous donor molecule.
• the on-target editing activity is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 10-fold, 10 2 -fold, 10 3 -fold, 10 4 -fold, 10 5 -fold, or 10 6 -fold.
• a modified cell obtained by the methods described herein.
• the cell is capable of engraftment.
• the cell is capable of giving rise to progeny cells after engraftment.
• the cell is capable of giving rise to progeny cells after an autologous engraftment.
• the cell is capable of giving rise to progeny cells for at least 12 months or at least 24 months after engraftment.
• the cell is selected from the group consisting of a hematopoietic stem cell, a progenitor cell, a CD34+ hematopoietic stem cell, a bone marrow cell, and a peripheral mononucleated cell.
• composition comprising any one of the modified cells described herein and a pharmaceutically acceptable carrier.
• an in vitro or ex vivo method of preparing the composition comprising mixing the cells with the pharmaceutically acceptable carrier.
• a polynucleotide molecule encoding any one of the OMNI-79 nuclease variant proteins described herein.
• the OMNI-79 variant compositions described herein may be delivered as a protein, DNA molecules, RNA molecules, Ribonucleoproteins (RNP), nucleic acid vectors, or any combination thereof.
• the RNA molecule comprises a chemical modification.
• suitable chemical modifications include 2’-0-methyl CM), 2’-0-methyl, 3’phosphorothioate (MS) or 2’-0-methyl, 3 ThioPACE (MSP), pseudouridine, and 1-methyl pseudo-uridine.
• the OMNI-79 variants and/or polynucleotides encoding same described herein, and/or additional molecules, such as a single-guide RNA molecule, crRNA molecule, tracrRNA molecules or a nucleotide molecule that encodes any one of them, may be delivered to a target cell by any suitable means.
• the target cell may be any type of cell e.g., eukaryotic or prokaryotic, in any environment e.g., isolated or not, maintained in culture, in vitro, ex vivo, in vivo or in planta.
• a target site in a target cell may be within the nucleus of the cell.
• compositions described herein may be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance.
• compositions may introduced into a cell as naked nucleic acids or proteins, as nucleic acids or proteins complexed with or packaged within an agent such as a liposome, exosome, or poloxamer, or can be delivered by recombinant viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)) or virus-like particles.
• viruses e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)
• the composition 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 capability.
• AAV adeno-associated virus
• a lentivirus such as a non-integrating lentivirus or a lentivirus lacking reverse transcription capability.
• Additional non-limiting examples include packaging the composition into liposomes, extracellular vesicles, or exosomes, which may be pseudotyped with vesicular stomatitis glycoprotein (VSVG) or conjugated to a cell-penetrating peptide, an antibody, a targeting moiety, or any combination thereof.
• VSVG vesicular stomatitis glycoprotein
• the composition to be delivered includes mRNA of the nuclease and RNA of the guide. In some embodiments, the composition to be delivered includes mRNA of the nuclease, RNA of the guide and a donor template. In some embodiments, the composition to be delivered includes the CRISPR nuclease and guide RNA. In some embodiments, the composition to be delivered includes the CRISPR nuclease, guide RNA and a donor template for gene editing via, for example, homology directed repair.
• the lentivirus includes mRNA of the nuclease and a guide RNA molecule, e.g.
• the composition delivered to a cell includes mRNA of the nuclease, a guide RNA molecule and a donor template molecule.
• the lentivirus includes the nuclease protein variant and a guide RNA molecule.
• the composition delivered to a cell includes the nuclease protein variant, a guide RNA molecule and/or donor template for homology directed repair.
• the composition delivered to a cell includes mRNA of the nuclease variant, a DNA-targeting crRNA molecule, and a tracrRNA molecule
• the composition delivered to a cell includes mRNA of the nuclease variant, DNA-targeting crRNA molecule, and a tracrRNA molecule, and a donor template molecule
• the composition delivered to a cell includes the nuclease protein variant, DNA-targeting crRNA molecule, and a tracrRNA molecule.
• the composition delivered to a cell includes the nuclease protein variant, DNA-targeting crRNA molecule, and a tracrRNA molecule, and DNA donor template molecule for homology directed repair.
• Any suitable viral vector system may be used to deliver such compositions.
• Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids and/or OMNI-79 nuclease variant protein in cells (e.g., mammalian cells, plant cells, etc.) and target tissues. Such methods can also be used to administer nucleic acids encoding and/or OMNI-79 nuclease variant protein to cells in vitro.
• nucleic acids and/or an OMNI- 79 nuclease variant protein are administered for in vivo or ex vivo gene therapy uses.
• Non-viral vector delivery systems include naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
• a delivery vehicle such as a liposome or poloxamer.
• Methods of non-viral delivery of nucleic acids and/or proteins include electroporation, lipofection, microinjection, biolistics, particle gun acceleration, virosomes, virus-like particles, exosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, artificial virions, and agent-enhanced uptake of nucleic acids or can be delivered to plant cells by bacteria or viruses (e.g., Agrobacterium, Rhizobium sp. NGR234, Sinorhizoboiummeliloti, Mesorhizobium loti, tobacco mosaic virus, potato virus X, cauliflower mosaic virus and cassava vein mosaic virus.
• bacteria or viruses e.g., Agrobacterium, Rhizobium sp. NGR234, Sinorhizoboiummeliloti, Mesorhizobium loti, tobacco mosaic virus, potato virus X, cauliflower mosaic virus and cassava vein mosaic virus.
• Non-viral vectors such as transposon-based systems e.g.
• recombinant Sleeping Beauty transposon systems or recombinant PiggyBac transposon systems may also be delivered to a target cell and utilized for transposition of a polynucleotide sequence of a molecule of the composition or a polynucleotide sequence encoding a molecule of the composition in the target cell.
• 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 for example U.S. Patent No. 6,008,336).
• Lipofection is described in e.g., U.S. Patent No. 5,049,386, U.S. Patent No. 4,946,787; and U.S. Patent No. 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam.TM., Lipofectin.TM. and Lipofectamine.TM. RNAiMAX).
• Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those disclosed in PCT International Publication Nos. WO/1991/017424 and WO/1991/016024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).
• lipidmucleic acid complexes including targeted liposomes such as immunolipid complexes
• Boese 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); 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).
• Additional methods of delivery include the use of packaging the nucleic acids to be delivered into EnGenelC delivery vehicles (EDVs).
• EDVs EnGenelC delivery vehicles
• These EDVs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV.
• the antibody brings the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis. Once in the cell, the contents are released (see MacDiamid et al (2009) Nature Biotechnology 27(7) p. 643).
• RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
• Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo).
• Conventional viral based systems for the delivery of nucleic acids include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer.
• OMNI-79 variant or a nucleic acid expressing the variant, as well as any associated nucleic acids may be delivered by a non-integrating lentivirus.
• RNA delivery with lentivirus is utilized.
• the lentivirus includes mRNA of the nuclease and a guide RNA molecule, e.g. a single-guide RNA molecule or crRNA molecule, which is used to target the nuclease to a target site.
• the lentivirus includes mRNA of the nuclease, guide RNA molecule and a donor template molecule.
• the lentivirus includes the nuclease protein variant and a guide RNA molecule.
• the lentivirus includes the nuclease protein variant, a guide RNA molecule and/or donor template molecule for homology directed repair.
• the lentivirus includes mRNA of the nuclease variant, a DNA-targeting crRNA molecule, and a tracrRNA molecule.
• the lentivirus includes mRNA of the nuclease variant, DNA-targeting crRNA molecule, and a tracrRNA molecule, and a donor template molecule.
• the lentivirus includes the nuclease protein variant, DNA- targeting crRNA molecule, and a tracrRNA molecule.
• the lentivirus includes the nuclease protein variant, DNA-targeting crRNA molecule, and a tracrRNA molecule, and DNA donor template molecule for homology directed repair.
• compositions described herein may be delivered to a target cell using a non-integrating lentiviral particle method, e.g. a LentiFlash® system.
• a non-integrating lentiviral particle method e.g. a LentiFlash® system.
• Such a method may be used to deliver mRNA or other types of RNAs into the target cell, such that delivery of the RNAs to the target cell results in assembly of the compositions described herein inside of the target cell.
• a non-integrating lentiviral particle method e.g. a LentiFlash® system.
• Such a method may be used to deliver mRNA or other types of RNAs into the target cell, such that delivery of the RNAs to the target cell results in assembly of the compositions described herein inside of the target cell.
• Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system depends on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
• Widely used retroviral vectors include those based upon 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); PCT International Publication No. WO/1994/026877A1).
• At least six viral vector approaches are currently available for gene transfer in clinical trials, which utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent.
• 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 a gene therapy trial. (Blaese et al., Science (1995)). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al., Immunol Immunother. (1997); Dranoff et al., Hum. Gene Ther. (1997).
• Packaging cells are used to form virus particles that are capable of infecting a host cell.
• Such cells include 293 cells, which package adenovirus, AAV, and .psi.2 cells or PA317 cells, which package retrovirus.
• Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle.
• the vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed.
• the missing viral functions are supplied in trans by the packaging cell line.
• AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome.
• ITR inverted terminal repeat
• Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
• the cell line is also infected with adenovirus as a helper.
• the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
• the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additionally, AAV can be produced at clinical scale using baculovirus systems (see U.S. Patent No. 7,479,554.
• a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus.
• the ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al., Proc. Natl. Acad. Sci.
• 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 human epidermal growth factor receptor.
• This principle can be extended to other virus-target cell pairs, in which the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand for the cell-surface receptor.
• filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor.
• Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
• vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
• Ex vivo cell transfection for diagnostics, research, or for gene therapy is well known to those of skill in the art.
• cells are isolated from the subject organism, transfected with an RNA composition, and re-infused back into the subject organism (e.g., patient).
• RNA composition e.g., RNA-derived RNA-derived RNA-derived RNA-derived RNA-derived RNA-derived RNA-derived RNA-derived RNA-derived RNA composition
• RNA composition e.g., RNA composition
• RNA composition e.g., RNA composition
• re-infused back into the subject organism e.g., patient
• Various cell types 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 (3 rd ed. 1994)) and the references cited therein for a discussion of how to isolate and culture cells from patients).
• Suitable cells include but 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-Agl4, 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 Spodopterafugiperda (Sf), or fungal cells such as Saccharomyces, Pichia and Schizosaccharomyces.
• COS CHO
• CHO e.g., CHO— S, CHO-K1,
• the cell line is a CHO- Kl, MDCK or HEK293 cell line.
• primary cells may be isolated and used ex vivo for reintroduction into the subject to be treated following treatment with nuclease systems (e.g. CRISPR/Cas).
• Suitable primary cells include peripheral blood mononuclear cells (PBMC), and other blood cell subsets such as, but not limited to, CD4+ T cells or CD8+ T cells.
• Suitable cells also include stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neuronal stem cells and mesenchymal stem cells.
• stem cells are used in ex vivo procedures for cell transfection and gene therapy.
• the advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow.
• Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN-. gamma, and TNF-alpha are known (as a non-limiting example see, Inaba et al., J. Exp. Med. 176: 1693-1702 (1992)).
• Stem cells are isolated for transduction and differentiation using known methods.
• stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+(panB cells), GR-1 (granulocytes), and lad (differentiated antigen presenting cells) (as a non-limiting example see Inaba et al., J. Exp. Med. 176: 1693-1702 (1992)).
• stem cells that have been modified may also be used in some embodiments.
• any one of the OMNI-79 variant described herein may be suitable for genome editing in post-mitotic cells or any cell which is not actively dividing, e.g., arrested cells.
• Examples of post-mitotic cells which may be edited using an OMNI-79 variant of the present invention include, but are not limited to, myocyte, a cardiomyocyte, a hepatocyte, an osteocyte and a neuron.
• Vectors e.g., retroviruses, liposomes, etc.
• 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 any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
• Vectors suitable for introduction of transgenes into immune cells include non-integrating lentivirus vectors. See, for example, U.S. Patent Publication No. 2009/0117617.
• compositions are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington’s Pharmaceutical Sciences, 17 th ed., 1989).
• a variant OMNI-79 nuclease is utilized to affect a DNA break at a target site to induce cellular repair mechanisms, for example, but not limited to, non-homologous end-joining (NHEJ) or homology-directed repair (HDR).
• NHEJ non-homologous end-joining
• HDR homology-directed repair
• HDR refers to a mechanism for repairing 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” (nucleic acid template or donor template used interchangeably herein) to repair the sequence where the doublestranded or single break occurred (e.g., DNA target sequence). This results in the transfer of genetic information from, for example, the nucleic acid template to the DNA target sequence.
• HDR may result in alteration of the DNA target sequence (e.g., insertion, deletion, mutation) if the nucleic acid template sequence differs from the DNA target sequence and part or all of the nucleic acid template polynucleotide or oligonucleotide is incorporated into the DNA target sequence.
• an entire nucleic acid template polynucleotide, a portion of the nucleic acid template polynucleotide, or a copy of the nucleic acid template is integrated at the site of the DNA target sequence.
• the terms "nucleic acid template” and “donor”, refer to a nucleotide sequence that is inserted or copied into a genome.
• the nucleic acid template comprises a nucleotide sequence, e.g., of one or more nucleotides, that will be added to or will template a change in the target nucleic acid or may be used to modify the target sequence.
• a nucleic acid template sequence may be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value there between or there above), preferably between about 100 and 1,000 nucleotides in length (or any integer there between), more preferably between about 200 and 500 nucleotides in length.
• a nucleic acid template may be a single stranded nucleic acid, a double stranded nucleic acid.
• the nucleic acid template comprises a nucleotide sequence, e.g., of one or more nucleotides, that corresponds to wild type sequence of the target nucleic acid, e.g., of the target position.
• the nucleic acid template comprises a ribonucleotide sequence, e.g., of one or more ribonucleotides, that corresponds to wild type sequence of the target nucleic acid, e.g., of the target position.
• the nucleic acid template comprises modified ribonucleotides.
• donor sequence also called a "donor sequence,” donor template” or “donor”
• donor sequence is typically not identical to the genomic sequence where it is placed.
• a donor sequence can contain a non-homologous sequence flanked by two regions of homology to allow for efficient HDR at the location of interest.
• donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin.
• a donor molecule can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a donor nucleic acid molecule and flanked by regions of homology to sequence in the region of interest.
• the donor polynucleotide can be DNA or RNA, single- stranded and/or doublestranded and can be introduced into a cell in linear or circular form. See, e.g., U.S. Patent Publication Nos. 2010/0047805; 2011/0281361; 2011/0207221; and 2019/0330620. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3’ terminus of a linear molecule and/or self- complementary oligonucleotides are ligated to one or both ends.
• Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified intemucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.
• a geneediting composition comprises: (1) an RNA molecule comprising a guide sequence to affect a double strand break in a gene prior to repair and (2) a donor RNA template for repair, and the RNA molecule comprising the guide sequence is a first RNA molecule and the donor RNA template is a second RNA molecule.
• the guide RNA molecule and template RNA molecule are connected as part of a single molecule.
• a donor sequence may also be an oligonucleotide and be used for gene correction or targeted alteration of an endogenous sequence.
• the oligonucleotide may be introduced to the cell on a vector, may be electroporated into the cell, or may be introduced via other methods known in the art.
• the oligonucleotide can be used to correct' a mutated sequence in an endogenous gene (e.g., the sickle mutation in beta globin), or may be used to insert sequences with a desired purpose into an endogenous locus.
• a polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance.
• donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with or packaged within an agent such as a liposome, exosome, or pol oxamer, or can be delivered by recombinant viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)) or virus-like particles.
• Non-viral vectors such as transposon-based systems, e.g.
• the donor is generally inserted so that its expression is driven by the endogenous promoter at the integration site, namely the promoter that drives expression of the endogenous gene into which the donor is inserted.
• the donor may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue specific promoter.
• the donor molecule may be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed.
• a transgene as described herein may be inserted into an endogenous locus such that some (N-terminal and/or C-terminal to the transgene) or none of the endogenous sequences are expressed, for example as a fusion with the transgene.
• the transgene (e.g., with or without additional coding sequences such as forthe endogenous gene) is integrated into any endogenous locus, for example a safe-harbor locus, for example a CCR5 gene, a CXCR4 gene, a PPPlR12c (also known as AAVS1) gene, an albumin gene or a Rosa gene.
• a safe-harbor locus for example a CCR5 gene, a CXCR4 gene, a PPPlR12c (also known as AAVS1) gene, an albumin gene or a Rosa gene. See, e.g., U.S. Patent Nos. 7,951,925 and 8,110,379; U.S. Publication Nos.
• the endogenous sequences When endogenous sequences (endogenous or part of the transgene) are expressed with the transgene, the endogenous sequences may be full-length sequences (wild-type or mutant) or partial sequences. Preferably the endogenous sequences are functional. Non-limiting examples of the function of these full length or partial sequences include increasing the serum half-life of the polypeptide expressed by the transgene (e.g., therapeutic gene) and/or acting as a carrier.
• exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.
• 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 lacking in the cell or in the individual or an alternate version of a gene encoding a protein), a regulatory sequence and/or a sequence that encodes a structural nucleic acid such as a microRNA or siRNA.
• a gene encoding a protein e.g., a coding sequence encoding a protein that is lacking in the cell or in the individual or an alternate version of a gene encoding a protein
• a regulatory sequence e.g., a coding sequence encoding a protein that is lacking in the cell or in the individual or an alternate version of a gene encoding a protein
• a regulatory sequence e.g., a coding sequence encoding a protein that is lacking in the cell or in the individual or an alternate version of a gene encoding a protein
• a regulatory sequence e.g.,
• a DNA-targeting RNA molecule comprises a guide sequence portion.
• the “guide sequence portion” of an RNA molecule refers to a nucleotide sequence that is capable of hybridizing to a specific target DNA sequence, e.g., the guide sequence portion has a nucleotide sequence which is partially or fully complementary to the DNA sequence being targeted along the length of the guide sequence portion.
• 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 in length, or approximately 17-50,
• the entire length of the guide sequence portion is fully complementary to the DNA sequence being targeted along the length of the guide sequence portion.
• the guide sequence portion may be part of an RNA molecule that can form a complex with a CRISPR nuclease with the guide sequence portion serving as the DNA targeting portion of the CRISPR complex.
• the RNA molecule is capable of targeting the CRISPR nuclease to the specific target DNA sequence.
• RNA molecule can be custom designed to target any desired sequence. Accordingly, a molecule comprising 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 comprising a guide sequence portion, and the term “spacer” is synonymous with a “guide sequence portion.”
• the disclosed methods comprise a method of modifying a nucleotide sequence at a target site in a cell-free system or the genome of a cell comprising introducing into the cell the composition of any one of the embodiments described herein.
• the cell is a eukaryotic cell, preferably a mammalian cell or a plant cell.
• genome modifying occurs within the nucleus of a cell.
• the disclosed methods comprise a use of any one of the compositions described herein for the treatment of a subject afflicted with a disease associated with a genomic mutation comprising modifying a nucleotide sequence at a target site in the genome of the subj ect.
• the disclosed methods comprise a method of treating subject having a mutation disorder comprising targeting any one of the compositions described herein to an allele associated with the mutation disorder.
• the mutation disorder is related to a disease or disorder selected from any of a neoplasia, age-related macular degeneration, schizophrenia, neurological, neurodegenerative, or movement disorder, 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, cell dysregulation and oncology diseases and disorders, inflammation and immune-related diseases and disorders, metabolic, liver, kidney and protein diseases and disorders, muscular and skeletal diseases and disorders, dermatological diseases and disorders, neurological and neuronal diseases and disorders, and ocular diseases and disorders.
• a disease or disorder selected from any of a neoplasia, age-related macular degeneration, schizophrenia, neurological, neurodegenerative, or movement disorder, Fragile X Syndrome, secretase-related disorders, prion- related disorders, ALS, addiction, autism, Alzheimer’s Disease, neutr
• Certain embodiments of the invention target a nuclease to a specific genetic locus associated with a disease or disorder as a form of gene editing, method of treatment, or therapy.
• a novel nuclease disclosed herein may be specifically targeted to a pathogenic mutant allele of the gene using a custom designed guide RNA molecule.
• the guide RNA molecule is preferably designed by first considering the PAM requirement of the nuclease, which as shown herein is also dependent on the system in which the gene editing is being performed.
• a guide RNA molecule designed to target an OMNI- 79 nuclease to a target site is designed to contain a spacer sequence complementary to a DNA strand of a DNA double-stranded region that neighbors a OMNI-79 PAM sequence, e.g. “NGG.”
• the guide RNA molecule is further preferably designed to contain a spacer region (i.e. the region of the guide RNA molecule having complementarity to the target allele) of sufficient and preferably optimal length in order to increase specific activity of the nuclease and reduce off-target effects.
• the guide RNA molecule may be designed to target the nuclease to a specific region of a mutant allele, e.g. near the start codon, such that upon DNA damage caused by the nuclease a non-homologous end joining (NHEJ) pathway is induced and leads to silencing of the mutant allele by introduction of frameshift mutations.
• NHEJ non-homologous end joining
• the guide RNA molecule may be designed to target a specific pathogenic mutation of a mutated allele, such that upon DNA damage caused by the nuclease a homology directed repair (HDR) pathway is induced and leads to template mediated correction of the mutant allele.
• HDR homology directed repair
• Non-limiting examples of specific genes which may be targeted for alteration to treat a disease or disorder are presented herein below.
• Specific disease-associated genes and mutations that induce a mutation disorder are described in the literature.
• Such mutations can be used to design a DNA-targeting RNA molecule to target a CRISPR composition to an allele of the disease associated gene, where the CRISPR composition causes DNA damage and induces a DNA repair pathway to alter the allele and thereby treat the mutation disorder.
• Mutations in the ELANE gene are associated with neutropenia. Accordingly, without limitation, embodiments of the invention that target ELANE may be used in methods of treating subjects afflicted with neutropenia. Guide RNA molecules which target the ELANE gene and are useful for treating neutropenia are disclosed in PCT International Application No. PCT/US2020/059186, incorporated herein by reference.
• CXCR4 is a co-receptor for the human immunodeficiency virus type 1 (HIV-1) infection. Accordingly, without limitation, embodiments of the invention that target CXCR4 may be used in methods of treating subjects afflicted with HIV-1 or conferring resistance to HIV-1 infection in a subject.
• HIV-1 human immunodeficiency virus type 1
• PD-1 disruption enhances CAR-T cell mediated killing of tumor cells and PD-1 may be a target in other cancer therapies. Accordingly, without limitation, embodiments of the invention that target PD-1 may be used in methods of treating subjects afflicted with cancer. In an embodiment, the treatment is CAR-T cell therapy with T cells that have been modified according to the invention to be PD-1 deficient.
• BCL11A is a gene that plays a role in the suppression of hemoglobin production. Globin production may be increased to treat diseases such as thalassemia or sickle cell anemia by inhibiting BCL11A. See for example, PCT International Publication No. WO 2017/077394 A2; U.S. Publication No. US2011/0182867A1; Humbert et al. Sci. Transl. Med. (2019); and Canver et al. Nature (2015). Accordingly, without limitation, embodiments of the invention that target an enhancer of BCL11 A may be used in methods of treating subjects afflicted with beta thalassemia or sickle cell anemia.
• Embodiments of the invention may also be used for targeting any disease-associated gene, for studying, altering, or treating any of the diseases or disorders listed in Table A or Table B below. Indeed, any disease-associated with a genetic locus may be studied, altered, or treated by using the nucleases disclosed herein to target the appropriate disease-associated gene, for example, those listed in U.S. Publication No. 2018/0282762A1 and European Patent No. EP3079726B1.
• each of the verbs, “comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.
• Other terms as used herein are meant to be defined by their well-known meanings in the art.
• the term “targeting sequence” or “targeting molecule” refers a nucleotide sequence or molecule comprising a nucleotide sequence that is capable of hybridizing to a specific target sequence, e.g., the targeting sequence has a nucleotide sequence which is at least partially complementary to the sequence being targeted along the length of the targeting sequence.
• the targeting sequence or targeting molecule may be part of an RNA molecule that can form a complex with a CRISPR nuclease, either alone or in combination with other RNA molecules, with the targeting sequence serving as the targeting portion of the CRISPR complex.
• the RNA molecule alone or in combination with an additional one or more RNA molecules (e.g. a tracrRNA molecule), is capable of targeting the CRISPR nuclease to the specific target sequence.
• a guide sequence portion of a CRISPR RNA molecule or single-guide RNA molecule may serve as a targeting molecule.
• a targeting sequence can be custom designed to target any desired sequence.
• targets refers to preferentially hybridizing a targeting sequence of a targeting molecule to a nucleic acid having a targeted nucleotide sequence. It is understood that the term “targets” encompasses variable hybridization efficiencies, such that there is preferential targeting of the nucleic acid having the targeted nucleotide sequence, but unintentional off-target hybridization in addition to on-target hybridization might also occur. It is understood that where an RNA molecule targets a sequence, a complex of the RNA molecule and a CRISPR nuclease molecule targets the sequence for nuclease activity.
• wild-type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms. Accordingly, as used herein, where a sequence of amino acids or nucleotides refers to a wild-type sequence, a variant refers to variant of that sequence, e.g., comprising substitutions, deletions, insertions.
• an engineered CRISPR nuclease is a variant CRISPR nuclease comprising at least one amino acid modification (e.g., substitution, deletion, and/or insertion), also referred to as a “mutation,” compared to the wiki-type OMNI-79 nuclease of SEQ ID NO: 1.
• nucleic acid molecules or polypeptides may mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
• mutant or “variant” are used interchangeably and indicate a molecule that is non-naturally occurring or engineered.
• amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and both the D- or L-, optical isomers, and amino acid analogs and peptidomimetics.
• genomic DNA refers to linear and/or chromosomal DNA and/or to plasmid or other extrachromosomal DNA sequences present in the cell or cells of interest.
• the cell of interest is a eukaryotic cell.
• the cell of interest is a prokaryotic cell.
• the methods produce double-stranded breaks (DSBs) at pre-determined target sites in a genomic DNA sequence, resulting in mutation, insertion, and/or deletion of DNA sequences at the target site(s) in a genome.
• Eukaryotic cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells.
• modified cells refers to cells in which a double strand break is affected by a complex of an RNA molecule and the CRISPR nuclease variant as a result of hybridization with the target sequence, i.e. on-target hybridization.
• modified cells may further encompass cells in which a repair or correction of a mutation was affected following the double strand break induced by the variant.
• the modified cell may be any type of cell e.g., eukaryotic or prokaryotic, in any environment e.g., isolated or not, maintained in culture, in vitro, ex vivo, in vivo or in planta.
• This invention provides a modified cell or cells obtained by use of any of the variants or methods described herein. In an embodiment these modified cell or cells are capable of giving rise to progeny cells. In an embodiment these modified cell or cells are capable of giving rise to progeny cells after engraftment.
• the modified cells may be hematopoietic stem cell (HSC), or any cell suitable for an allogenic cell transplant or autologous cell transplant.
• HSC hematopoietic stem cell
• the variants and methods described herein may also be utilized to generate chimeric antigen receptor T (CAR-T) cells.
• This invention also provides a composition comprising these modified cells and a pharmaceutically acceptable carrier. Also provided is an in vitro or ex vivo method of preparing this, comprising mixing the cells with the pharmaceutically acceptable carrier.
• nuclease refers to an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acid.
• a nuclease may be isolated or derived from a natural source. The natural source may be any living organism. Alternatively, a nuclease may be a modified or a synthetic protein which retains the phosphodiester bond cleaving activity.
• PAM protospacer adjacent motif
• the terms “protospacer adjacent motif’ or “PAM” as used herein refers to a nucleotide sequence of a target DNA located in proximity to the targeted DNA sequence and recognized by the CRISPR nuclease.
• the PAM sequence may differ depending on the nuclease identity.
• wild-type Streptococcus pyogenes Cas9 recognizes a “NGG” PAM sequence.
• a skilled artisan will appreciate that single-guide RNA molecules or crRNA:tracrRNA complexes capable of complexing with a CRISPR nuclease such as to associate with a target genomic DNA sequence of interest next to a protospacer adjacent motif (PAM).
• PAM protospacer adjacent motif
• sequence or molecule has an X% “sequence identity” to another sequence or molecule if X% of bases or amino acids between the sequences of molecules are the same and in the same relative position.
• sequence identity X% of bases or amino acids between the sequences of molecules are the same and in the same relative position.
• a first nucleotide sequence having at least a 95% sequence identity with a second nucleotide sequence will have at least 95% of bases, in the same relative position, identical with the other sequence.
• nuclear localization sequence and "NLS” are used interchangeably to indicate an amino acid sequence/peptide that directs the transport of a protein with which it is associated from the cytoplasm of a cell across the nuclear envelope barrier.
• the term “NLS” is intended to encompass not only the nuclear localization sequence of a particular peptide, but also derivatives thereof that are capable of directing translocation of a cytoplasmic polypeptide across the nuclear envelope barrier.
• NLSs are capable of directing nuclear translocation of a polypeptide when attached to the N-terminus, the C-terminus, or both the N- and C-termini of the polypeptide.
• an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, but other types of NLS are known.
• Non- limiting examples of NLSs include an NLS sequence derived from: the SV40 virus large T-antigen, nucleoplasmin, c-myc, the hRNPAl M9 NLS, the IBB domain from importin-alpha, myoma T protein, human p53, mouse c- abl IV, influenza vims NS1, Hepatitis virus delta antigen, mouse Mxl protein, human poly(ADP- ribose) polymerase, and the steroid hormone receptors (human) glucocorticoid.
• NLSs include an NLS sequence derived from: the SV40 virus large T-antigen, nucleoplasmin, c-myc, the hRNPAl M9 NLS, the IBB domain from importin-alpha, myoma T protein, human p53, mouse c- abl IV, influenza vims NS1, Hepatitis virus delta antigen, mouse Mxl protein, human poly(ADP- ribose)
• CRISPR system refers to a CRISPR endonuclease system that includes a CRISPR nuclease protein, such as the mutants or variants described herein, and a suitable guide RNA molecule or guide RNA complex, e.g. a single-guide RNA or a crRNA:tracrRNA complex, for targeting 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.
• a suitable guide RNA molecule or guide RNA complex e.g. a single-guide RNA or a crRNA:tracrRNA complex
• wild-type CRISPR endonuclease system refers to a CRISPR endonuclease system that includes wild-type CRISPR protein and a suitable guide RNA molecule or guide RNA complex, e.g. a single-guide RNA or a crRNA:tracrRNA complex, for targeting 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.
• a suitable guide RNA molecule or guide RNA complex e.g. a single-guide RNA or a crRNA:tracrRNA complex
• “maintained on-target editing activity” refers to the ability of an OMNL79 variant to target a DNA target site that is targeted by a guide RNA molecule associated with, and thereby programming, the OMNI-79 variant.
• the OMNI-79 variant maintains on-target editing activity of a DNA target at a percent editing level greater than or equal to the percent editing level of a wild-type OMNI-79 nuclease for the DNA target.
• the OMNI-79 variant maintains on-target editing activity of a DNA target of at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% the level of percent editing of a wild-type OMNI-79 nuclease for the DNA target.
• each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiment.
• any of the RNA molecules or compositions of the present invention may be utilized in any of the methods of the present invention.
• all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.
• the open reading frame of the wild-type OMNI-79 CRIPSR nuclease was codon optimized for human cell line expression (SEQ ID NO: 1) and cloned into a dual expression plasmid (pShuttle) that enables both bacterial and mammalian expression using a T7 or a CMV promoter, respectively.
• a full gene library with combinatorial random mutations along the full length OMNI-79 open reading frame (ORF) was constructed using incorporated oligos with an NNK degenerate codon at each position in the OMNI-79 sequence, resulting in a library having an average of 2.6 amino acid substitutions per ORF.
• a positive selection bacterial system was designed.
• a positive selection plasmid was electroporated into Escherichia coli strain BW25141 (1DE3) to create the positive selection bacterial strain.
• the positive selection plasmid contains a T7-expressed single-guide RNA (sgRNA) and an embedded on-target site.
• the sequence of the target site which is a sequence located within Exon 5 of the human Serpina gene, and the spacer and scaffold sequences of the guide RNA molecule, are listed in Table 1.
• the positive selection plasmid also contains a chloramphenicol resistance cassette and expresses the E.
• transformed bacteria are plated on selective TB plates containing carbenicillin and 15mM arabinose and incubated overnight at 37° C. The next morning the surviving pool is collected, plasmids are isolated, and re-transformed into the positive selection bacterial strain for another round of selection. Seven rounds of positive selection were performed, and after the final round single bacterial colonies were randomly picked and fully sequenced.
• OMNI-79 CRISPR nuclease variants that were highly enriched after the bacterial selection were isolated and both showed an increased activity relative to the wild-type OMNI-79 CRISPR nuclease in HeLa cells as indicated in Table 3.
• OMNI-79 Variant 5570 also showed high activity when tested in Neuro-2a mouse neuroblastoma (mN2A) cells using an mSARMl target (see Table 3).
• V5570 and V5603 are highly active variants of the CRISPR OMNI-79 nuclease (see Table 3, above).
• the editing activity of variant V5570 was tested on several targeted hLDLR gene sites using DNA transfection in HeLa cells (Table 6).
• OMNI-79 variant V5570 showed higher editing activity in comparison to wild-type OMNI-79 (Fig. 1, Table 8).
• OMNI-79 variants V5570 and V5603 each contain two mutations.
• single-mutation variants were generated and their activity was tested. Specifically, the activity of four single-mutation variants were tested via DNA transfection in HeLa cells on three targeted genomic sites: hSERP_gl2R, hLDLR_g46 and hLDLR_g76 (Table 6). Only variants containing either a S1005R or S1005K mutation showed higher editing activity in comparison to wild-type OMNI-79 (Figs. 2A-2C, Table 8). These results demonstrate that the mutations at position 1005 are responsible for the higher activity observed for variants V5570 and V5603.
• OMNI-79 nuclease and its variant nucleases were expressed in a mammalian cell system (HeLa) by DNA transfection together with an sgRNA-expressing plasmid. Each sgRNA is composed of a tracrRNA portion and a spacer portion. The spacer 3’ genomic sequence contains the expected PAM relevant for OMNI- 79 nuclease. All assays were performed in triplicate. ‘OMNI nuclease only’ (i.e. no guide) transfected cells served as a negative control. Cell lysates were used for site specific genomic DNA amplification and NGS analysis.
• OMNI-79 V5570 was tested as part of a ribonucleoprotein (RNP) complex in HepG2 cells targeting several sites in the LDLR gene. In all cases the editing as calculated by NGS was high, ranging from 58.5% to up to 84.3% (Fig. 4, Table 6). Also, OMNI- 79 V5570 was tested in transfection and electroporation experiments by delivering an mRNA molecule which encoded the variant nuclease. Again, in all cases a high level of editing was observed across three (3) different genes (Fig. 5, Fig. 7, Table 7). Finally, OMNI-79 V5570 was delivered using viral transduction (Fig. 6, Table 8).
• RNP ribonucleoprotein
• OMNI-79 nuclease open reading frame was codon optimized to bacteria (Table 1) and cloned into pNNC plasmid with the following elements - SV40 NLS - OMNI-79 ORF bacterial optimized - HA tag - SV40 NLS - 8 His-tag (Table 4).
• the OMNI-79 construct was expressed in KRX cells (PROMEGA). Cells were grown in TB, 0.4% Glycerol with addition of 6.66 mM Rhamnose, 0.05% glucose and carb antibiotics. Induction was performed in mid-log phase, after 4 hours by temperature reduction to 18°C.
• HiScribe T7 High Yield RNA Synthesis Kit (NEB# E2040S) was used with Nl- Methylpseudouridine-5'-Triphosphate and CleanCap to produce OMNI-79 V5570-encoding mRNA, following the manufacture’s instructions. The yield was approximately 150 pg mRNA.
• HeLa cells were seeded to be 70-90% confluent at the time of transfection. Lipofectamine 3000 was used to transfect the IVT mRNA with synthetic gRNAs, per the manufacturer’s instructions. 72 hours after transfection, cells were dissociated with trypsin and genomic extract was prepared with QuickExtract solution according to manufacturer’s instructions. InDeis percentage analysis was performed by NGS.
• complexes were assembled by mixing 105 pmol of purified protein with 124 pmol of sgRNA and 100 pM Cas9 electroporation enhancer (IDT). After 10 minutes of incubation at 25°C, the RNP complexes were mixed with 4xl0 5 pre-washed HepG2 cells and electroporated using a Lonza SF Cell Line 4D-NucleofectorTM X Kit with the DS-123 program for HepG2 cells, according to the manufacturer’s instructions. Cells were replated in 12- well tissue culture plates and incubated in a TC incubator (37°C, 5% CO2). Cells were harvested 72 hours post-electroporation. Cell lysis and genomic DNA extraction was performed using Quick extract (Lucigen) and endogenous genomic regions were amplified using specific primers to measure on target activity by NGS (Fig. 4, Table 3).
• IDT Cas9 electroporation enhancer
• AAV-DJ viruses that harbored OMNI- 79 V5570 and corresponding sgRNA molecules were produced and purified (VectorBuilder). HepG2 and Hepal-6 cells were infected by AAV-DJ harboring corresponding sgRNAs at an of MOI IxlO 5 and 3xl0 5 , respectively. Cells with the addition of viral particles were incubated in TC incubator (37°C, 5% CO2) overnight. The next day cells were washed and incubated with fresh medium for another 48 hours. Cells were harvested 72 hours post-infection.

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